Spontaneous, elongation factor G independent translocation of Escherichia coli ribosomes.
In a classical translocation experiment, deacylated RNA is bound to the ribosomal peptidyl-tRNA site (P site) and N-acetyl-phenylalanyl-tRNA (AcPhe-tRNA) to the aminoacyl-tRNA site (A site); upon addition of elongation factor (EF-G) and GTP, AcPhe-tRNA is translocated from the A to the P site. Here, we demonstrate a model reaction for a spontaneous, EF-G independent translocation. If AcPhe-tRNA is bound to the P site and Phe-tRNA to the A site at 15 mM Mg2+, then at 37 degrees C up to 60% of the AcPhe2-tRNA formed is found at the P site without the addition of EF-G. We demonstrate the following: 1) the spontaneous translocation is not merely illusory as a result of Phe-tRNA binding directly to the P site; 2) it is not mimicked by release of AcPhe2-tRNA from an A site and rebinding to a P site of another ribosome; 3) it is not caused by an EF-G contaminant present in the 70 S preparation, since without EF-G the spontaneous translocation works equally well in the presence of guanyl-5'-yl imidodiphosphate or fusidic acid; 4) AcPhe2-tRNA evidently has a higher affinity for the P site than AcPhe-tRNA, thus promoting the spontaneous translocation; and 5) peptide-bond formation favors the subsequent translocation. Addition of EF-G increases the initial rate by a factor of 13. Furthermore, at 15 mM Mg2+, 37 degrees C and in the presence of EF-G and GTP, Phe-tRNA cannot be translocated from the A to the P site, if the P site is occupied by deacylated tRNA. With the spontaneous translocation system, all reactions of the elongation cycle are cooperatively interconnected; i.e. upon binding of aminoacyl-tRNA to the A site, a significant portion of the ribosomes performs a complete round of the elongation cycle without the addition of elongation factor EF-G.
D004926:Escherichia coli / Q000235:genetics*; D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D014178:Translocation, Genetic
D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors; D012346:RNA, Transfer, Amino Acyl; C006383:tRNA, N-acetylphenylalanine-