Thermodynamic Analysis of Conserved Loop−Stem Interactions in P1−P2 Frameshifting RNA Pseudoknots from Plant Luteoviridae†
Identifieur interne : 002233 ( Istex/Corpus ); précédent : 002232; suivant : 002234Thermodynamic Analysis of Conserved Loop−Stem Interactions in P1−P2 Frameshifting RNA Pseudoknots from Plant Luteoviridae†
Auteurs : Paul L. Nixon ; Peter V. Cornish ; Saritha V. Suram ; David P. GiedrocSource :
- Biochemistry [ 0006-2960 ] ; 2002.
Abstract
The RNA genomes of plant luteovirids beet western yellows virus (BWYV), potato leaf roll virus (PLRV), and pea enation mosaic virus (PEMV RNA1; PEMV-1) contain a short mRNA pseudoknotted motif overlapping the P1 and P2 open reading frames required for programmed −1 mRNA ribosomal frameshifting. The relationship between structure, stability, and function is poorly understood in these RNA systems. A m5-C8-substituted BWYV RNA is employed to establish that the BWYV P1−P2 pseudoknot is protonated at cytidine 8 in loop L1 (δN3H+ = 12.98 ppm), which stabilizes a C+·(G−C) major groove base triple by Δ(ΔG37)protonation = 3.1 (±0.4) kcal mol-1. The stabilities of both the PLRV and PEMV-1 P1−P2 pseudoknots are also strongly pH-dependent, with Δ(ΔG37)protonation = 2.1 (±0.2) kcal mol-1 for the PEMV-1 pseudoknot despite a distinct structural context. As previously found for the BWYV pseudoknot [Nixon and Giedroc (2000) J. Mol. Biol. 296, 659], both the PLRV and PEMV-1 RNAs are stabilized by ΔH ≥ 30 kcal mol-1 in excess of secondary structure predictions, attributed to loop L2−stem S1 minor groove triplex interactions. BWYV RNAs containing single 2‘-deoxy or A → G substitutions that disrupt L2−S1 hydrogen bonding are strongly destabilized with Δ(ΔG37)folding (pH = 7.0) ranging from ≈1.8 (±0.3) to ≥4.0 kcal mol-1, relative to the wild-type BWYV RNA. These findings suggest that each member of this family of pseudoknots adopts a tightly folded structure that maximizes the cooperativity and complementarity of L1−S2 and L2−S1 loop−stem interactions required in part to offset the low intrinsic stability of the short three base pair pseudoknot stem S2.
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DOI: 10.1021/bi025843c
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Frameshifting RNA Pseudoknots from Plant <hi rend="italic">Luteoviridae</hi><ref type="bib" target="#bi025843cAF2"><hi rend="superscript">†</hi></ref></title><author><name sortKey="Nixon, Paul L" sort="Nixon, Paul L" uniqKey="Nixon P" first="Paul L." last="Nixon">Paul L. Nixon</name><affiliation><mods:affiliation>Department of Biochemistry and Biophysics, Center for Advanced Biomolecular Research, Texas A&M University,College Station, Texas 77843-2128</mods:affiliation></affiliation><affiliation><mods:affiliation> Current address: Department of Biochemistry, University of TexasSouthwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas,TX 75390-9038.</mods:affiliation></affiliation></author><author><name sortKey="Cornish, Peter V" sort="Cornish, Peter V" uniqKey="Cornish P" first="Peter V." last="Cornish">Peter V. Cornish</name><affiliation><mods:affiliation>Department of Biochemistry and Biophysics, Center for Advanced Biomolecular Research, Texas A&M University,College Station, Texas 77843-2128</mods:affiliation></affiliation></author><author><name sortKey="Suram, Saritha V" sort="Suram, Saritha V" uniqKey="Suram S" first="Saritha V." last="Suram">Saritha V. Suram</name><affiliation><mods:affiliation>Department of Biochemistry and Biophysics, Center for Advanced Biomolecular Research, Texas A&M University,College Station, Texas 77843-2128</mods:affiliation></affiliation></author><author><name sortKey="Giedroc, David P" sort="Giedroc, David P" uniqKey="Giedroc D" first="David P." last="Giedroc">David P. Giedroc</name><affiliation><mods:affiliation>Department of Biochemistry and Biophysics, Center for Advanced Biomolecular Research, Texas A&M University,College Station, Texas 77843-2128</mods:affiliation></affiliation><affiliation><mods:affiliation> Corresponding author. E-mail: giedroc@tamu.edu. Telephone: 979-845-4231. Fax: 979-845-4946.</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j" type="main">Biochemistry</title><title level="j" type="abbrev">Biochemistry</title><idno type="ISSN">0006-2960</idno><idno type="eISSN">1520-4995</idno><imprint><publisher>American Chemical Society</publisher><date type="e-published">2002</date><date type="published">2002</date><biblScope unit="vol">41</biblScope><biblScope unit="issue">34</biblScope><biblScope unit="page" from="10665">10665</biblScope><biblScope unit="page" to="10674">10674</biblScope></imprint><idno type="ISSN">0006-2960</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0006-2960</idno></seriesStmt></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract">The RNA genomes of plant luteovirids beet western yellows virus (BWYV), potato leaf roll virus (PLRV), and pea enation mosaic virus (PEMV RNA1; PEMV-1) contain a short mRNA pseudoknotted motif overlapping the P1 and P2 open reading frames required for programmed −1 mRNA ribosomal frameshifting. The relationship between structure, stability, and function is poorly understood in these RNA systems. A m5-C8-substituted BWYV RNA is employed to establish that the BWYV P1−P2 pseudoknot is protonated at cytidine 8 in loop L1 (δN3H+ = 12.98 ppm), which stabilizes a C+·(G−C) major groove base triple by Δ(ΔG37)protonation = 3.1 (±0.4) kcal mol-1. The stabilities of both the PLRV and PEMV-1 P1−P2 pseudoknots are also strongly pH-dependent, with Δ(ΔG37)protonation = 2.1 (±0.2) kcal mol-1 for the PEMV-1 pseudoknot despite a distinct structural context. As previously found for the BWYV pseudoknot [Nixon and Giedroc (2000) J. Mol. Biol. 296, 659], both the PLRV and PEMV-1 RNAs are stabilized by ΔH ≥ 30 kcal mol-1 in excess of secondary structure predictions, attributed to loop L2−stem S1 minor groove triplex interactions. BWYV RNAs containing single 2‘-deoxy or A → G substitutions that disrupt L2−S1 hydrogen bonding are strongly destabilized with Δ(ΔG37)folding (pH = 7.0) ranging from ≈1.8 (±0.3) to ≥4.0 kcal mol-1, relative to the wild-type BWYV RNA. These findings suggest that each member of this family of pseudoknots adopts a tightly folded structure that maximizes the cooperativity and complementarity of L1−S2 and L2−S1 loop−stem interactions required in part to offset the low intrinsic stability of the short three base pair pseudoknot stem S2.</div></front></TEI><istex><corpusName>acs</corpusName><keywords><teeft><json:string>pseudoknot</json:string><json:string>bwyv</json:string><json:string>kcal</json:string><json:string>pseudoknots</json:string><json:string>frameshifting</json:string><json:string>plrv</json:string><json:string>bwyv pseudoknot</json:string><json:string>biol</json:string><json:string>biochemistry</json:string><json:string>protonated</json:string><json:string>rna</json:string><json:string>helical</json:string><json:string>protonation</json:string><json:string>cytidine</json:string><json:string>base pair</json:string><json:string>giedroc</json:string><json:string>datum</json:string><json:string>plrv pseudoknot</json:string><json:string>base triple</json:string><json:string>helical junction</json:string><json:string>ribosomal</json:string><json:string>pseudoknotted</json:string><json:string>sequential</json:string><json:string>triplex</json:string><json:string>luteoviral</json:string><json:string>mrna</json:string><json:string>mutant</json:string><json:string>amino proton</json:string><json:string>solid line</json:string><json:string>tertiary</json:string><json:string>pseudoknotted conformation</json:string><json:string>imino proton</json:string><json:string>ribosomal frameshifting</json:string><json:string>minor groove triplex</json:string><json:string>bwyv rna</json:string><json:string>unmodified bwyv pseudoknot</json:string><json:string>thermodynamic</json:string><json:string>groove</json:string><json:string>exchangeable proton region</json:string><json:string>hydrogen bond</json:string><json:string>luteoviral frameshifting pseudoknots biochemistry</json:string><json:string>thermodynamic evidence</json:string><json:string>tertiary structure</json:string><json:string>thermodynamic parameter</json:string><json:string>enation mosaic virus</json:string><json:string>thermodynamic analysis</json:string><json:string>single protonatable group</json:string><json:string>amino</json:string><json:string>proton</json:string><json:string>hairpin</json:string><json:string>global stability</json:string><json:string>individual transition</json:string><json:string>entire dependence</json:string><json:string>potato leaf roll virus</json:string><json:string>triple base pair</json:string><json:string>buffer salt</json:string><json:string>nucleic acid</json:string><json:string>varian inova</json:string><json:string>free cytidine</json:string><json:string>complex point</json:string><json:string>substitution</json:string><json:string>triple</json:string></teeft></keywords><author><json:item><name>NIXON Paul L.</name><affiliations><json:string>Department of Biochemistry and Biophysics, Center for Advanced Biomolecular Research, Texas A&M University,College Station, Texas 77843-2128</json:string><json:string>Current address: Department of Biochemistry, University of TexasSouthwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas,TX 75390-9038.</json:string></affiliations></json:item><json:item><name>CORNISH Peter V.</name><affiliations><json:string>Department of Biochemistry and Biophysics, Center for Advanced Biomolecular Research, Texas A&M University,College Station, Texas 77843-2128</json:string></affiliations></json:item><json:item><name>SURAM Saritha V.</name><affiliations><json:string>Department of Biochemistry and Biophysics, Center for Advanced Biomolecular Research, Texas A&M University,College Station, Texas 77843-2128</json:string></affiliations></json:item><json:item><name>GIEDROC David P.</name><affiliations><json:string>Department of Biochemistry and Biophysics, Center for Advanced Biomolecular Research, Texas A&M University,College Station, Texas 77843-2128</json:string><json:string>Corresponding author. E-mail: giedroc@tamu.edu. Telephone: 979-845-4231. Fax: 979-845-4946.</json:string></affiliations></json:item></author><arkIstex>ark:/67375/TPS-76DVCBS5-1</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>research-article</json:string></originalGenre><abstract>The RNA genomes of plant luteovirids beet western yellows virus (BWYV), potato leaf roll virus (PLRV), and pea enation mosaic virus (PEMV RNA1; PEMV-1) contain a short mRNA pseudoknotted motif overlapping the P1 and P2 open reading frames required for programmed −1 mRNA ribosomal frameshifting. The relationship between structure, stability, and function is poorly understood in these RNA systems. A m5-C8-substituted BWYV RNA is employed to establish that the BWYV P1−P2 pseudoknot is protonated at cytidine 8 in loop L1 (δN3H+ = 12.98 ppm), which stabilizes a C+·(G−C) major groove base triple by Δ(ΔG37)protonation = 3.1 (±0.4) kcal mol-1. The stabilities of both the PLRV and PEMV-1 P1−P2 pseudoknots are also strongly pH-dependent, with Δ(ΔG37)protonation = 2.1 (±0.2) kcal mol-1 for the PEMV-1 pseudoknot despite a distinct structural context. As previously found for the BWYV pseudoknot [Nixon and Giedroc (2000) J. Mol. Biol. 296, 659], both the PLRV and PEMV-1 RNAs are stabilized by ΔH ≥ 30 kcal mol-1 in excess of secondary structure predictions, attributed to loop L2−stem S1 minor groove triplex interactions. BWYV RNAs containing single 2‘-deoxy or A → G substitutions that disrupt L2−S1 hydrogen bonding are strongly destabilized with Δ(ΔG37)folding (pH = 7.0) ranging from ≈1.8 (±0.3) to ≥4.0 kcal mol-1, relative to the wild-type BWYV RNA. These findings suggest that each member of this family of pseudoknots adopts a tightly folded structure that maximizes the cooperativity and complementarity of L1−S2 and L2−S1 loop−stem interactions required in part to offset the low intrinsic stability of the short three base pair pseudoknot stem S2.</abstract><qualityIndicators><score>10</score><pdfWordCount>6601</pdfWordCount><pdfCharCount>39263</pdfCharCount><pdfVersion>1.2</pdfVersion><pdfPageCount>10</pdfPageCount><pdfPageSize>612 x 792 pts (letter)</pdfPageSize><pdfWordsPerPage>660</pdfWordsPerPage><pdfText>true</pdfText><refBibsNative>true</refBibsNative><abstractWordCount>257</abstractWordCount><abstractCharCount>1660</abstractCharCount><keywordCount>0</keywordCount></qualityIndicators><title>Thermodynamic Analysis of Conserved Loop−Stem Interactions in P1−P2 Frameshifting RNA Pseudoknots from Plant Luteoviridae†</title><genre><json:string>research-article</json:string></genre><host><title>Biochemistry</title><language><json:string>unknown</json:string></language><issn><json:string>0006-2960</json:string></issn><eissn><json:string>1520-4995</json:string></eissn><volume>41</volume><issue>34</issue><pages><first>10665</first><last>10674</last></pages><genre><json:string>journal</json:string></genre></host><ark><json:string>ark:/67375/TPS-76DVCBS5-1</json:string></ark><categories><wos><json:string>1 - science</json:string><json:string>2 - biochemistry & molecular biology</json:string></wos><scienceMetrix><json:string>1 - health sciences</json:string><json:string>2 - biomedical research</json:string><json:string>3 - biochemistry & molecular biology</json:string></scienceMetrix><scopus><json:string>1 - Life Sciences</json:string><json:string>2 - Biochemistry, Genetics and Molecular Biology</json:string><json:string>3 - Biochemistry</json:string></scopus><inist><json:string>1 - sciences appliquees, technologies et medecines</json:string><json:string>2 - sciences biologiques et medicales</json:string><json:string>3 - sciences biologiques fondamentales et appliquees. psychologie</json:string><json:string>4 - phytopathologie. zoologie agricole. protection des cultures et des forets</json:string></inist></categories><publicationDate>2002</publicationDate><copyrightDate>2002</copyrightDate><doi><json:string>10.1021/bi025843c</json:string></doi><id>1AC18E5B035C5DF34B8387A5362A306048727D65</id><score>1</score><fulltext><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-76DVCBS5-1/fulltext.pdf</uri></json:item><json:item><extension>zip</extension><original>false</original><mimetype>application/zip</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-76DVCBS5-1/bundle.zip</uri></json:item><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-76DVCBS5-1/fulltext.txt</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/ark:/67375/TPS-76DVCBS5-1/fulltext.tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main">Thermodynamic Analysis of Conserved Loop−Stem Interactions in P1−P2
Frameshifting RNA Pseudoknots from Plant <hi rend="italic">Luteoviridae</hi><ref type="bib" target="#bi025843cAF2"><hi rend="superscript">†</hi></ref></title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher>American Chemical Society</publisher><availability><licence>Copyright © 2002 American Chemical Society</licence><p>American Chemical Society</p></availability><date type="published">2002</date><date type="Copyright" when="2002">2002</date></publicationStmt><notesStmt><note type="content-type" source="research-article" scheme="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</note><note type="publication-type" scheme="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</note></notesStmt><sourceDesc><biblStruct type="article"><analytic><title level="a" type="main">Thermodynamic Analysis of Conserved Loop−Stem Interactions in P1−P2
Frameshifting RNA Pseudoknots from Plant <hi rend="italic">Luteoviridae</hi><ref type="bib" target="#bi025843cAF2"><hi rend="superscript">†</hi></ref></title><author xml:id="author-0000"><persName><surname>Nixon</surname><forename type="first">Paul L.</forename></persName><affiliation><orgName type="division">Department of Biochemistry and Biophysics</orgName><orgName type="institution">Center for Advanced Biomolecular Research</orgName><orgName type="institution">Texas A&M University</orgName><orgName type="institution">College Station</orgName><address><addrLine>Texas 77843-2128</addrLine></address></affiliation><note place="foot" n="bi025843cAF3"><ref>‡</ref><p>
Current address: Department of Biochemistry, University of Texas
Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas,
TX 75390-9038.</p></note></author><author xml:id="author-0001"><persName><surname>Cornish</surname><forename type="first">Peter V.</forename></persName><affiliation><orgName type="division">Department of Biochemistry and Biophysics</orgName><orgName type="institution">Center for Advanced Biomolecular Research</orgName><orgName type="institution">Texas A&M University</orgName><orgName type="institution">College Station</orgName><address><addrLine>Texas 77843-2128</addrLine></address></affiliation></author><author xml:id="author-0002"><persName><surname>Suram</surname><forename type="first">Saritha V.</forename></persName><affiliation><orgName type="division">Department of Biochemistry and Biophysics</orgName><orgName type="institution">Center for Advanced Biomolecular Research</orgName><orgName type="institution">Texas A&M University</orgName><orgName type="institution">College Station</orgName><address><addrLine>Texas 77843-2128</addrLine></address></affiliation></author><author xml:id="author-0003" role="corresp"><persName><surname>Giedroc</surname><forename type="first">David P.</forename></persName><affiliation><orgName type="division">Department of Biochemistry and Biophysics</orgName><orgName type="institution">Center for Advanced Biomolecular Research</orgName><orgName type="institution">Texas A&M University</orgName><orgName type="institution">College Station</orgName><address><addrLine>Texas 77843-2128</addrLine></address></affiliation><affiliation role="corresp"> Corresponding author. E-mail: giedroc@tamu.edu. Telephone: 979-845-4231. Fax: 979-845-4946.</affiliation></author><idno type="istex">1AC18E5B035C5DF34B8387A5362A306048727D65</idno><idno type="ark">ark:/67375/TPS-76DVCBS5-1</idno><idno type="DOI">10.1021/bi025843c</idno></analytic><monogr><title level="j" type="main">Biochemistry</title><title level="j" type="abbrev">Biochemistry</title><idno type="acspubs">bi</idno><idno type="coden">bichaw</idno><idno type="pISSN">0006-2960</idno><idno type="eISSN">1520-4995</idno><imprint><publisher>American Chemical Society</publisher><date type="e-published">2002</date><date type="published">2002</date><biblScope unit="vol">41</biblScope><biblScope unit="issue">34</biblScope><biblScope unit="page" from="10665">10665</biblScope><biblScope unit="page" to="10674">10674</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><encodingDesc><schemaRef type="ODD" url="https://xml-schema.delivery.istex.fr/tei-istex.odd"></schemaRef><appInfo><application ident="pub2tei" version="1.0.41" when="2020-04-06"><label>pub2TEI-ISTEX</label><desc>A set of style sheets for converting XML documents encoded in various scientific publisher formats into a common TEI format.
<ref target="http://www.tei-c.org/">We use TEI</ref></desc></application></appInfo></encodingDesc><profileDesc><abstract><p>The RNA genomes of plant luteovirids beet western yellows virus (BWYV), potato leaf roll
virus (PLRV), and pea enation mosaic virus (PEMV RNA1; PEMV-1) contain a short mRNA pseudoknotted
motif overlapping the P1 and P2 open reading frames required for programmed −1 mRNA ribosomal
frameshifting. The relationship between structure, stability, and function is poorly understood in these
RNA systems. A m<hi rend="superscript">5</hi>-C<hi rend="subscript">8</hi>-substituted BWYV RNA is employed to establish that the BWYV P1−P2
pseudoknot is protonated at cytidine 8 in loop L1 (δ<hi rend="subscript">N</hi><hi rend="subscript"><hi rend="subscript">3</hi></hi><hi rend="subscript">H</hi><hi rend="subscript"><hi rend="superscript">+</hi></hi> = 12.98 ppm), which stabilizes a C<hi rend="superscript">+</hi>·(G−C)
major groove base triple by Δ(Δ<hi rend="italic">G</hi><hi rend="subscript">37</hi>)<hi rend="subscript">protonation</hi> = 3.1 (±0.4) kcal mol<hi rend="superscript">-1</hi>. The stabilities of both the PLRV
and PEMV-1 P1−P2 pseudoknots are also strongly pH-dependent, with Δ(Δ<hi rend="italic">G</hi><hi rend="subscript">37</hi>)<hi rend="subscript">protonation</hi> = 2.1 (±0.2)
kcal mol<hi rend="superscript">-1</hi> for the PEMV-1 pseudoknot despite a distinct structural context. As previously found for the
BWYV pseudoknot [Nixon and Giedroc (2000) <hi rend="italic">J. Mol. Biol. 296</hi>, 659], both the PLRV and PEMV-1
RNAs are stabilized by Δ<hi rend="italic">H</hi> ≥ 30 kcal mol<hi rend="superscript">-1</hi> in excess of secondary structure predictions, attributed to
loop L2−stem S1 minor groove triplex interactions. BWYV RNAs containing single 2‘-deoxy or A → G
substitutions that disrupt L2−S1 hydrogen bonding are strongly destabilized with Δ(Δ<hi rend="italic">G</hi><hi rend="subscript">37</hi>)<hi rend="subscript">folding</hi> (pH =
7.0) ranging from ≈1.8 (±0.3) to ≥4.0 kcal mol<hi rend="superscript">-1</hi>, relative to the wild-type BWYV RNA. These findings
suggest that each member of this family of pseudoknots adopts a tightly folded structure that maximizes
the cooperativity and complementarity of L1−S2 and L2−S1 loop−stem interactions required in part to
offset the low intrinsic stability of the short three base pair pseudoknot stem S2.
</p></abstract><textClass ana="subject"><keywords scheme="document-type-name"><term>Article</term></keywords></textClass><langUsage><language ident="en"></language></langUsage></profileDesc><revisionDesc><change when="2020-04-06" who="#istex" xml:id="pub2tei">formatting</change></revisionDesc></teiHeader></istex:fulltextTEI></fulltext><metadata><istex:metadataXml wicri:clean="corpus acs not found" wicri:toSee="no header"><istex:xmlDeclaration>version="1.0" encoding="UTF-8"</istex:xmlDeclaration><istex:document><article article-type="research-article" specific-use="acs2jats-1.1.23" dtd-version="1.1d1"><front><journal-meta><journal-id journal-id-type="acspubs">bi</journal-id><journal-id journal-id-type="coden">bichaw</journal-id><journal-title-group><journal-title>Biochemistry</journal-title><abbrev-journal-title>Biochemistry</abbrev-journal-title></journal-title-group><issn pub-type="ppub">0006-2960</issn><issn pub-type="epub">1520-4995</issn><publisher><publisher-name>American Chemical Society</publisher-name></publisher><self-uri>pubs.acs.org/biochemistry</self-uri></journal-meta><article-meta><article-id pub-id-type="doi">10.1021/bi025843c</article-id><article-categories><subj-group subj-group-type="document-type-name"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Thermodynamic Analysis of Conserved Loop−Stem Interactions in P1−P2
Frameshifting RNA Pseudoknots from Plant <italic toggle="yes">Luteoviridae</italic><xref rid="bi025843cAF2"><sup>†</sup></xref></article-title></title-group><contrib-group><contrib contrib-type="author"><name name-style="western"><surname>Nixon</surname><given-names>Paul L.</given-names></name><xref rid="bi025843cAF3"><sup>‡</sup></xref></contrib><contrib contrib-type="author"><name name-style="western"><surname>Cornish</surname><given-names>Peter V.</given-names></name></contrib><contrib contrib-type="author"><name name-style="western"><surname>Suram</surname><given-names>Saritha V.</given-names></name></contrib><contrib contrib-type="author" corresp="yes"><name name-style="western"><surname>Giedroc</surname><given-names>David P.</given-names></name><xref rid="bi025843cAF1">*</xref></contrib><aff>Department of Biochemistry and Biophysics, Center for Advanced Biomolecular Research, Texas A&M University,
College Station, Texas 77843-2128
</aff></contrib-group><author-notes><fn id="bi025843cAF3"><label>‡</label><p>
Current address: Department of Biochemistry, University of Texas
Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas,
TX 75390-9038.</p></fn><corresp id="bi025843cAF1">
Corresponding author. E-mail: giedroc@tamu.edu. Telephone:
979-845-4231. Fax: 979-845-4946.</corresp></author-notes><pub-date pub-type="epub"><day>2</day><month>08</month><year>2002</year></pub-date><pub-date pub-type="ppub"><day>27</day><month>08</month><year>2002</year></pub-date><volume>41</volume><issue>34</issue><fpage>10665</fpage><lpage>10674</lpage><history><date date-type="received"><day>20</day><month>03</month><year>2002</year></date><date date-type="rev-recd"><day>19</day><month>06</month><year>2002</year></date><date date-type="asap"><day>2</day><month>08</month><year>2002</year></date><date date-type="issue-pub"><day>27</day><month>08</month><year>2002</year></date></history><permissions><copyright-statement>Copyright © 2002 American Chemical Society</copyright-statement><copyright-year>2002</copyright-year><copyright-holder>American Chemical Society</copyright-holder></permissions><abstract><p>The RNA genomes of plant luteovirids beet western yellows virus (BWYV), potato leaf roll
virus (PLRV), and pea enation mosaic virus (PEMV RNA1; PEMV-1) contain a short mRNA pseudoknotted
motif overlapping the P1 and P2 open reading frames required for programmed −1 mRNA ribosomal
frameshifting. The relationship between structure, stability, and function is poorly understood in these
RNA systems. A m<sup>5</sup>-C<sub>8</sub>-substituted BWYV RNA is employed to establish that the BWYV P1−P2
pseudoknot is protonated at cytidine 8 in loop L1 (δ<sub>N</sub><sub><sub>3</sub></sub><sub>H</sub><sub><sup>+</sup></sub> = 12.98 ppm), which stabilizes a C<sup>+</sup>·(G−C)
major groove base triple by Δ(Δ<italic toggle="yes">G</italic><sub>37</sub>)<sub>protonation</sub> = 3.1 (±0.4) kcal mol<sup>-1</sup>. The stabilities of both the PLRV
and PEMV-1 P1−P2 pseudoknots are also strongly pH-dependent, with Δ(Δ<italic toggle="yes">G</italic><sub>37</sub>)<sub>protonation</sub> = 2.1 (±0.2)
kcal mol<sup>-1</sup> for the PEMV-1 pseudoknot despite a distinct structural context. As previously found for the
BWYV pseudoknot [Nixon and Giedroc (2000) <italic toggle="yes">J. Mol. Biol. 296</italic>, 659], both the PLRV and PEMV-1
RNAs are stabilized by Δ<italic toggle="yes">H</italic> ≥ 30 kcal mol<sup>-1</sup> in excess of secondary structure predictions, attributed to
loop L2−stem S1 minor groove triplex interactions. BWYV RNAs containing single 2‘-deoxy or A → G
substitutions that disrupt L2−S1 hydrogen bonding are strongly destabilized with Δ(Δ<italic toggle="yes">G</italic><sub>37</sub>)<sub>folding</sub> (pH =
7.0) ranging from ≈1.8 (±0.3) to ≥4.0 kcal mol<sup>-1</sup>, relative to the wild-type BWYV RNA. These findings
suggest that each member of this family of pseudoknots adopts a tightly folded structure that maximizes
the cooperativity and complementarity of L1−S2 and L2−S1 loop−stem interactions required in part to
offset the low intrinsic stability of the short three base pair pseudoknot stem S2.
</p></abstract><custom-meta-group><custom-meta><meta-name>document-id-old-9</meta-name><meta-value>bi025843c</meta-value></custom-meta></custom-meta-group></article-meta><notes id="bi025843cAF2"><label>†</label><p>
This work was supported by grants from the NIH (AI40187) and
the Texas Higher Education Coordinating Board Advanced Research
Program (010361-0278-1999). P.V.C. was supported in part by an NIH
Chemistry−Biology Interface Training Grant (T32 GM08523).</p></notes></front><body><sec id="d7e204"><title></title><p>Programmed −1 ribosomal mRNA frameshifting is employed by RNA viruses and retrotransposable elements to
synthesize structural proteins relative to replicative enzymes
encoded in overlapping open reading frames by way of a
single translation initiation event (for reviews, see refs <italic toggle="yes">1−4</italic>).
Specific sequence and structural requirements are known to
be required to stimulate high levels of translational reprogramming via −1 frameshifting of the elongating ribosome.
Originally proposed in the simultaneous slippage model (<italic toggle="yes"><xref rid="bi025843cb00005" ref-type="bibr"></xref></italic>),
the ribosome must encounter a “slippery site” of the general
sequence X XXY YYZ which stimulates both ribosomal
pausing and slippage (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi025843cb00006" ref-type="bibr"></xref>, <xref rid="bi025843cb00007" ref-type="bibr"></xref></named-content></italic>). While the presence of a slip
site in an mRNA imparts a low level of background −1
ribosomal frameshifting, high levels of recoding require the
presence of an RNA secondary structure, typically a hairpin-type RNA pseudoknot, optimally positioned six to eight
nucleotides downstream of the slip site (<italic toggle="yes"><xref rid="bi025843cb00004" ref-type="bibr"></xref></italic>).
</p><p>Although the mechanism by which pseudoknots stimulate
−1 ribosomal frameshifting remains elusive, a range of
pseudoknot topologies appear capable of stimulating recoding. These pseudoknots can be grouped into four basic
classes, each characterized by distinct structural and sequence
requirements for mRNA frameshifting (<italic toggle="yes"><xref rid="bi025843cb00004" ref-type="bibr"></xref></italic>). The P1−P2
frameshifting pseudoknot from the plant luteovirus beet
western yellows virus (BWYV)<xref rid="bi025843cb00001" ref-type="bibr"></xref> is representative of one such
class (Figure <xref rid="bi025843cf00001"></xref>). From polymer prediction theories (<italic toggle="yes"><xref rid="bi025843cb00008" ref-type="bibr"></xref></italic>), the
BWYV pseudoknot is predicted to be weakly folded in the
absence of additional stabilizing loop−stem interactions
beyond that established by Watson−Crick base stacking, base
pairing, and potential stacking of pseudoknot helical stems,
due principally to a short helical stem S2 (Figure <xref rid="bi025843cf00001"></xref>).
Consistent with this prediction, the crystallographic structure
of the BWYV pseudoknot reveals significant loop−stem
interactions previously not observed in simple H-type
pseudoknots (<italic toggle="yes"><xref rid="bi025843cb00009" ref-type="bibr"></xref></italic>). Three stacked adenosines found at the 3‘
end of loop L2 form a number of hydrogen-bonding
interactions to 2‘-OH groups and the minor groove faces of
a pair of conserved G-C base pairs derived from stem S1,
which are expected to be strongly stabilizing (<italic toggle="yes"><xref rid="bi025843cb00010" ref-type="bibr"></xref></italic>). A cytidine
from loop L1 (C<sub>8</sub>) was also found to make hydrogen-bonding
interactions with the major groove face of a G-C base pair
in stem S2 (G<sub>12</sub>-C<sub>26</sub>) to form a protonated C<sup>+</sup>·(G-C)
Hoogsteen-type base triple. The structure of this triple is
analogous to that found in the HDV ribozyme (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi025843cb00011" ref-type="bibr"></xref>, <xref rid="bi025843cb00012" ref-type="bibr"></xref></named-content></italic>), and
protonation contributes ≈3.5 kcal mol<sup>-1</sup> to the stability of
the folded RNA (<italic toggle="yes"><xref rid="bi025843cb00013" ref-type="bibr"></xref></italic>). The C<sup>+</sup>·(G-C) base triple as well as
the minor groove adenosine triplex motif has been shown
to be absolutely required to stimulate −1 frameshifting (<italic toggle="yes"><xref rid="bi025843cb00009" ref-type="bibr"></xref></italic>).
<fig id="bi025843cf00001" position="float" orientation="portrait"><label>1</label><caption><p>Secondary structural schematic representations of the plant luteoviral family of P1−P2 frameshifting pseudoknots from beet western yellows virus (BWYV), potato leaf roll virus (PLRV), and pea enation mosaic virus 1 (PEMV-1). Nucleotides involved in formation of the C<sup>+</sup>·(G-C) base triple in the BWYV and PLRV
pseudoknots are shown in bold italics; note that the nucleotide
sequences of loop L1 and stem S2 are different in PEMV-1 (see
text for details). The uridine at the helical junction (shaded) is
unpaired and extruded from the helix in the BWYV (<italic toggle="yes"><xref rid="bi025843cb00009" ref-type="bibr"></xref></italic>) and PEMV-1<sup>2</sup> pseudoknots.</p></caption><graphic xlink:href="bi025843cf00001.tif" position="float" orientation="portrait"></graphic></fig></p><p>BWYV is a member of plant <italic toggle="yes">Luteoviridae, </italic>a large group
of aphid-transmissible RNA viruses (for a review, see ref
<italic toggle="yes">14</italic>). The genomes of the polerovirus potato leaf roll virus
(PLRV) and the luteovirus barley yellow dwarf virus
(BYDV) (<italic toggle="yes"><xref rid="bi025843cb00022" ref-type="bibr"></xref></italic>), as well as RNA1 of pea enation mosaic virus
(PEMV-1), all encode P1−P2 pseudoknots expected to be
similar to that of BWYV (cf. Figure <xref rid="bi025843cf00001"></xref>); all have been shown
to stimulate modest levels of −1 ribosomal mRNA frameshifting in vitro and in vivo (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi025843cb00015" ref-type="bibr"></xref>−<xref rid="bi025843cb00016" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi025843cb00017" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi025843cb00018" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi025843cb00019" ref-type="bibr"></xref></named-content></italic>).<xref rid="bi025843cb00002" ref-type="bibr"></xref> Of particular interest
here is the PEMV-1 RNA, which incorporates base substitutions that would be predicted to impair formation of a
BWYV-like Hoogsteen C<sup>+</sup>·(G-C) base triple (Figure <xref rid="bi025843cf00001"></xref>).
</p><p>As first pointed out by Puglisi et al. (<italic toggle="yes"><xref rid="bi025843cb00020" ref-type="bibr"></xref></italic>), the pseudoknotted conformation is in equilibrium with the partially folded
stem S1 and stem S2 stem−loop structures in a manner
governed by the relative free energies of the different states
(for a review, see ref <italic toggle="yes">4</italic>). Plant <italic toggle="yes">Luteoviridae</italic> pseudoknots are
universally characterized by pseudoknot S2 stems that lack
sufficient length (three base pairs) to easily overcome the
entropic barrier to closing two pseudoknot loops (<italic toggle="yes"><xref rid="bi025843cb00008" ref-type="bibr"></xref></italic>). In this
paper, we present NMR and thermodynamic evidence
supporting the proposal that all luteovirid P1−P2 pseudoknots
adopt folded structures that maximize cooperativity and
complementarity of pseudoknot loop−stem interactions that
are required to stabilize the pseudoknotted conformation
relative to partially folded and functionally inactive stem−loop structures.
</p></sec><sec id="d7e336"><title>Materials and Methods</title><p><italic toggle="yes">RNA Synthesis and Purification.</italic> RNA transcripts corresponding to the minimal sequences of the PLRV and
PEMV-1 P1−P2 frameshifting RNA pseudoknots and
pseudoknot mutants (cf. Figure <xref rid="bi025843cf00001"></xref>) were synthesized using
in vitro transcription under control of the SP6 promoter from
fully double-stranded oligonucleotide templates essentially
as described previously (<italic toggle="yes"><xref rid="bi025843cb00021" ref-type="bibr"></xref></italic>). SP6 RNAP was purified from
<italic toggle="yes">Escherichia coli</italic> BL21(DE3) transformed with pSR3 using
the method described by Jorgensen et al. (<italic toggle="yes"><xref rid="bi025843cb00022" ref-type="bibr"></xref></italic>) but with the
final gel filtration column omitted. Wild-type, A23G, A24G,
and A25G BWYV RNAs were obtained by in vitro transcription of partially double-stranded templates by T7 RNAP
or fully double-stranded templates with SP6 RNAP as
previously described (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi025843cb00013" ref-type="bibr"></xref>, <xref rid="bi025843cb00023" ref-type="bibr"></xref></named-content></italic>). The m<sup>5</sup>-C<sub>8</sub>, deoxy-2‘-C<sub>14</sub>, and
deoxy-2‘-C<sub>15</sub> BWYV RNAs were obtained as 28-mers
(Figure <xref rid="bi025843cf00001"></xref>) from Dharmacon, deprotected as described by the
manufacturer, and purified using denaturing PAGE, electroelution, and C18 chromatography (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi025843cb00013" ref-type="bibr"></xref>, <xref rid="bi025843cb00023" ref-type="bibr"></xref></named-content></italic>).
</p><p><italic toggle="yes">Sample Preparation.</italic> RNAs for optical denaturation studies
were resuspended and dialyzed for ≈9 h against three
changes of the appropriate buffer. The first buffer change
contained 1 mM EDTA to remove divalent ions weakly
bound to the RNAs. Samples for analysis by differential
scanning calorimetry (DSC) were further dialyzed for more
than 16 h against the final change of dialysis buffer. Buffers
used for the calorimetry and optical denaturation studies were
made 0.5 M KCl and 10 mM in buffer component as
follows: acetate, pH 4.5 and 5.0; Mes, pH 5.5 and 6.0; Mops,
pH 6.5 and 7.0; Hepes, pH 7.5 and 8.0; Epps, pH 8.5; and
Ches, pH 9.0 (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi025843cb00013" ref-type="bibr"></xref>, <xref rid="bi025843cb00024" ref-type="bibr"></xref></named-content></italic>). NMR samples were prepared by
repeated ethanol precipitation in the appropriate buffer to
remove residual acrylamide prior to resuspension in a final
volume of 300 μL of 100 mM KCl and 10 mM potassium
phosphate, pH 6.0, lyophilized overnight, and loaded into
Shigemi tubes for NMR analysis.
</p><p><italic toggle="yes">Optical and Calorimetric Data Collection and Analysis.</italic>
Optical denaturation profiles were collected on a Cary 1
spectrophotometer equipped with a temperature controller
(1−3 μM RNA), and the resulting melting profiles (∂<italic toggle="yes">A</italic>/∂<italic toggle="yes">T</italic>
vs <italic toggle="yes">T</italic>) acquired at 260 and 280 nm were simultaneously fit
to a multiple sequential interacting transition unfolding model
using t-melt as described previously (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi025843cb00013" ref-type="bibr"></xref>, <xref rid="bi025843cb00025" ref-type="bibr"></xref></named-content></italic>). Only up-melts
were performed, and the temperature ramp rate was 0.3 °C/min from 5 to 95 °C. Reproducibility of melting profiles
was assessed from replicate melts. Calorimetrically monitored
unfolding profiles were collected on a MicroCal VP DSC
from 5 to 120 °C at a ramp rate of 1 °C/min as described
previously (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi025843cb00013" ref-type="bibr"></xref>, <xref rid="bi025843cb00025" ref-type="bibr"></xref></named-content></italic>) and analyzed using Origin to a model
corresponding to two or three sequential interacting two-state unfolding transitions assuming Δ<italic toggle="yes">C<sub>p</sub></italic>° = 0. In these fits,
the sum of the van't Hoff unfolding enthalpies (Δ<italic toggle="yes">H</italic><sub>vH</sub>) for
each transition was constrained to equal Δ<italic toggle="yes">H</italic><sub>cal</sub>, with ∑Δ<italic toggle="yes">H</italic><sub>vH</sub>
= ∑Δ<italic toggle="yes">H</italic><sub>cal</sub>. Total Δ<italic toggle="yes">H</italic><sub>cal</sub> was defined by the integrated area
under the reference-subtracted melting curve, with the
baseline interpolated using a linear baseline approximation
method conservatively estimated from the pre- and posttransition regions, using the optical melting profiles as a
guide (<italic toggle="yes"><xref rid="bi025843cb00013" ref-type="bibr"></xref></italic>).
</p><p><italic toggle="yes">NMR Data Collection and Analysis.</italic> NMR data on the
PEMV-1 and BWYV RNA samples were acquired on either
a Varian Inova 500 or Varian Inova 600 MHz spectrometer
(Biomolecular NMR Laboratory, Texas A&M University)
using WATERGATE water suppression. 1D spectra were
collected as 256 transients of 2560 complex points over a
sweep width of 12.5−15 kHz. The 2D spectra of the m<sup>5</sup>-C8-BWYV RNA were collected using 64 transients containing 2560 complex points in the directly detected dimension
over a 15 kHz sweep width with 340 complex points in the
indirectly detected dimension. Data were processed using
NMRPipe and visualized using NMRDraw (<italic toggle="yes"><xref rid="bi025843cb00026" ref-type="bibr"></xref></italic>) or with
Sparky, version 3 (SPARKY 3, University of California, San
Francisco). All NMR spectra were referenced to DSS.
</p><p><italic toggle="yes">Analysis of the pH Dependence of Unfolding.</italic> <italic toggle="yes">t</italic><sub>m,i</sub> and
Δ<italic toggle="yes">H</italic><sub>vH,i</sub> resolved for the first and second unfolding transitions
from optical and calorimetric melting profiles collected as a
function of pH were used to calculate Δ<italic toggle="yes">S</italic><sub>i</sub> from Δ<italic toggle="yes">S</italic><sub>i</sub> = Δ<italic toggle="yes">H</italic><sub>vH,i</sub>/<italic toggle="yes">t</italic><sub>m,i</sub> and Δ<italic toggle="yes">G</italic><sub>37,i</sub> = Δ<italic toggle="yes">H</italic><sub>vH,i</sub> − <italic toggle="yes">T</italic>Δ<italic toggle="yes">S</italic><sub>i</sub>, where <italic toggle="yes">T</italic> = 310 K. For a
simple RNA motif like a pseudoknot, unfolding along the
equilibrium coordinate is often well-ordered, with tertiary
structure unfolding prior to the unfolding of the helical stems,
which unfold in an order governed by their relative secondary
structural stabilities (<italic toggle="yes"><xref rid="bi025843cb00027" ref-type="bibr"></xref></italic>). Thus, tertiary structure can potentially stabilize secondary structure unfolding (<italic toggle="yes"><xref rid="bi025843cb00028" ref-type="bibr"></xref></italic>), which can
effectively increase the cooperativity of unfolding (cf.
Results). Since the thermodynamic parameters for the
unfolding of stem S1 were found to be independent of pH,
the sum of Δ<italic toggle="yes">G</italic><sub>37,i</sub> for the first and second unfolding
transitions (Δ<italic toggle="yes">G</italic><sub>37,obs</sub> = Δ<italic toggle="yes">G</italic><sub>1</sub> + Δ<italic toggle="yes">G</italic><sub>2</sub>) fully takes this obligatory
coupling into account. The pH dependence of Δ<italic toggle="yes">G</italic><sub>37,obs</sub> was
analyzed using a model in which the entire change in Δ<italic toggle="yes">G</italic><sub>37,obs</sub>
as a function of pH is attributed to protonation of a single
ionizable group on the RNA characterized by unique p<italic toggle="yes">K</italic><sub>a</sub>
according to<xref rid="bi025843ce00001"></xref><disp-formula content-type="pre-labeled" id="bi025843ce00001"><!--%@md;sys;6q@%&Dgr;%@ital@%G%@rsf@%%@sb@%37,obs%@sbx@% = &Dgr;%@ital@%G%@rsf@%%@sb@%37%@sbx@%%@ex@%unprotonated%@exx@% + %@bf@%[S_EL2;quad]\
%@fn;(;vis;full;auto@%&Dgr;%@ital@%G%@rsf@%%@sb@%37%@sbx@%%@ex@%protonated%@exx@% − &Dgr;%@ital@%G%@rsf@%%@sb@%37%@sbx@%%@ex@%unprotonated%@exx@%%@fnx;);vis;full@%%@fn;[;vis;full;auto@%10%@ex@%p%@ital@%K%@rsf@%%@sb@%a%@sbx@%$-$pH%@exx@%/%@fn;(;vis;full;unlock@%1 + 10%@ex@%p%@ital@%K%@rsf@%%@sb@%a%@sbx@%$-$pH%@exx@%%@fnx;);vis;full@%%@fnx;];vis;full@%%@mx@%[S_EL2;quad]
--><graphic xlink:href="bi025843ce00001.gif" position="anchor" orientation="portrait"></graphic></disp-formula>
where Δ<italic toggle="yes">G</italic><sub>37</sub><sup>protonated</sup> is the Δ<italic toggle="yes">G</italic><sub>1</sub> + Δ<italic toggle="yes">G</italic><sub>2</sub> for the protonated RNA
and Δ<italic toggle="yes">G</italic><sub>37</sub><sup>unprotonated</sup> is the Δ<italic toggle="yes">G</italic><sub>1</sub> + Δ<italic toggle="yes">G</italic><sub>2</sub> for the unprotonated
RNA. This analysis is analogous to that performed previously
(<italic toggle="yes">t</italic><sub>m,1</sub><sup>-1</sup> vs [H<sup>+</sup>]) (<italic toggle="yes"><xref rid="bi025843cb00013" ref-type="bibr"></xref></italic>), except that it more fully accounts for
the degree to which protonation stabilizes the RNA.
</p></sec><sec id="d7e612"><title>Results</title><p><italic toggle="yes">NMR Structural and Thermodynamic Analysis of the m<sup>5</sup></italic><sup></sup><italic toggle="yes">-C<sub>8</sub></italic><italic toggle="yes">-BWYV Pseudoknot. </italic>Previously, evidence in support of
the protonation of N3 of C<sub>8</sub> in solution to form a C<sub>8</sub><sup>+</sup>·(G<sub>12</sub>-C<sub>26</sub>) base triple in the BWYV pseudoknot was provided by
a thermodynamic analysis of the unfolding of U<sub>8</sub> and A<sub>12</sub>-U<sub>26</sub> substitution mutant BWYV pseudoknots (<italic toggle="yes"><xref rid="bi025843cb00013" ref-type="bibr"></xref></italic>). Direct
verification of the protonation state of C<sub>8</sub> at pH 6.0 was
obtained with a synthetic RNA containing 5-methylcytidine
in place of C<sub>8</sub>. As described in Figure <xref rid="bi025843cf00002"></xref>A, analysis of a region
of the short mixing time H<sub>2</sub>O NOESY spectrum of the m<sup>5</sup>-C<sub>8</sub>-BWYV RNA (Figure <xref rid="bi025843cf00002"></xref>A) is fully compatible with the
proposed C<sub>8</sub><sup>+</sup>·(G<sub>12</sub>-C<sub>26</sub>) base triple shown in Figure <xref rid="bi025843cf00002"></xref>B.
<fig id="bi025843cf00002" position="float" orientation="portrait"><label>2</label><caption><p>(A) 60 ms WATERGATE NOESY spectrum of the m<sup>5</sup>-C<sub>8</sub>-BWYV pseudoknot (0.8 mM) acquired on a Varian Inova 600
spectrometer at 10 °C, pH 6.0. The methyl protons of m<sup>5</sup>-C<sub>8</sub>
resonate at 1.73 ppm and exhibit strong cross-peaks to a pair of
downfield-shifted amino protons (9.25 and 11.02 ppm) as expected
for a cytidine protonated at N3. These m<sup>5</sup>-C<sub>8</sub> amino protons have
strong cross-peaks to an imino proton at 12.98 ppm, assigned to
m<sup>5</sup>-C<sub>8</sub><sup>+</sup> H3. Also highlighted are a pair of hydrogen-bonded amino
protons (7.85 and 8.15 ppm) which give strong cross-peaks to two
imino protons, m<sup>5</sup>-C<sub>8</sub><sup>+</sup> H3 (12.98) and another assigned to G<sub>12</sub> H1
(13.77 ppm) of the accepting C<sub>12</sub>-G<sub>26</sub> base pair. (B) Schematic
representation of the m<sup>5</sup>-C<sub>8</sub><sup>+</sup>·(G<sub>12</sub>-C<sub>26</sub>) Hoogsteen base triple from
the BWYV pseudoknot.
</p></caption><graphic xlink:href="bi025843cf00002.tif" position="float" orientation="portrait"></graphic></fig></p><p>Analysis of the thermodynamics of unfolding of the m<sup>5</sup>-C<sub>8</sub>-BWYV pseudoknot using optical spectroscopy reveals
melting profiles essentially identical to those observed
previously (<italic toggle="yes"><xref rid="bi025843cb00013" ref-type="bibr"></xref></italic>) for the unmodified BWYV pseudoknot
(Figure <xref rid="bi025843cf00003"></xref>). The unfolding of the m<sup>5</sup>-C<sub>8</sub>-BWYV pseudoknot
is modeled well by three sequential two-state unfolding
transitions where transition 1 is assigned to the unfolding of
the loop−stem interactions (F → PK), transition 2 to the
unfolding of the less stable stem S2 (PK → S1), with
transition 3 corresponding to the unfolding of the more stable
stem S1 (S1 → U) (Figure <xref rid="bi025843cf00003"></xref>). Fitting of the optically
monitored denaturation profiles using a sequential interacting
three-transition unfolding model suggests little enthalpic
destabilization of the loop−stem tertiary structure interactions
results upon m<sup>5</sup>-C<sub>8</sub> substitution [Δ<italic toggle="yes">H</italic><sub>F</sub><sub>→</sub><sub>PK</sub> = 33 (±5) vs 32
(±7) kcal mol<sup>-1</sup> for BWYV] (Figure <xref rid="bi025843cf00003"></xref>, legend) (<italic toggle="yes"><xref rid="bi025843cb00013" ref-type="bibr"></xref></italic>).
<fig id="bi025843cf00003" position="float" orientation="portrait"><label>3</label><caption><p>(A) Optically monitored unfolding of the m<sup>5</sup>-C<sub>8</sub>-BWYV
pseudoknot at 260 nm (·) and 280 nm (○). The solid lines
superimposed on 20% of the experimental data represent a
simultaneous fit of the data to a model of three sequential,
interacting unfolding transitions. The three unfolding transitions
(1, 2, and 3) are attributed to the unfolding of the pseudoknot tertiary
structure, stem S2, and stem S1, respectively (<italic toggle="yes"><xref rid="bi025843cb00013" ref-type="bibr"></xref></italic>). (B) Individual
transitions which comprise the composite fit to the data shown in
panel A dA<sub>260</sub>/dT (), dA<sub>280</sub>/dT (---). This melting profile is
qualitatively similar to the unmodified BWYV pseudoknot (<italic toggle="yes"><xref rid="bi025843cb00013" ref-type="bibr"></xref></italic>).
Calculated values for the fit are as follows: <italic toggle="yes">t</italic><sub>m1</sub> = 57.3 °C, Δ<italic toggle="yes">H</italic><sub>1</sub> =
33.6 kcal mol<sup>-1</sup> (Δ<italic toggle="yes">H</italic><sub>BWYV</sub> = 32 ± 7 kcal mol<sup>-1</sup>); <italic toggle="yes">t</italic><sub>m2</sub> = 81.1 °C,
Δ<italic toggle="yes">H</italic><sub>2</sub> = 29 kcal mol<sup>-1</sup> (Δ<italic toggle="yes">H</italic><sub>INN</sub><sub>-</sub><sub>HB</sub> = 26 kcal mol<sup>-1</sup>); <italic toggle="yes">t</italic><sub>m3</sub> = 97.3
°C, Δ<italic toggle="yes">H</italic><sub>3</sub> = 47 kcal mol<sup>-1</sup> (Δ<italic toggle="yes">H</italic><sub>INN</sub><sub>-</sub><sub>HB</sub> = 50 kcal mol<sup>-1</sup>). (C)
Analysis of the dependence on solution pH of unfolding of the m<sup>5</sup>-C<sub>8</sub>-BWYV pseudoknot (○) versus the unmodified BWYV pseudoknot (·). Data shown are the average (±SD) Δ<italic toggle="yes">G</italic><sub>37,obs</sub> (Δ<italic toggle="yes">G</italic><sub>1</sub> + Δ<italic toggle="yes">G</italic><sub>2</sub>)
from four independent experiments for the first two unfolding
transitions (F → PK and PK → S1). The solid line through the
experimental data represents a fit to a model which attributes the
entire dependence of Δ<italic toggle="yes">G</italic><sub>37</sub> on solution pH to a single protonatable
group on the RNA (see Materials and Methods). The parameters
derived from the fits are compiled in Table <xref rid="bi025843ct00001"></xref>.</p></caption><graphic xlink:href="bi025843cf00003.tif" position="float" orientation="portrait"></graphic></fig></p><p>The total change of Δ<italic toggle="yes">G</italic><sub>37</sub> as a function of pH for unfolding
of the pseudoknot including the loop−stem interactions (F
→ PK) and stem S2 (PK → S1) can be used to determine
the macroscopic p<italic toggle="yes">K</italic><sub>a</sub> for those functional group(s) which
contribute to the stability of the m<sup>5</sup>-C<sub>8</sub>-BWYV pseudoknot
(<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi025843cb00013" ref-type="bibr"></xref>, <xref rid="bi025843cb00024" ref-type="bibr"></xref></named-content></italic>). Note that this p<italic toggle="yes">K</italic><sub>a</sub> does not necessarily correspond
to the microscopic p<italic toggle="yes">K</italic><sub>a</sub> for N3 of m<sup>5</sup>-C<sub>8</sub> under these solution
conditions. The p<italic toggle="yes">K</italic><sub>a</sub> of N3 for free 5-methylcytidine is 0.1−0.2 unit higher than free cytidine (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi025843cb00029" ref-type="bibr"></xref>, <xref rid="bi025843cb00030" ref-type="bibr"></xref></named-content></italic>). Fitting the change
of Δ<italic toggle="yes">G</italic><sub>37(F</sub><sub>→</sub><sub>S1)</sub> as a function of pH for this RNA to a model
in which the entire change in Δ<italic toggle="yes">G</italic><sub>37</sub> is attributed to a single
protonatable group resolves an apparent p<italic toggle="yes">K</italic><sub>a</sub> = 6.7 (±0.4)
for the N3 of 5-methylcytidine in the context of the BWYV
pseudoknot, only slightly lower than that of the unmodified
BWYV pseudoknot [p<italic toggle="yes">K</italic><sub>a</sub> = 7.3 (±0.4)]. The fully protonated
m<sup>5</sup>-C<sub>8</sub>-BWYV pseudoknot is also ≈1.1 kcal mol<sup>-1</sup> less stable
than the unmodified BWYV pseudoknot at pH 7.0, most of
which is associated with the relative stabilities of the
protonated forms (Table <xref rid="bi025843ct00001"></xref>).
<table-wrap id="bi025843ct00001" position="float" orientation="portrait"><label>1</label><caption><p>Thermodynamic Parameters for the pH-Dependent Unfolding of the Luteoviral Pseudoknots<italic toggle="yes"><sup>a</sup></italic><sup></sup></p></caption><oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="6"><oasis:colspec colnum="1" colname="1"></oasis:colspec><oasis:colspec colnum="2" colname="2"></oasis:colspec><oasis:colspec colnum="3" colname="3"></oasis:colspec><oasis:colspec colnum="4" colname="4"></oasis:colspec><oasis:colspec colnum="5" colname="5"></oasis:colspec><oasis:colspec colnum="6" colname="6"></oasis:colspec><oasis:tbody><oasis:row><oasis:entry namest="1" nameend="1">RNA</oasis:entry><oasis:entry namest="2" nameend="2">Δ<italic toggle="yes">G</italic><sub>37</sub><sup>protonated</sup>
(kcal mol<sup>-1</sup>)</oasis:entry><oasis:entry namest="3" nameend="3">Δ<italic toggle="yes">G</italic><sub>37</sub><sup>unprotonated</sup>
(kcal mol<sup>-1</sup>)</oasis:entry><oasis:entry namest="4" nameend="4">ΔΔ<italic toggle="yes">G</italic><sub>37</sub><sup>protonation</sup>
(kcal mol<sup>-1</sup>)</oasis:entry><oasis:entry namest="5" nameend="5">p<italic toggle="yes">K</italic><sub>a</sub></oasis:entry><oasis:entry namest="6" nameend="6">Δ<italic toggle="yes">G</italic><sub>37</sub><sup>7.0</sup>
(kcal mol<sup>-1</sup>)<italic toggle="yes"><sup>b</sup></italic><sup></sup></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">BWYV
</oasis:entry><oasis:entry colname="2">6.8 (±0.3)
</oasis:entry><oasis:entry colname="3">3.7 (±0.3)
</oasis:entry><oasis:entry colname="4">3.1 (±0.4)
</oasis:entry><oasis:entry colname="5">7.3 (±0.3)
</oasis:entry><oasis:entry colname="6">5.8 (±0.3)
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">dC<sub>14 </sub>BWYV
</oasis:entry><oasis:entry colname="2">6.4 (±0.3)
</oasis:entry><oasis:entry colname="3">3.8 (±0.2)
</oasis:entry><oasis:entry colname="4">2.6 (±0.4)
</oasis:entry><oasis:entry colname="5">5.7 (±0.3)
</oasis:entry><oasis:entry colname="6">4.0 (±0.2)
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">m<sup>5</sup>-C<sub>8</sub>-BWYV
</oasis:entry><oasis:entry colname="2">5.8 (±0.3)
</oasis:entry><oasis:entry colname="3">4.0 (±0.2)
</oasis:entry><oasis:entry colname="4">1.8 (±0.4)
</oasis:entry><oasis:entry colname="5">6.7 (±0.4)
</oasis:entry><oasis:entry colname="6">4.7 (±0.5)
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">PLRV<italic toggle="yes"><sup>c</sup></italic><sup></sup></oasis:entry><oasis:entry colname="2">3.2 (±0.2)
</oasis:entry><oasis:entry colname="3">2.1 (±0.1)
</oasis:entry><oasis:entry colname="4">1.1 (±0.1)
</oasis:entry><oasis:entry colname="5">6.9 (±0.3)
</oasis:entry><oasis:entry colname="6">2.5 (±0.5)
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">PEMV-1
</oasis:entry><oasis:entry colname="2">4.9 (±0.2)
</oasis:entry><oasis:entry colname="3">2.8 (±0.1)
</oasis:entry><oasis:entry colname="4">2.1 (±0.2)
</oasis:entry><oasis:entry colname="5">7.1 (±0.2)
</oasis:entry><oasis:entry colname="6">3.7 (±0.4)
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">C<sub>11</sub>-G<sub>30</sub> PEMV-1
</oasis:entry><oasis:entry colname="2">3.4 (±0.2)
</oasis:entry><oasis:entry colname="3">1.1 (±0.3)
</oasis:entry><oasis:entry colname="4">2.3 (±0.3)
</oasis:entry><oasis:entry colname="5">8.0 (±0.2)
</oasis:entry><oasis:entry colname="6">3.5 (±0.1)</oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table><table-wrap-foot><p><italic toggle="yes"><sup>a</sup></italic><sup></sup> Summed from Δ<italic toggle="yes">G</italic><sub>37</sub> derived for unfolding transitions 1 and 2. Δ<italic toggle="yes">G</italic><sub>37</sub> for transition 3 was found to be independent of pH and was 7.7 (±0.5),
7.2 (±0.2), 7.8 (±0.3), 7.8 (±0.3), 5.6 (±0.1), and 5.7 (±0.1) kcal mol<sup>-1</sup> for the BWYV, dC<sub>14</sub> BWYV, m<sup>5</sup>-C<sub>8</sub>-BWYV, PLRV, PEMV-1, and
C<sub>11</sub>-G<sub>30</sub> PEMV RNAs.<italic toggle="yes"><sup>b</sup></italic><sup></sup> Experimentally determined, not fitted.<italic toggle="yes"><sup>c</sup></italic><sup></sup> All Δ<italic toggle="yes">G</italic><sub>37</sub> values for the PLRV pseudoknot refer to those derived from unfolding
transition 1 (Δ<italic toggle="yes">G</italic><sub>1</sub>), not the sum Δ<italic toggle="yes">G</italic><sub>1</sub> + Δ<italic toggle="yes">G</italic><sub>2</sub>;<xref rid="bi025843cb00003" ref-type="bibr"></xref> this particularly underestimates ΔΔ<italic toggle="yes">G</italic><sub>37</sub><sup>protonation</sup> since these unfolding transitions are obligatorily
coupled (see text for details).</p></table-wrap-foot></table-wrap></p><p><italic toggle="yes">Thermodynamic Evidence for Conservation of Structure
in the PLRV Pseudoknot</italic>. On the basis of sequence (Figure
<xref rid="bi025843cf00001"></xref>) and functional similarities (<italic toggle="yes"><xref rid="bi025843cb00027" ref-type="bibr"></xref></italic>) to the BWYV pseudoknot,
the PLRV pseudoknot was expected to be stabilized by the
presence of loop L2−stem S1 minor groove and loop L1−stem S2 major groove triplex interactions. Consistent with
these expectations, the calorimetrically monitored unfolding
of the PLRV pseudoknot as a function of pH (Figure <xref rid="bi025843cf00004"></xref>)
reveals a total Δ<italic toggle="yes">H</italic> ≈ 120 kcal mol<sup>-1</sup>, essentially identical
to the unfolding enthalpy of the BWYV pseudoknot, and in
large excess of the 63−70 kcal mol<sup>-1</sup> of enthalpy anticipated
for unfolding of the helical stems of the PLRV pseudoknot
based on INN-HB rules (<italic toggle="yes"><xref rid="bi025843cb00027" ref-type="bibr"></xref></italic>). Deconvolution of the calorimetrically monitored unfolding of the PLRV pseudoknot
upon application of the same sequential interacting model
reveals that the loop−stem interactions of the PLRV
pseudoknot contribute Δ<italic toggle="yes">H</italic> = 32 kcal mol<sup>-1</sup> to the stability
of the folded pseudoknot, while protonation of the C<sub>8</sub><sup>+</sup>·(G<sub>12</sub>-C<sub>27</sub>) triple base pair contributes ≥2.5 kcal mol<sup>-1</sup> in stability
at 37 °C by DSC (Figure <xref rid="bi025843cf00004"></xref>, legend), coupled with an elevated
p<italic toggle="yes">K</italic><sub>a</sub> = 6.9 (±0.3) (Table <xref rid="bi025843ct00001"></xref>).<xref rid="bi025843cb00003" ref-type="bibr"></xref> These findings are fully
compatible with detailed NMR analysis of the PLRV
pseudoknot, which reveals a 2D NOE spectrum that is
essentially superimposable on the BWYV pseudoknot (P.
Nixon and D. Giedroc, unpublished observations).
<fig id="bi025843cf00004" position="float" orientation="portrait"><label>4</label><caption><p>Calorimetrically monitored unfolding of the PLRV P1−P2 pseudoknot at pH 6.0, 7.0, and 8.0 in 0.5 M KCl and 10 mM buffer salt (see Materials and Methods). Sample concentrations were 46.0, 45.1, and 47.9 μM, respectively. For clarity, only 5% of the collected data points are shown in each panel with both the composite fit (solid line) and the individual transitions (dotted lines) superimposed on the data. Calculated thermodynamic parameters (in kcal mol<sup>-1</sup>) for the unfolding of the PLRV pseudoknot obtained
from fitting to a model of three sequential interacting transitions
are as follows: pH 6.0, Δ<italic toggle="yes">H</italic><sub>F</sub><sub>→</sub><sub>PK</sub> = 33.0, Δ<italic toggle="yes">H</italic><sub>total</sub> = 123, Δ<italic toggle="yes">G</italic><sub>37(total)</sub>
= 14.7, Δ<italic toggle="yes">G</italic><sub>37(F</sub><sub>-</sub><sub>PK)</sub> = 3.3; pH 7.0, Δ<italic toggle="yes">H</italic><sub>F</sub><sub>→</sub><sub>PK</sub> = 33.2, Δ<italic toggle="yes">H</italic><sub>total</sub> = 122,
Δ<italic toggle="yes">G</italic><sub>37(total)</sub> = 13.1, ΔG<sub>37(F</sub><sub>-</sub><sub>PK)</sub> = 2.4; pH 8.0, Δ<italic toggle="yes">H</italic><sub>F</sub><sub>→</sub><sub>PK</sub> = 30.7, Δ<italic toggle="yes">H</italic><sub>total</sub>
= 121, Δ<italic toggle="yes">G</italic><sub>37(total)</sub> = 11.4, Δ<italic toggle="yes">G</italic><sub>37(F</sub><sub>-</sub><sub>PK)</sub> = 1.3.</p></caption><graphic xlink:href="bi025843cf00004.tif" position="float" orientation="portrait"></graphic></fig></p><p><italic toggle="yes">Thermodynamic Evidence for Conservation of Structure
in the PEMV-1 Pseudoknot. </italic>The PEMV-1 pseudoknot
incorporates a transversion in the sequence of loop L1 where
the loop L1 cytidine (C<sub>8</sub> in the BWYV pseudoknot) is moved
to the 3‘ loop position with a uridine in place of the conserved
cytidine at the 5‘ side of loop L1. In addition, the putative
accepting pair for the Hoogsteen C<sup>+</sup>·(G-C) triple (C<sub>13</sub>-G<sub>28</sub>)
is inverted relative to the other members of the luteovirus
family pseudoknots (G<sub>12</sub>-C<sub>26</sub> in the BWYV pseudoknot)
(Figure <xref rid="bi025843cf00001"></xref>). Given the previously described functional (<italic toggle="yes"><xref rid="bi025843cb00016" ref-type="bibr"></xref></italic>)
and thermodynamic studies (<italic toggle="yes"><xref rid="bi025843cb00013" ref-type="bibr"></xref></italic>), these sequence changes
were expected to abolish the pH dependence of the unfolding
of the PEMV-1 pseudoknot.
</p><p>The calorimetrically monitored unfolding of the PEMV-1
pseudoknot (Figure <xref rid="bi025843cf00005"></xref>) reveals that, contrary to these expectations, the RNA is characterized by an unfolding enthalpy in
excess of the INN-HB predictions (Δ<italic toggle="yes">H </italic>≈ 109 kcal mol<sup>-1</sup>)
and a strongly pH-dependent stability, with Δ(Δ<italic toggle="yes">G</italic>)<sub>37</sub> for
protonation of 2.1 (±0.2) kcal mol<sup>-1</sup> (Table <xref rid="bi025843ct00001"></xref>). It is
interesting to note that, at pH values below 7.0, the unfolding
of the tertiary structure and stem S2 of the PEMV-1
pseudoknot become coincident or so tightly coupled that they
are best modeled by a single, cooperative transition. The Δ<italic toggle="yes">H
</italic>of 52 kcal mol<sup>-1</sup> is near that determined for the sum of
tertiary structure and S2 unfolding resolved at pH 7.0 and
8.0 (Δ<italic toggle="yes">H</italic><sub>F</sub><sub>→</sub><sub>PK+PK</sub><sub>→</sub><sub>S1</sub> = 55.8 ± 1.0 kcal mol<sup>-1</sup>). Optically
monitored melts are also characterized by significant pH
dependence (melts not shown) (Table <xref rid="bi025843ct00001"></xref>). A simultaneous
analysis of pH dependence of the optical and calorimetric
data like that applied to analyze the BWYV and PLRV RNA
melts reveals a macroscopic p<italic toggle="yes">K</italic><sub>a</sub> of 7.1 (±0.2) (Figure <xref rid="bi025843cf00006"></xref>).
This likely corresponds to apparent p<italic toggle="yes">K</italic><sub>a</sub> of the N<sub>3</sub>H<sup>+</sup> imino
proton of C<sub>10</sub> in loop L1.
<fig id="bi025843cf00005" position="float" orientation="portrait"><label>5</label><caption><p>Calorimetrically monitored unfolding of the PEMV-1 pseudoknot at pH 6.0, 7.0, and 8.0 in 500 mM KCl and 10 mM buffer salt (see Materials and Methods). Sample concentrations were 85.0, 88.8, and 83.7 μM, respectively. For clarity, only 5% of the collected data points are shown in each panel with both the composite fit (solid line) and the individual transitions (dotted lines) superimposed on the data. Calculated thermodynamic parameters (in kcal mol<sup>-1</sup>) for the unfolding of the PEMV-1 pseudoknot
obtained from fitting to a model of three sequential interacting
transitions are as follows: pH 6.0, Δ<italic toggle="yes">H</italic><sub>F</sub><sub>→</sub><sub>S1</sub> = 52.0, Δ<italic toggle="yes">H</italic><sub>total</sub> = 112,
Δ<italic toggle="yes">G</italic><sub>37(total)</sub> = 11.9, Δ<italic toggle="yes">G</italic><sub>37(F</sub><sub>-</sub><sub>S1)</sub> = 4.7; pH 7.0, Δ<italic toggle="yes">H</italic><sub>F</sub><sub>→</sub><sub>PK</sub> = 37.0, Δ<italic toggle="yes">H</italic><sub>total</sub>
= 109, Δ<italic toggle="yes">G</italic><sub>37(total)</sub> = 10.2, ΔG<sub>37(F</sub><sub>-</sub><sub>PK)</sub> = 2.4; pH 8.0, Δ<italic toggle="yes">H</italic><sub>F</sub><sub>→</sub><sub>PK</sub> =
30.0, Δ<italic toggle="yes">H</italic><sub>total</sub> = 107, Δ<italic toggle="yes">G</italic><sub>37(total)</sub> = 9.3, Δ<italic toggle="yes">G</italic><sub>37(F</sub><sub>-</sub><sub>PK)</sub> = 1.3.</p></caption><graphic xlink:href="bi025843cf00005.tif" position="float" orientation="portrait"></graphic></fig><fig id="bi025843cf00006" position="float" orientation="portrait"><label>6</label><caption><p>pH dependence on the unfolding of the wild-type (·) and mutant C<sub>11</sub>-G<sub>30</sub> (○) PEMV-1 pseudoknots. The average (±SD)
Δ<italic toggle="yes">G</italic><sub>1</sub> + Δ<italic toggle="yes">G</italic><sub>2</sub> values derived from four independent optical melting
experiments acquired at each pH value, except that Δ<italic toggle="yes">G</italic><sub>1</sub> + Δ<italic toggle="yes">G</italic><sub>2</sub>
determined at pH 6.0, 7.0 and 8.0 for the wild-type PEMV-1
pseudoknot also incorporates results from DSC (cf. Figure <xref rid="bi025843cf00005"></xref>). The
solid line through each set of data corresponds to a fit to an equation
which describes the entire dependence as attributable to a single
protonatable group (see Materials and Methods). The parameters
obtained from this analysis are compiled in Table <xref rid="bi025843ct00001"></xref>.</p></caption><graphic xlink:href="bi025843cf00006.tif" position="float" orientation="portrait"></graphic></fig></p><p><italic toggle="yes">NMR and Thermodynamic Evidence for a C<sub>10</sub></italic><italic toggle="yes"><sup>+</sup></italic><sup></sup><italic toggle="yes">·(C<sub>13</sub></italic><italic toggle="yes">-G<sub>28</sub></italic><italic toggle="yes">)
Base Triple in the PEMV-1 Pseudoknot.</italic> Since the tertiary
structure of the PEMV-1 pseudoknot is strongly dependent
on solution pH, three possibilities exist for the participation
of C<sub>10</sub> in a protonated L1−S2 triple base pair, analogous to
that of the BWYV and PLRV pseudoknots (Figure <xref rid="bi025843cf00007"></xref>). One
is a C<sub>10</sub><sup>+</sup>·(G<sub>11</sub>-C<sub>30</sub>) triple which would be isostructural with
the BWYV C<sub>8</sub><sup>+</sup>·(G<sub>12</sub>-C<sub>26</sub>) base triple (Figure <xref rid="bi025843cf00007"></xref>A), simply
moved away from the helical junction to the base of S2
(Figure <xref rid="bi025843cf00007"></xref>B). Two other base triple structures are possible
(Figure <xref rid="bi025843cf00007"></xref>C,D). The C<sub>10</sub><sup>+</sup>·(A<sub>12</sub>-U<sub>29</sub>) triple (Figure <xref rid="bi025843cf00007"></xref>C) seems
unlikely due to formation of only a single protonated C<sub>10</sub>
N<sub>3</sub>H<sup>+</sup>···O4 U<sub>29</sub> hydrogen bond and no hydrogen bonds to
the C<sub>10</sub><sup>+</sup> amino protons, a structure difficult to rationalize
with the strongly downfield-shifted, nondegenerate chemical
shifts of these two protons (cf. Figure <xref rid="bi025843cf00008"></xref>). The other possibility
(Figure <xref rid="bi025843cf00007"></xref>D) is that the entire C<sub>10</sub> nucleotide is “flipped over”,
analogous to that previously observed for the biotin-binding
pseudoknot (<italic toggle="yes"><xref rid="bi025843cb00031" ref-type="bibr"></xref></italic>), which would enforce a local change in the
polarity of the polynucleotide backbone; in this arrangement,
C<sub>10</sub> is in a position to form the same set of hydrogen bonds
of the C<sub>8</sub><sup>+</sup>·(G<sub>12</sub>-C<sub>26</sub>) base pair in the BWYV pseudoknot with
the Hoogsteen face of the <italic toggle="yes">inverted</italic> C<sub>13</sub>-G<sub>28</sub> base pair at the
helical junction. NMR spectroscopy and thermodynamic
analysis of the wild-type PEMV-1 pseudoknot and two
variant RNAs, U<sub>10</sub> and C<sub>11</sub>-G<sub>30</sub>, were carried out to distinguish among these possibilities.
<fig id="bi025843cf00007" position="float" orientation="portrait"><label>7</label><caption><p>Schematic representation of the possible protonated triple base pairs that could be formed between a protonated C<sub>10</sub> (L1) and
the stem S2 base pairs in the PEMV-1 pseudoknot (panels B−D)
compared to that of the wild-type BWYV base triple (panel A).
See text for details.
</p></caption><graphic xlink:href="bi025843cf00007.tif" position="float" orientation="portrait"></graphic></fig></p><p>Figure <xref rid="bi025843cf00008"></xref> (top spectrum) reveals that the wild-type PEMV-1
pseudoknot is characterized by the correct number of imino
proton resonances consistent with a pseudoknotted structure;<xref rid="bi025843cb00002" ref-type="bibr"></xref>
furthermore, this RNA exhibits a pair of downfield-shifted
amino protons indicative of the presence of a protonated
cytidine, assigned to C<sub>10</sub>.<xref rid="bi025843cb00002" ref-type="bibr"></xref> Although there are some significant chemical shift changes, the exchangeable proton region
of the base pair inverted C<sub>11</sub>-G<sub>30</sub> PEMV-1 RNA yields
substantially the same spectrum, including the presence of
the downfield-shifted amino protons associated with protonated C<sub>10</sub> (Figure <xref rid="bi025843cf00008"></xref>, middle spectrum). Thus, it is unlikely
that the closing G<sub>11</sub>-C<sub>30</sub> base pair is the triple base pair
acceptor, ruling out the model in Figure <xref rid="bi025843cf00007"></xref>B. Consistent with
this, the unfolding of the C<sub>11</sub>-G<sub>30</sub> PEMV-1 pseudoknot, while
lowered as a result of the terminal base pair inversion,
remains dependent on pH, with a macroscopic p<italic toggle="yes">K</italic><sub>a</sub> of 8.0
(±0.2) (Figure <xref rid="bi025843cf00006"></xref>, Table <xref rid="bi025843ct00001"></xref>). Interestingly, the stabilities of
the wild-type and C<sub>11</sub>-G<sub>30</sub> PEMV-1 pseudoknots are indistinguishable at pH 7.0 (Table <xref rid="bi025843ct00001"></xref>).
<fig id="bi025843cf00008" position="float" orientation="portrait"><label>8</label><caption><p>1D NMR spectra (256 transients) of the exchangeable proton region of the wild-type (1.1 mM), C<sub>11</sub>-G<sub>30</sub> (1.5 mM), and
U<sub>10</sub> (0.5 mM) PEMV-1 RNAs at 1 °C, pH 6.0, and 0.10 M KCl.
Complete proton resonance assignments for the PEMV-1 RNA
pseudoknot have been deposited in the BMRB (deposition 5278).<xref rid="bi025843cb00002" ref-type="bibr"></xref>
The minor resonances at ≈12.2 and ≈13.4 ppm in the PEMV-1
spectrum derive from a small amount of contaminating 3‘ truncated
RNA which only folds into an S1 hairpin (cf. the U<sub>10</sub> spectrum).
The downfield shoulder on the peak labeled G11 (≈11.8 ppm)
apparently derives from a distinct environment around the base of
stem S2 in a minor conformation (data not shown), also observed
in the analogous region of the phage T2 pseudoknot (<italic toggle="yes"><xref rid="bi025843cb00038" ref-type="bibr"></xref></italic>).</p></caption><graphic xlink:href="bi025843cf00008.tif" position="float" orientation="portrait"></graphic></fig></p><p>In contrast, the U<sub>10</sub> PEMV-1 RNA does not adopt a folded
RNA pseudoknot since only three imino protons are observed
under these conditions, the expected number if the five base
pair stem S1 is folded with the imino protons of the two
terminal base pairs exchanging rapidly with solvent (Figure
<xref rid="bi025843cf00008"></xref>, bottom spectrum). The surprising conclusion is that the
PEMV-1 pseudoknot conserves the stabilizing features of
the BWYV/PLRV C<sup>+</sup>·(G-C) triple base pair (Figure <xref rid="bi025843cf00007"></xref>D) in
a distinct structural context. Formation of this base triple
appears to play a key role in nucleating other loop−stem
interactions, e.g., a minor groove triplex, since substitution
of C<sub>10</sub> lowers the stability of the pseudoknot to a extent below
that of the S1 hairpin (Figure <xref rid="bi025843cf00008"></xref>, bottom spectrum).
</p><p><italic toggle="yes">Characterization of Loop L2 Substitution Mutants of the
BWYV Pseudoknot.</italic> The other prominent structural feature
of <italic toggle="yes">Luteoviridae</italic> P1−P2 pseudoknots is a minor groove triplex
motif, in which loop L2 adenosines, particularly those at the
3‘ end of loop L2, make functionally important (<italic toggle="yes"><xref rid="bi025843cb00016" ref-type="bibr"></xref></italic>)
hydrogen-bonding interactions into the minor groove side
of the G-C base pairs at the base of stem S1, near the helical
junction (<italic toggle="yes">9). </italic>Structural and thermodynamic characterizations
of A<sub>23</sub> → G<sub>23</sub>, A<sub>24</sub> → G<sub>24</sub>, and A<sub>25</sub> → G<sub>25</sub> and two 2‘-deoxy
BWYV substitution mutants, 2‘-dC<sub>14</sub> and 2‘-dC<sub>15</sub>, were
undertaken to quantify the extent to which individual
hydrogen-bonding interactions contribute to the stability of
the BWYV pseudoknot.
</p><p>1D NMR spectra of the exchangeable proton region for
each of these RNAs are shown in Figure <xref rid="bi025843cf00009"></xref>, compared to the
spectrum of the wild-type BWYV pseudoknot (pH 6.0, 0.10
M K<sup>+</sup>, 10 °C). Although only limited resonance assignments
are available for the BWYV RNA, the presence or absence
of the C<sub>8</sub><sup>+</sup> amino protons (see also Figure <xref rid="bi025843cf00002"></xref>), coupled with
the presence of the slowly exchanging 2‘-OH of C<sub>14</sub>, provides
a qualitative indication of the extent to which the folded
pseudoknotted conformation is the primary conformer under
these conditions. These data reveal that substitution of A<sub>25</sub>
with G<sub>25</sub> and replacement of the 2‘-OH of C<sub>14</sub> with a proton,
i.e., the stabilizing elements closest to the helical junction,
are tolerated by the pseudoknotted conformation. Consistent
with this, thermal melting profiles of the 2‘-dC<sub>14</sub> RNA are
well described by three unfolding transitions (Figure <xref rid="bi025843cf00010"></xref>A);
however, the RNA is destabilized by a Δ(Δ<italic toggle="yes">G</italic><sub>37</sub>) ≈ 1.8 kcal
mol<sup>-1</sup> (pH 7.0, 0.5 M K<sup>+</sup>) (Table <xref rid="bi025843ct00001"></xref>) and is characterized by
a downward-shifted p<italic toggle="yes">K</italic><sub>a</sub> of 5.7 (±0.4) relative to the wild-type RNA (Figure <xref rid="bi025843cf00010"></xref>B). Thermal melts of the G<sub>25</sub> RNA also
show the presence of three unfolding transitions (data not
shown). In contrast, the G<sub>24</sub> and G<sub>23</sub> BWYV RNAs give
nearly identical NMR spectra and appear to be largely
unfolded, with the G<sub>24</sub> and the 2‘-dC<sub>15</sub> RNA best described
as a mixture of folded and S1 hairpin conformers. Thus,
disruption of minor groove interactions one S1 base pair or
one L2 nucleotide removed from the helical junction is
comparatively more destabilizing than are identical substitutions nearer the helical junction (see Discussion).
<fig id="bi025843cf00009" position="float" orientation="portrait"><label>9</label><caption><p>1D NMR spectra (2048 transients) of the exchangeable proton region of the wild-type BWYV (0.6 mM), dC<sub>14</sub> (0.7 mM),
dC<sub>15</sub> (1.1 mM), A<sub>25</sub>G (0.9 mM), A<sub>24</sub>G (1.1 mM), and A<sub>23</sub>G (1.2
mM) RNAs at 10 °C, pH 6.0, and 0.1 M KCl.</p></caption><graphic xlink:href="bi025843cf00009.tif" position="float" orientation="portrait"></graphic></fig><fig id="bi025843cf00010" position="float" orientation="portrait"><label>10</label><caption><p>(A) Representative thermal melting profiles (dA<sub>260</sub>/dT) obtained for dC<sub>14</sub> BWYV RNA at pH 6.0 (·) and pH 8.0 (○)
in 0.5 M KCl and 10 mM buffer salt (see Materials and Methods).
The smooth curves superimposed on the data represent a simultaneous fit (both dA<sub>260</sub>/dT and dA<sub>280</sub>/dT were used; dA<sub>280</sub>/dT is not
shown for clarity) to a model of three sequential, interacting
unfolding transitions (transitions 1−3) assigned to the unfolding
of the pseudoknot tertiary structure, stem S2, and stem S1,
respectively (<italic toggle="yes"><xref rid="bi025843cb00013" ref-type="bibr"></xref></italic>). (B) pH dependence on the unfolding of the dC<sub>14</sub>
BWYV pseudoknot. The average (±SD) Δ<italic toggle="yes">G</italic><sub>1</sub> + Δ<italic toggle="yes">G</italic><sub>2</sub> values (37
°C) derived from four independent optical melting experiments
acquired at each pH value are shown. The solid line through the
data corresponds to a fit to an equation which describes the entire
dependence as attributable to a single protonatable group. The
apparent p<italic toggle="yes">K</italic><sub>a</sub> determined from this analysis is 5.7 (±0.3) (Table
<xref rid="bi025843ct00001"></xref>). The pH dependence of the unfolding of the wild-type BWYV
pseudoknot is shown (dashed line; cf. Figure <xref rid="bi025843cf00002"></xref>) for comparison.</p></caption><graphic xlink:href="bi025843cf00010.tif" position="float" orientation="portrait"></graphic></fig></p></sec><sec id="d7e2029"><title>Discussion</title><p>In the work described here, we provide thermodynamic
and structural evidence that all members of the <italic toggle="yes">Luteoviridae</italic>
P1−P2 mRNA frameshifting pseudoknots contain a common
collection of loop−stem tertiary structural interactions that
make integral contributions to the stability of the
pseudoknotted conformation relative to the partially unfolded S1 hairpin.
This conclusion is fully compatible with functional studies
of mutant BWYV (<italic toggle="yes"><xref rid="bi025843cb00016" ref-type="bibr"></xref></italic>), PLRV (<italic toggle="yes"><xref rid="bi025843cb00017" ref-type="bibr"></xref></italic>), and PEMV-1<sup>2</sup> pseudoknots; any disruption of these stabilizing tertiary structural
interactions was found to be generally deleterious to function.
</p><p><italic toggle="yes">The C<sup>+</sup></italic><sup></sup><italic toggle="yes">·(G-C) Base Triple.</italic> The C<sup>+</sup>·(G-C) major groove
base triple motif is a common feature of all luteoviral
pseudoknots, despite nucleotide sequence changes in the
PEMV-1 RNA which would be expected to abolish this
interaction. In all cases, the global stability of the pseudoknot
is markedly pH-dependent with a p<italic toggle="yes">K</italic><sub>a</sub> of unfolding markedly
elevated relative to that of free cytidine, such that significant
protonation of the loop L2 cytidine N3 occurs at neutral pH.
The thermodynamic driving force for the elevated p<italic toggle="yes">K</italic><sub>a</sub> is not
known with certainty although the structures of the BWYV
(<italic toggle="yes"><xref rid="bi025843cb00009" ref-type="bibr"></xref></italic>) and PEMV-1<sup>2</sup> pseudoknots reveal that while one face of
the protonated cytidine is largely exposed to solvent, the other
is nearby a patch of significant negative electrostatic potential
deep in the major groove of S2. The same is true of the
C<sup>+</sup>·(G-C) base triple in the genomic HDV ribozyme (<italic toggle="yes"><xref rid="bi025843cb00011" ref-type="bibr"></xref></italic>).
Protonation may therefore be important for enhancing
electrostatic complementarity in the major groove of S2.
Regardless of the molecular origin, protonation of the loop
L1 cytidine makes a significant contribution to the global
stability with a Δ(Δ<italic toggle="yes">G</italic><sub>37</sub>)<sub>folding</sub> of 3.1 (±0.3) and ≥2.5 kcal
mol<sup>-1</sup> for the BWYV and PLRV<sup>3</sup> pseudoknots, respectively,
and slightly less, 2.1 (±0.3) kcal mol<sup>-1</sup>, to the stability of
the PEMV-1 RNA.
</p><p>As anticipated, replacement of C<sub>8</sub> with m<sup>5</sup>-C<sub>8</sub> in the
BWYV pseudoknot is fully compatible with formation of
the pseudoknot (Figure <xref rid="bi025843cf00002"></xref>), with Δ(Δ<italic toggle="yes">G</italic><sub>37</sub>)<sub>folding</sub> of ≈1.1 (±0.5)
kcal mol<sup>-1</sup> at pH 7.0 (Table <xref rid="bi025843ct00001"></xref>) relative to that of the
unmethylated RNA. Although the effect is small (Figure <xref rid="bi025843cf00003"></xref>),
at least some of the difference in stability between the
BWYV and m<sup>5</sup>-C<sub>8</sub>-BWYV pseudoknots at pH 7.0 is due to
a small downward shift in the macroscopic p<italic toggle="yes">K</italic><sub>a</sub> for pseudoknot
unfolding (Table <xref rid="bi025843ct00001"></xref>), in a direction opposite to expectations
based on the relative p<italic toggle="yes">K</italic><sub>a</sub>'s of free cytidine (p<italic toggle="yes">K</italic><sub>a</sub> ≈ 4.2) and
free 5-methylcytidine (p<italic toggle="yes">K</italic><sub>a</sub> ≈ 4.4) (<italic toggle="yes"><xref rid="bi025843cb00030" ref-type="bibr"></xref></italic>). It is important to
recognize that the p<italic toggle="yes">K</italic><sub>a</sub> for (un)folding is an apparent p<italic toggle="yes">K</italic><sub>a</sub>
that measures the microscopic p<italic toggle="yes">K</italic><sub>a</sub> superimposed on changes
in base stacking and hydrophobicity and electrostatics (<italic toggle="yes"><xref rid="bi025843cb00032" ref-type="bibr"></xref></italic>),
as well as other coupled protonation/deprotonation equilibria,
which might influence the global unfolding equilibrium.
</p><p><italic toggle="yes">Thermodynamic and Structural Analysis of Loop L2
Mutants in the BWYV Pseudoknot: Cooperativity of Weak
Interactions. </italic>From the thermodynamic data alone, both the
PLRV and PEMV-1 pseudoknots were expected to contain
a comprehensive collection of loop−stem minor groove
interactions; the solution structure of the PEMV-1 pseudoknot
is consistent with this expectation.<xref rid="bi025843cb00002" ref-type="bibr"></xref> In addition, the NMR
spectra of both the BWYV (Figure <xref rid="bi025843cf00009"></xref>) and the PEMV-1
(Figure <xref rid="bi025843cf00008"></xref>) pseudoknots reveal that the 2‘-hydroxyl protons
of C<sub>14</sub> in BWYV and the analogous nucleotide (C<sub>15</sub>) in
PEMV-1 are significantly downfield-shifted and exchange
very slowly with solvent,<xref rid="bi025843cb00002" ref-type="bibr"></xref> evidence that they are involved
in donating hydrogen bonds to L2 adenosines in solution.
In an effort to define the extent to which each of these
interactions contributes to the stability of the pseudoknotted
conformation, A<sub>23</sub>G, A<sub>24</sub>G, and A<sub>25</sub>G BWYV RNAs, as well
as two 2‘-deoxy RNAs, dC<sub>14</sub> and dC<sub>15</sub>, were characterized.
</p><p>We find that the dC<sub>14</sub> BWYV RNA is indeed folded and
destabilized to an extent compatible with previous studies
of the <italic toggle="yes">T. thermophilus</italic> P4−P6 group I intron domain (<italic toggle="yes"><xref rid="bi025843cb00010" ref-type="bibr"></xref></italic>)
[Δ(Δ<italic toggle="yes">G</italic><sub>37</sub>)<sub>folding</sub> ≈ 1.2 kcal mol<sup>-1</sup>], with Δ(Δ<italic toggle="yes">G</italic><sub>37</sub>)<sub>folding</sub> ≈ 1.8
(±0.5) kcal mol<sup>-1</sup> at pH 7.0, coupled with a significant
reduction in the apparent p<italic toggle="yes">K</italic><sub>a</sub> of C<sub>8</sub> (Figure <xref rid="bi025843cf00010"></xref>, Table <xref rid="bi025843ct00001"></xref>). In
strong contrast, the dC<sub>15</sub> RNA is substantially unfolded under
these conditions, with only a minor population of molecules
adopting a pseudoknotted conformation as defined by the
intensity of the protonated C<sub>8</sub><sup>+</sup> amino protons (Figure <xref rid="bi025843cf00009"></xref>).
The same is true of the A<sub>25</sub>G RNA. In contrast, the structure
of the A<sub>24</sub>G and A<sub>23</sub>G RNAs are significantly perturbed with
little evidence of a stable folded pseudoknot. Knowledge of
the relative stabilities of the pseudoknot and partially folded
S1 hairpin reveals a Δ(Δ<italic toggle="yes">G</italic><sub>37</sub>)<sub>folding</sub> of ≈7 kcal mol<sup>-1</sup> at low
pH vs ≈4 kcal mol<sup>-1</sup> at elevated pH (Table <xref rid="bi025843ct00001"></xref>). Thus, a lower
limit for Δ(Δ<italic toggle="yes">G</italic><sub>37</sub>)<sub>folding</sub> which characterizes these A → G base
substitutions as well as the replacement of a single 2‘-OH
with a 2‘-H group in C<sub>15</sub> is ≥4 kcal mol<sup>-1</sup> under these
solution conditions, compatible with previous findings for
globally stabilizing A → G substitutions the P4−P6 domain
of the group I intron (<italic toggle="yes"><xref rid="bi025843cb00010" ref-type="bibr"></xref></italic>).
</p><p><italic toggle="yes">Implications for RNA Folding.</italic> In the absence of stabilizing
interactions associated specifically with A<sub>23</sub>, A<sub>24</sub>, and the G<sub>6</sub>-C<sub>15</sub> S1 base pair, a large-scale perturbation of complementarity of loop−stem interactions results, which, when not
allowed to form, subsequently abolishes the formation of
other stabilizing interactions nearer the helical junction and
perhaps through stem S2. This has the effect of coupling
the formation of the minor groove triplex with the formation
of the relatively weak three base pair stem S2, which itself
is further stabilized by protonation of an L1 nucleotide.
Molecular dynamics simulations carried out at elevated
temperature (400 K) provide additional evidence for strong
<italic toggle="yes">temporal</italic> coupling of disruption of the C<sub>8</sub><sup>+</sup>·G<sub>12</sub> Hoogsteen
pair with that of the minor groove triplex (<italic toggle="yes"><xref rid="bi025843cb00033" ref-type="bibr"></xref></italic>); along the
folding coordinate, this would obligatorily couple formation
of the minor groove triplex with the C<sub>8</sub><sup>+</sup>·(G<sub>12</sub>-C<sub>26</sub>) base triple.
Thus, A<sub>23</sub> and A<sub>24</sub> may therefore play important roles in
nucleating a strong cooperativity of subsequent interactions
along the kinetic folding reaction coordinate as well.
</p><p><italic toggle="yes">Functional Implications. </italic>These and previous studies reveal
a strong correlation between the formation of the major C<sup>+</sup>·(G-C) triple base pair and a minor groove triplex and the
ability of <italic toggle="yes">Luteoviridae</italic> P1−P2 pseudoknots to stimulate
programmed −1 ribosomal frameshifting in vitro (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi025843cb00016" ref-type="bibr"></xref>, <xref rid="bi025843cb00017" ref-type="bibr"></xref></named-content></italic>).<xref rid="bi025843cb00002" ref-type="bibr"></xref>
Indeed, it is not difficult to understand how mutations that
shift the folding equilibrium toward the S1 hairpin would
abolish stimulation of frameshifting activity. However, what
is not so clear is how small changes in the stability of a
loop−stem tertiary structural motif nearer the helical junction,
for example, could account for the large reductions in
frameshifting efficiency. The role of the protonated C<sup>+</sup>·(G-C) triple in stimulating ribosomal frameshifting is a case in
point. If it were simply the protonation state or stability
derived from this interaction which were responsible for
stimulation of frameshifting efficiencies, in vitro frameshifting assays would not likely have detected this, since these
experiments are typically carried out at pH 7.8,<xref rid="bi025843cb00004" ref-type="bibr"></xref> a pH at
which the luteoviral pseudoknots are predicted to be near
minimal stability (Table <xref rid="bi025843ct00001"></xref>). Despite this, mutations which
disrupt the pH dependence of the C<sup>+</sup>·(G-C) base triple but
do not abolish pseudoknot formation, e.g., U<sub>8</sub> BWYV
pseudoknot, result in an apparently complete loss in the
ability to stimulate frameshifting (<italic toggle="yes"><xref rid="bi025843cb00016" ref-type="bibr"></xref></italic>) with a relatively small
loss of thermodynamic stability at elevated pH (<italic toggle="yes"><xref rid="bi025843cb00013" ref-type="bibr"></xref></italic>). A similar
argument could also be made for the A<sub>25</sub>G substitution, which
is also functionally completely inactive (<italic toggle="yes"><xref rid="bi025843cb00016" ref-type="bibr"></xref></italic>).
</p><p>Other studies suggest that there is unlikely to be simple
relationship between the stability of the pseudoknot relative
to the partially folded hairpin conformation and efficiency
of stimulating −1 frameshifting, beyond that which is
required to ensure that the pseudoknotted structure dominates
the population ensemble (<italic toggle="yes"><xref rid="bi025843cb00004" ref-type="bibr"></xref></italic>).<xref rid="bi025843cb00005" ref-type="bibr"></xref> Similar conclusions have been
reached from studies of the HIV-1 <italic toggle="yes">gag-pol</italic> and HTLV-II
<italic toggle="yes">gag-pro</italic> stem−loop structures, which showed little to no
correlation between functional activity and predicted RNA
stem−loop stability (<italic toggle="yes"><xref rid="bi025843cb00034" ref-type="bibr"></xref></italic>). On the other hand, recent evidence
from the MMTV <italic toggle="yes">gag-pro</italic>, IBV-MMTV chimeric, and SRV-1
<italic toggle="yes">gag-pro</italic> pseudoknot systems suggests that weakly stabilizing
structural determinants positioned at the helical junction may
play a far more significant role in stimulation of frameshifting
activity as the pseudoknot abuts the elongating ribosome than
will global stability (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi025843cb00035" ref-type="bibr"></xref>−<xref rid="bi025843cb00036" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi025843cb00037" ref-type="bibr"></xref></named-content></italic>).<xref rid="bi025843cb00005" ref-type="bibr"></xref> It will be necessary to
elucidate the kinetics of pseudoknot unfolding and refolding,
as well as the structural nature of the transient interactions
of the pseudoknot with the ribosome, before a detailed
understanding of the mechanism of translational regulation
of gene expression by programmed −1 ribosomal frameshifting can be obtained.
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Giedroc, unpublished observations.</comment></mixed-citation></ref></ref-list></back></article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo><title>Thermodynamic Analysis of Conserved Loop−Stem Interactions in P1−P2 Frameshifting RNA Pseudoknots from Plant Luteoviridae†</title></titleInfo><titleInfo contentType="CDATA"><title>Thermodynamic Analysis of Conserved Loop−Stem Interactions in P1−P2 Frameshifting RNA Pseudoknots from Plant Luteoviridae†</title></titleInfo><name type="personal"><namePart type="family">NIXON</namePart><namePart type="given">Paul L.</namePart><affiliation>Department of Biochemistry and Biophysics, Center for Advanced Biomolecular Research, Texas A&M University,College Station, Texas 77843-2128</affiliation><affiliation> Current address: Department of Biochemistry, University of TexasSouthwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas,TX 75390-9038.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="family">CORNISH</namePart><namePart type="given">Peter V.</namePart><affiliation>Department of Biochemistry and Biophysics, Center for Advanced Biomolecular Research, Texas A&M University,College Station, Texas 77843-2128</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="family">SURAM</namePart><namePart type="given">Saritha V.</namePart><affiliation>Department of Biochemistry and Biophysics, Center for Advanced Biomolecular Research, Texas A&M University,College Station, Texas 77843-2128</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal" displayLabel="corresp"><namePart type="family">GIEDROC</namePart><namePart type="given">David P.</namePart><affiliation>Department of Biochemistry and Biophysics, Center for Advanced Biomolecular Research, Texas A&M University,College Station, Texas 77843-2128</affiliation><affiliation> Corresponding author. E-mail: giedroc@tamu.edu. Telephone: 979-845-4231. Fax: 979-845-4946.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="research-article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</genre><originInfo><publisher>American Chemical Society</publisher><dateCreated encoding="w3cdtf">2002-08-02</dateCreated><dateIssued encoding="w3cdtf">2002-08-27</dateIssued><copyrightDate encoding="w3cdtf">2002</copyrightDate></originInfo><note type="footnote" ID="bi025843cAF2"> This work was supported by grants from the NIH (AI40187) and the Texas Higher Education Coordinating Board Advanced Research Program (010361-0278-1999). P.V.C. was supported in part by an NIH Chemistry−Biology Interface Training Grant (T32 GM08523).</note><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract>The RNA genomes of plant luteovirids beet western yellows virus (BWYV), potato leaf roll virus (PLRV), and pea enation mosaic virus (PEMV RNA1; PEMV-1) contain a short mRNA pseudoknotted motif overlapping the P1 and P2 open reading frames required for programmed −1 mRNA ribosomal frameshifting. The relationship between structure, stability, and function is poorly understood in these RNA systems. A m5-C8-substituted BWYV RNA is employed to establish that the BWYV P1−P2 pseudoknot is protonated at cytidine 8 in loop L1 (δN3H+ = 12.98 ppm), which stabilizes a C+·(G−C) major groove base triple by Δ(ΔG37)protonation = 3.1 (±0.4) kcal mol-1. The stabilities of both the PLRV and PEMV-1 P1−P2 pseudoknots are also strongly pH-dependent, with Δ(ΔG37)protonation = 2.1 (±0.2) kcal mol-1 for the PEMV-1 pseudoknot despite a distinct structural context. As previously found for the BWYV pseudoknot [Nixon and Giedroc (2000) J. Mol. Biol. 296, 659], both the PLRV and PEMV-1 RNAs are stabilized by ΔH ≥ 30 kcal mol-1 in excess of secondary structure predictions, attributed to loop L2−stem S1 minor groove triplex interactions. BWYV RNAs containing single 2‘-deoxy or A → G substitutions that disrupt L2−S1 hydrogen bonding are strongly destabilized with Δ(ΔG37)folding (pH = 7.0) ranging from ≈1.8 (±0.3) to ≥4.0 kcal mol-1, relative to the wild-type BWYV RNA. These findings suggest that each member of this family of pseudoknots adopts a tightly folded structure that maximizes the cooperativity and complementarity of L1−S2 and L2−S1 loop−stem interactions required in part to offset the low intrinsic stability of the short three base pair pseudoknot stem S2.</abstract><note type="footnote" ID="bi025843cAF2"> This work was supported by grants from the NIH (AI40187) and the Texas Higher Education Coordinating Board Advanced Research Program (010361-0278-1999). 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Giedroc, unpublished observations.</title></titleInfo><note type="content-in-line">E. Sulistijo, B. Cannon, C. Theimer, and D. Giedroc, unpublished observations.</note></relatedItem><identifier type="istex">1AC18E5B035C5DF34B8387A5362A306048727D65</identifier><identifier type="ark">ark:/67375/TPS-76DVCBS5-1</identifier><identifier type="DOI">10.1021/bi025843c</identifier><accessCondition type="use and reproduction" contentType="restricted">Copyright © 2002 American Chemical Society</accessCondition><recordInfo><recordContentSource authority="ISTEX" authorityURI="https://loaded-corpus.data.istex.fr" valueURI="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-X5HBJWF8-J">acs</recordContentSource><recordOrigin>Converted from (version 1.2.10) to MODS version 3.6.</recordOrigin><recordCreationDate encoding="w3cdtf">2020-04-10</recordCreationDate></recordInfo></mods><json:item><extension>json</extension><original>false</original><mimetype>application/json</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-76DVCBS5-1/record.json</uri></json:item></metadata><annexes><json:item><extension>gif</extension><original>true</original><mimetype>image/gif</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-76DVCBS5-1/annexes.gif</uri></json:item><json:item><extension>tiff</extension><original>true</original><mimetype>image/tiff</mimetype><uri>https://api.istex.fr/document/1AC18E5B035C5DF34B8387A5362A306048727D65/annexes/tiff</uri></json:item></annexes><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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