Thermodynamic characterization of the cooperativity of 40S complex formation during the initiation of eukaryotic protein synthesis.
Identifieur interne : 004257 ( Main/Exploration ); précédent : 004256; suivant : 004258Thermodynamic characterization of the cooperativity of 40S complex formation during the initiation of eukaryotic protein synthesis.
Auteurs : K M Parkhurst ; R E Hileman ; D. Saha ; N K Gupta ; L J ParkhurstSource :
- Biochemistry [ 0006-2960 ] ; 1994.
Descripteurs français
- KwdFr :
- ARN de transfert de la méthionine (métabolisme), ARN messager (métabolisme), Animaux, Biosynthèse des protéines, Codon, Colorants fluorescents, Données de séquences moléculaires, Facteur-2 d'initiation eucaryote (métabolisme), Facteurs d'échange de nucléotides guanyliques, Guanosine triphosphate (métabolisme), Polarisation de fluorescence, Protéines (métabolisme), Relation structure-activité, Ribosomes (métabolisme), Sites de fixation, Séquence nucléotidique, Thermodynamique.
- MESH :
- métabolisme : ARN de transfert de la méthionine, ARN messager, Facteur-2 d'initiation eucaryote, Guanosine triphosphate, Protéines, Ribosomes.
- Animaux, Biosynthèse des protéines, Codon, Colorants fluorescents, Données de séquences moléculaires, Facteurs d'échange de nucléotides guanyliques, Polarisation de fluorescence, Relation structure-activité, Sites de fixation, Séquence nucléotidique, Thermodynamique.
English descriptors
- KwdEn :
- Animals, Base Sequence, Binding Sites, Codon, Eukaryotic Initiation Factor-2 (metabolism), Fluorescence Polarization, Fluorescent Dyes, Guanine Nucleotide Exchange Factors, Guanosine Triphosphate (metabolism), Molecular Sequence Data, Protein Biosynthesis, Proteins (metabolism), RNA, Messenger (metabolism), RNA, Transfer, Met (metabolism), Ribosomes (metabolism), Structure-Activity Relationship, Thermodynamics.
- MESH :
- chemical , metabolism : Eukaryotic Initiation Factor-2, Guanosine Triphosphate, Proteins, RNA, Messenger, RNA, Transfer, Met.
- chemical : Codon, Fluorescent Dyes, Guanine Nucleotide Exchange Factors.
- metabolism : Ribosomes.
- Animals, Base Sequence, Binding Sites, Fluorescence Polarization, Molecular Sequence Data, Protein Biosynthesis, Structure-Activity Relationship, Thermodynamics.
Abstract
The first step in mammalian protein synthesis is the formation of the 40S initiation complex, composed of the 40S ribosomal subunit (R), mRNA (M, here, a 10-mer oligoribonucleotide analogue containing the initiation codon), and the quaternary complex (Q, composed of eIF-2, GTP, Met-tRNA(fMet), and the ancillary protein factor Co-eIF-2C). The interdependence of the binding of R, M, and Q in forming the 40S complex is currently unclear. We have determined the thermodynamic parameters that characterize these interactions. The binary constants for R+M and Q+M were determined spectroscopically, measuring changes in the anisotropy of the fluorescence emission of 3'-fluorescein labeled M. The other binary constant, for Q+R, and the ternary constant were determined from Millipore filtration assays using radiolabeled Met-tRNA(fMet). The association constants for the binary reactions were as follows: Ka(Q,M) < or = 0.14 x 10(6) M-1, Ka(R,M) = 1.78 x 10(6) M-1, and Ka(Q,R) = 0.94 x 10(6) M-1. The binding of Q to R.M was markedly greater than that of Q to R [Ka(Q,R.M)/Ka(Q,R) > 62]. High cooperativity for this interaction occurs in either a single-site model or in lattice models for the binding of M to R. Data obtained using five other RNA 10-mers, each with the sequence altered at the AUG codon, suggest that this cooperativity is AUG dependent. The data are consistent with a scheme in which mRNA and Q bind independently to the 40S ribosome, but when the AUG codon is properly aligned with Q, a conformational change results in a 2.4 kcal/mol stabilization of the complex.
DOI: 10.1021/bi00254a028
PubMed: 7999777
Affiliations:
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Saha</name></author><author><name sortKey="Gupta, N K" sort="Gupta, N K" uniqKey="Gupta N" first="N K" last="Gupta">N K Gupta</name></author><author><name sortKey="Parkhurst, L J" sort="Parkhurst, L J" uniqKey="Parkhurst L" first="L J" last="Parkhurst">L J Parkhurst</name></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="1994">1994</date><idno type="RBID">pubmed:7999777</idno><idno type="pmid">7999777</idno><idno type="doi">10.1021/bi00254a028</idno><idno type="wicri:Area/PubMed/Corpus">002848</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Corpus" wicri:corpus="PubMed">002848</idno><idno type="wicri:Area/PubMed/Curation">002848</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Curation">002848</idno><idno type="wicri:Area/PubMed/Checkpoint">002705</idno><idno type="wicri:explorRef" wicri:stream="Checkpoint" wicri:step="PubMed">002705</idno><idno type="wicri:Area/Ncbi/Merge">002951</idno><idno type="wicri:Area/Ncbi/Curation">002951</idno><idno type="wicri:Area/Ncbi/Checkpoint">002951</idno><idno type="wicri:doubleKey">0006-2960:1994:Parkhurst K:thermodynamic:characterization:of</idno><idno type="wicri:Area/Main/Merge">004319</idno><idno type="wicri:Area/Main/Curation">004257</idno><idno type="wicri:Area/Main/Exploration">004257</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">Thermodynamic characterization of the cooperativity of 40S complex formation during the initiation of eukaryotic protein synthesis.</title><author><name sortKey="Parkhurst, K M" sort="Parkhurst, K M" uniqKey="Parkhurst K" first="K M" last="Parkhurst">K M Parkhurst</name><affiliation><nlm:affiliation>Department of Chemistry, University of Nebraska--Lincoln, 68588-0304.</nlm:affiliation><wicri:noCountry code="subField">68588-0304</wicri:noCountry></affiliation></author><author><name sortKey="Hileman, R E" sort="Hileman, R E" uniqKey="Hileman R" first="R E" last="Hileman">R E Hileman</name></author><author><name sortKey="Saha, D" sort="Saha, D" uniqKey="Saha D" first="D" last="Saha">D. Saha</name></author><author><name sortKey="Gupta, N K" sort="Gupta, N K" uniqKey="Gupta N" first="N K" last="Gupta">N K Gupta</name></author><author><name sortKey="Parkhurst, L J" sort="Parkhurst, L J" uniqKey="Parkhurst L" first="L J" last="Parkhurst">L J Parkhurst</name></author></analytic><series><title level="j">Biochemistry</title><idno type="ISSN">0006-2960</idno><imprint><date when="1994" type="published">1994</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Animals</term><term>Base Sequence</term><term>Binding Sites</term><term>Codon</term><term>Eukaryotic Initiation Factor-2 (metabolism)</term><term>Fluorescence Polarization</term><term>Fluorescent Dyes</term><term>Guanine Nucleotide Exchange Factors</term><term>Guanosine Triphosphate (metabolism)</term><term>Molecular Sequence Data</term><term>Protein Biosynthesis</term><term>Proteins (metabolism)</term><term>RNA, Messenger (metabolism)</term><term>RNA, Transfer, Met (metabolism)</term><term>Ribosomes (metabolism)</term><term>Structure-Activity Relationship</term><term>Thermodynamics</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>ARN de transfert de la méthionine (métabolisme)</term><term>ARN messager (métabolisme)</term><term>Animaux</term><term>Biosynthèse des protéines</term><term>Codon</term><term>Colorants fluorescents</term><term>Données de séquences moléculaires</term><term>Facteur-2 d'initiation eucaryote (métabolisme)</term><term>Facteurs d'échange de nucléotides guanyliques</term><term>Guanosine triphosphate (métabolisme)</term><term>Polarisation de fluorescence</term><term>Protéines (métabolisme)</term><term>Relation structure-activité</term><term>Ribosomes (métabolisme)</term><term>Sites de fixation</term><term>Séquence nucléotidique</term><term>Thermodynamique</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Eukaryotic Initiation Factor-2</term><term>Guanosine Triphosphate</term><term>Proteins</term><term>RNA, Messenger</term><term>RNA, Transfer, Met</term></keywords><keywords scheme="MESH" type="chemical" xml:lang="en"><term>Codon</term><term>Fluorescent Dyes</term><term>Guanine Nucleotide Exchange Factors</term></keywords><keywords scheme="MESH" qualifier="metabolism" xml:lang="en"><term>Ribosomes</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>ARN de transfert de la méthionine</term><term>ARN messager</term><term>Facteur-2 d'initiation eucaryote</term><term>Guanosine triphosphate</term><term>Protéines</term><term>Ribosomes</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Animals</term><term>Base Sequence</term><term>Binding Sites</term><term>Fluorescence Polarization</term><term>Molecular Sequence Data</term><term>Protein Biosynthesis</term><term>Structure-Activity Relationship</term><term>Thermodynamics</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Animaux</term><term>Biosynthèse des protéines</term><term>Codon</term><term>Colorants fluorescents</term><term>Données de séquences moléculaires</term><term>Facteurs d'échange de nucléotides guanyliques</term><term>Polarisation de fluorescence</term><term>Relation structure-activité</term><term>Sites de fixation</term><term>Séquence nucléotidique</term><term>Thermodynamique</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The first step in mammalian protein synthesis is the formation of the 40S initiation complex, composed of the 40S ribosomal subunit (R), mRNA (M, here, a 10-mer oligoribonucleotide analogue containing the initiation codon), and the quaternary complex (Q, composed of eIF-2, GTP, Met-tRNA(fMet), and the ancillary protein factor Co-eIF-2C). The interdependence of the binding of R, M, and Q in forming the 40S complex is currently unclear. We have determined the thermodynamic parameters that characterize these interactions. The binary constants for R+M and Q+M were determined spectroscopically, measuring changes in the anisotropy of the fluorescence emission of 3'-fluorescein labeled M. The other binary constant, for Q+R, and the ternary constant were determined from Millipore filtration assays using radiolabeled Met-tRNA(fMet). The association constants for the binary reactions were as follows: Ka(Q,M) < or = 0.14 x 10(6) M-1, Ka(R,M) = 1.78 x 10(6) M-1, and Ka(Q,R) = 0.94 x 10(6) M-1. The binding of Q to R.M was markedly greater than that of Q to R [Ka(Q,R.M)/Ka(Q,R) > 62]. High cooperativity for this interaction occurs in either a single-site model or in lattice models for the binding of M to R. Data obtained using five other RNA 10-mers, each with the sequence altered at the AUG codon, suggest that this cooperativity is AUG dependent. The data are consistent with a scheme in which mRNA and Q bind independently to the 40S ribosome, but when the AUG codon is properly aligned with Q, a conformational change results in a 2.4 kcal/mol stabilization of the complex.</div></front></TEI><affiliations><list></list><tree><noCountry><name sortKey="Gupta, N K" sort="Gupta, N K" uniqKey="Gupta N" first="N K" last="Gupta">N K Gupta</name><name sortKey="Hileman, R E" sort="Hileman, R E" uniqKey="Hileman R" first="R E" last="Hileman">R E Hileman</name><name sortKey="Parkhurst, K M" sort="Parkhurst, K M" uniqKey="Parkhurst K" first="K M" last="Parkhurst">K M Parkhurst</name><name sortKey="Parkhurst, L J" sort="Parkhurst, L J" uniqKey="Parkhurst L" first="L J" last="Parkhurst">L J Parkhurst</name><name sortKey="Saha, D" sort="Saha, D" uniqKey="Saha D" first="D" last="Saha">D. 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