GTP CONSUMPTION OF ELONGATION-FACTOR TU DURING TRANSLATION OF HETEROPOLYMERIC MESSENGER-RNAS

被引:171
作者
RODNINA, MV [1 ]
WINTERMEYER, W [1 ]
机构
[1] UNIV WITTEN HERDECKE,INST MOLEK BIOL,D-58448 WITTEN,GERMANY
关键词
RIBOSOMES; FRAMESHIFT; PROTEIN SYNTHESIS; TRANSLOCATION; TRANSFER-RNA;
D O I
10.1073/pnas.92.6.1945
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
The stoichiometry of elongation factor Tu (EF-Tu) and GTP in the complex with aminoacyl-tRNA and the consumption of GTP during peptide bond formation on the ribosome were studied in the Escherichia coli system. The ribosomes were programmed either with two different heteropolymeric mRNAs coding for Met-Phe-Thr-Ile... (mMFTI) or Met-Phe-Phe-Gly... (mMFFG) or with poly(U). The composition of the complex of EF-Tu, GTP, and Phe-tRNA(Phe) was studied by gel chromatography. With equimolar amounts of factor and Phe-tRNA(Phe), a pentameric complex, (EF-Tu . GTP)(2) . Phe-tRNA(Phe), was observed, whereas the classical ternary complex, EF-Tu . GTP . Phe-tRNA(Phe), was found only when Phe-tRNA(Phe) was in excess. Upon binding of the purified pentameric complex to ribosomes carrying fMet-tRNA(fMet) in the peptidyl site and exposing a Phe codon in the aminoacyl site, only one out of two GTPs of the pentameric complex was hydrolyzed per Phe-tRNA bound and peptide bond formed, regardless of the mRNA used. In the presence of EF-G, the stoichiometry of one GTP hydrolyzed per peptide bond formed was found on mMFTI when one or two elongation cycles were completed. In contrast, on mMFFG, which contains two contiguous Phe codons, UUU-UUC, two GTP molecules of the pentameric complex were hydrolyzed per Phe incorporated into dipeptide, whereas the incorporation of the second Phe to form tripeptide consumed only one GTP. Thus, generally one GTP is hydrolyzed by EF-Tu per aminoacyl-tRNA bound and peptide bond formed, and more than one GTP is hydrolyzed only when a particular mRNA sequence, such as a homopolymeric stretch, is translated. The role of the additional GTP hydrolysis is not known; it may be related to frameshifting of peptidyl-tRNA during translocation.
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页码:1945 / 1949
页数:5
相关论文
共 23 条
[1]  
ATKINS JF, 1991, ANNU REV GENET, V25, P210
[2]   HOW MANY EF-TU MOLECULES PARTICIPATE IN AMINOACYL-TRANSFER RNA-BINDING [J].
BENSCH, K ;
PIEPER, U ;
OTT, G ;
SCHIRMER, N ;
SPRINZL, M ;
PINGOUD, A .
BIOCHIMIE, 1991, 73 (7-8) :1045-1050
[3]   KINETIC-PROPERTIES OF ESCHERICHIA-COLI RIBOSOMES WITH ALTERED FORMS OF S12 [J].
BILGIN, N ;
CLAESENS, F ;
PAHVERK, H ;
EHRENBERG, M .
JOURNAL OF MOLECULAR BIOLOGY, 1992, 224 (04) :1011-1027
[4]   SELECTION OF THE MESSENGER-RNA TRANSLATION INITIATION REGION BY ESCHERICHIA-COLI RIBOSOMES [J].
CALOGERO, RA ;
PON, CL ;
CANONACO, MA ;
GUALERZI, CO .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1988, 85 (17) :6427-6431
[5]   HOW MANY EF-TU MOLECULES PARTICIPATE IN AMINOACYL-TRANSFER-RNA BINDING AND PEPTIDE-BOND FORMATION IN ESCHERICHIA-COLI TRANSLATION [J].
EHRENBERG, M ;
ROJAS, AM ;
WEISER, J ;
KURLAND, CG .
JOURNAL OF MOLECULAR BIOLOGY, 1990, 211 (04) :739-749
[6]  
EHRENBERG M, 1993, TRANSLATIONAL APPARATUS, P305
[7]  
EHRENBERG M, 1990, RIBOSOME, P373
[8]  
Ehrenberg M., 1990, RIBOSOMES PROTEIN SY, P101
[9]  
GORDON J, 1969, J BIOL CHEM, V244, P5680
[10]   MUTANTS OF ELONGATION-FACTOR TU PROMOTE RIBOSOMAL FRAMESHIFTING AND NONSENSE READTHROUGH [J].
HUGHES, D ;
ATKINS, JF ;
THOMPSON, S .
EMBO JOURNAL, 1987, 6 (13) :4235-4239