GTP consumption of elongation factor Tu during translation of heteropolymeric mRNAs.
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.
Rodnina
M V
MV
Institut für Molekularbiologie, Universität Witten/Herdecke, Germany.
Wintermeyer
W
W
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002250:Carbon Radioisotopes; D004926:Escherichia coli / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010444:Peptide Elongation Factor Tu / Q000302:isolation & purification / Q000378:metabolism*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000302:isolation & purification / Q000378:metabolism*; D012360:RNA, Transfer, Phe / Q000302:isolation & purification / Q000378:metabolism*; D012363:RNA, Transfer, Thr / Q000378:metabolism; D016364:Reading Frames; D012270:Ribosomes / Q000378:metabolism; D014316:Tritium
D002250:Carbon Radioisotopes; D012333:RNA, Messenger; D012346:RNA, Transfer, Amino Acyl; D012360:RNA, Transfer, Phe; D012363:RNA, Transfer, Thr; D014316:Tritium; D006160:Guanosine Triphosphate; D010444:Peptide Elongation Factor Tu