Evidence that Uncharged tRNA Can Inhibit a Programmed Translational Frameshift in Escherichia coli
Identifieur interne : 001A45 ( Main/Exploration ); précédent : 001A44; suivant : 001A46Evidence that Uncharged tRNA Can Inhibit a Programmed Translational Frameshift in Escherichia coli
Auteurs : Wenwu Gao [États-Unis] ; Hieronim Jakubowski [États-Unis] ; Emanuel Goldman [États-Unis]Source :
- Journal of Molecular Biology [ 0022-2836 ] ; 1995.
English descriptors
- KwdEn :
- Teeft :
- Amino, Amino acids, Analog, Assay, Biol, Codon, Cognate, Coli, Curran, Curran yarus, Escherichia, Escherichia coli, Excess trnatrp, Frameshift, Frameshift frequency, Frameshifting, Goldman, Hydroxamate, Hyperaccurate ribosomes, Jakubowski, Lysine, Natl acad, Overproduced, Plasmid, Protein synthesis, Ptrp, Ribosomal, Ribosome, Shift site, Sipley, Sipley goldman, Synthetase, Translational, Translational frameshift, Trna, Trna species, Trnatrp, Tryptophan, Tryptophan hydroxamate, Uncharged, Uncharged trna, Uncharged trna decreases, Uncharged trnatrp, Yarus.
Abstract
Abstract: In the modified release factor 2 (RF2) programmed translational frameshift (with a sense codon replacing the wild-type in-frame UGA codon at the shift site), ribosomes shift+1 into the reading frame for an out-of-frame reporter fused to the frameshift sequence. Partitioning of ribosomes between the out-of-frame shift and in-frame reading depends on the codon at the shift site and on the levels of tRNA decoding the in-frame codon. Overexpression of a tRNA species cognate to the in-frame codon at the shift site significantly reduces the frequency of frame-shifting, presumably by facilitating in-frame reading, which would reduce production of the out-of-frame reporter. However, since overexpression of a tRNA increases levels of both charged and uncharged tRNA, it is possible that uncharged cognate tRNA might be able to reduce the frequency of the frameshift, by entering the A site on the ribosome. To test this, we manipulated charged and uncharged tRNA levelsin vivo, using the tryptophan analog tryptophan hydroxamate, which increases the proportion of uncharged tRNATrpby competing with cognate amino acid tryptophan for tryptophanyl-tRNA synthetase, thereby reducing protein synthesis. We report here that a slight but reproducible reduction in the relative frequency of the frameshift is observed when tryptophan hydroxamate is added to cells containing the modified RF2 shift with UGG (Trp codon) at the shift site. When tRNATrpis overexpressed from another plasmid, the shift frequency drops three- to fourfold, as expected, however, this reduction is still seen in the presence of the analog. Thus, under conditions when most of the tRNATrpis apparently uncharged, excess tRNATrpstill causes a significant reduction in the frameshift when UGG is at the shift site, providing evidence that uncharged cognate tRNA also can inhibit this frameshift.
Url:
DOI: 10.1006/jmbi.1995.0428
Affiliations:
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Microbiology and Molecular Genetics, Graduate School of Biomedical Sciences, 185 South Orange Avenue, Newark, NJ 07103</wicri:regionArea><placeName><region type="state">New Jersey</region></placeName></affiliation></author><author><name sortKey="Jakubowski, Hieronim" sort="Jakubowski, Hieronim" uniqKey="Jakubowski H" first="Hieronim" last="Jakubowski">Hieronim Jakubowski</name><affiliation wicri:level="2"><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Microbiology and Molecular Genetics, Graduate School of Biomedical Sciences, 185 South Orange Avenue, Newark, NJ 07103</wicri:regionArea><placeName><region type="state">New Jersey</region></placeName></affiliation></author><author><name sortKey="Goldman, Emanuel" sort="Goldman, Emanuel" uniqKey="Goldman E" first="Emanuel" last="Goldman">Emanuel Goldman</name><affiliation wicri:level="2"><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Microbiology and Molecular Genetics, Graduate School of Biomedical Sciences, 185 South Orange Avenue, Newark, NJ 07103</wicri:regionArea><placeName><region type="state">New Jersey</region></placeName></affiliation></author></analytic><monogr></monogr><series><title level="j">Journal of Molecular Biology</title><title level="j" type="abbrev">YJMBI</title><idno type="ISSN">0022-2836</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1995">1995</date><biblScope unit="volume">251</biblScope><biblScope unit="issue">2</biblScope><biblScope unit="page" from="210">210</biblScope><biblScope unit="page" to="216">216</biblScope></imprint><idno type="ISSN">0022-2836</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0022-2836</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>aminoacyl-tRNA synthetase</term><term>hyperaccurate ribosomes</term><term>release factor 2</term><term>ribosomal A site</term><term>tryptophan hydroxamate</term></keywords><keywords scheme="Teeft" xml:lang="en"><term>Amino</term><term>Amino acids</term><term>Analog</term><term>Assay</term><term>Biol</term><term>Codon</term><term>Cognate</term><term>Coli</term><term>Curran</term><term>Curran yarus</term><term>Escherichia</term><term>Escherichia coli</term><term>Excess trnatrp</term><term>Frameshift</term><term>Frameshift frequency</term><term>Frameshifting</term><term>Goldman</term><term>Hydroxamate</term><term>Hyperaccurate ribosomes</term><term>Jakubowski</term><term>Lysine</term><term>Natl acad</term><term>Overproduced</term><term>Plasmid</term><term>Protein synthesis</term><term>Ptrp</term><term>Ribosomal</term><term>Ribosome</term><term>Shift site</term><term>Sipley</term><term>Sipley goldman</term><term>Synthetase</term><term>Translational</term><term>Translational frameshift</term><term>Trna</term><term>Trna species</term><term>Trnatrp</term><term>Tryptophan</term><term>Tryptophan hydroxamate</term><term>Uncharged</term><term>Uncharged trna</term><term>Uncharged trna decreases</term><term>Uncharged trnatrp</term><term>Yarus</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Abstract: In the modified release factor 2 (RF2) programmed translational frameshift (with a sense codon replacing the wild-type in-frame UGA codon at the shift site), ribosomes shift+1 into the reading frame for an out-of-frame reporter fused to the frameshift sequence. Partitioning of ribosomes between the out-of-frame shift and in-frame reading depends on the codon at the shift site and on the levels of tRNA decoding the in-frame codon. Overexpression of a tRNA species cognate to the in-frame codon at the shift site significantly reduces the frequency of frame-shifting, presumably by facilitating in-frame reading, which would reduce production of the out-of-frame reporter. However, since overexpression of a tRNA increases levels of both charged and uncharged tRNA, it is possible that uncharged cognate tRNA might be able to reduce the frequency of the frameshift, by entering the A site on the ribosome. To test this, we manipulated charged and uncharged tRNA levelsin vivo, using the tryptophan analog tryptophan hydroxamate, which increases the proportion of uncharged tRNATrpby competing with cognate amino acid tryptophan for tryptophanyl-tRNA synthetase, thereby reducing protein synthesis. We report here that a slight but reproducible reduction in the relative frequency of the frameshift is observed when tryptophan hydroxamate is added to cells containing the modified RF2 shift with UGG (Trp codon) at the shift site. When tRNATrpis overexpressed from another plasmid, the shift frequency drops three- to fourfold, as expected, however, this reduction is still seen in the presence of the analog. Thus, under conditions when most of the tRNATrpis apparently uncharged, excess tRNATrpstill causes a significant reduction in the frameshift when UGG is at the shift site, providing evidence that uncharged cognate tRNA also can inhibit this frameshift.</div></front></TEI><affiliations><list><country><li>États-Unis</li></country><region><li>New Jersey</li></region></list><tree><country name="États-Unis"><region name="New Jersey"><name sortKey="Gao, Wenwu" sort="Gao, Wenwu" uniqKey="Gao W" first="Wenwu" last="Gao">Wenwu Gao</name></region><name sortKey="Goldman, Emanuel" sort="Goldman, Emanuel" uniqKey="Goldman E" first="Emanuel" last="Goldman">Emanuel Goldman</name><name sortKey="Jakubowski, Hieronim" sort="Jakubowski, Hieronim" uniqKey="Jakubowski H" first="Hieronim" last="Jakubowski">Hieronim Jakubowski</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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