Rodnina M V, Wintermeyer W
Institut für Molekularbiologie, Universität Witten/Herdecke, Germany.
Proc Natl Acad Sci U S A. 1995 Mar 14;92(6):1945-9. doi: 10.1073/pnas.92.6.1945.
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.
在大肠杆菌系统中，研究了延伸因子Tu（EF-Tu）与GTP在与氨酰tRNA形成的复合物中的化学计量关系，以及核糖体上肽键形成过程中GTP的消耗情况。核糖体用两种不同的编码Met-Phe-Thr-Ile...（mMFTI）或Met-Phe-Phe-Gly...（mMFFG）的异聚体mRNA或聚（U）进行编程。通过凝胶色谱法研究了EF-Tu、GTP和苯丙氨酰tRNA（Phe）复合物的组成。当因子和苯丙氨酰tRNA（Phe）等摩尔时，观察到一种五聚体复合物，即(EF-Tu.GTP)2.Phe-tRNA(Phe)，而经典的三元复合物EF-Tu.GTP.Phe-tRNA(Phe)仅在苯丙氨酰tRNA（Phe）过量时才会出现。将纯化的五聚体复合物与在肽酰位点携带甲酰甲硫氨酰tRNA（fMet）且在氨酰位点暴露苯丙氨酸密码子的核糖体结合后，无论使用何种mRNA，每结合一个苯丙氨酰tRNA并形成肽键，五聚体复合物中的两个GTP中只有一个会被水解。在EF-G存在的情况下，当完成一个或两个延伸循环时，在mMFTI上发现每形成一个肽键水解一个GTP的化学计量关系。相比之下，在含有两个相邻苯丙氨酸密码子UUU-UUC的mMFFG上，每掺入一个苯丙氨酸形成二肽，五聚体复合物的两个GTP分子会被水解，而掺入第二个苯丙氨酸形成三肽仅消耗一个GTP。因此，一般来说，每结合一个氨酰tRNA并形成一个肽键，EF-Tu会水解一个GTP，只有当翻译特定的mRNA序列（如同聚体片段）时，才会水解不止一个GTP。额外的GTP水解的作用尚不清楚；它可能与转位过程中肽酰tRNA的移码有关。
