Ribosomes synthesize proteins according to the information encoded in mRNA. During this process, both the incoming amino acid and the nascent peptide are bound to tRNA molecules. Three binding sites for tRNA in the ribosome are known: the A-site for aminoacyl-tRNA, the P-site for peptidyl-tRNA and the E-site for the deacylated tRNA leaving the ribosome. Here, we present a study of Escherichia coli ribosomes with the E-site binding destabilized by mutation C2394G of the 23S rRNA. Expression of the mutant 23S rRNA in vivo caused increased frameshifting and stop codon readthrough. The progression of these ribosomes through the ribosomal elongation cycle in vitro reveals ejection of deacylated tRNA during the translocation step or shortly after. E-site compromised ribosomes can undergo translocation, although in some cases it is less efficient and results in a frameshift. The mutation affects formation of the P/E hybrid site and leads to a loss of stimulation of the multiple turnover GTPase activity of EF-G by deacylated tRNA bound to the ribosome.

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Abstract5S rRNA extends from the central protuberance of the large ribosomal subunit, through the A-site finger, and down to the GTPase-associated center. Here, we present a structure-function analysis of seven 5S rRNA alleles which are sufficient for viability in the yeast Saccharomyces cerevisiae when expressed in the absence of wild-type 5S rRNAs, and extend this analysis using a large bank of mutant alleles that show semidominant phenotypes in the presence of wild-type 5S rRNA. This analysis supports the hypothesis that 5S rRNA serves to link together several different functional centers of the ribosome. Data are also presented which suggest that in eukaryotic genomes selection has favored the maintenance of multiple alleles of 5S rRNA, and that these may provide cells with a mechanism to post-transcriptionally regulate gene expression.

Translocation catalyzed by elongation factor G occurs after the peptidyltransferase reaction on the large ribosomal subunit. Deacylated tRNA in the P-site stimulates multiple turnover GTPase activity of EF-G. We suggest that the allosteric signal from the peptidyltransferase center that activates EF-G may involve the alteration in the conformation of elongation factor binding center of the ribosome. The latter consists of the moveable GTPase-associated center and the sarcin-ricin loop that keeps its position on the ribosome during translation elongation. The position of the GTPase-associated center was altered by mutagenesis. An insertion of additional base pair at positions C1030/G1124 was lethal and affected function of EF-G, but not that of EF-Tu. Structure probing revealed a putative allosteric signal pathway connecting the P-site with the binding site of the elongation factors. The results are consistent with the different structural requirements for EF-G and EF-Tu function, where the integrity of the path between the peptidyltransferase center and both GTPase-associated center and sarcin-ricin loop is important for EF-G binding.The ribosome is a large molecular machine for protein synthesis. During the elongation EF 1 -Tu brings aminoacyl-tRNA to the ribosomal A-site, and its GTPase activity is stimulated by recognition of the mRNA codon by the aminoacyl-tRNA anticodon at the decoding center of the small subunit. EF-G acts after completion of peptide transfer at the peptidyltransferase center of the large subunit.Before the peptidyltransferase reaction, the growing peptide is attached to the 3Ј-end of the tRNA bound to the P-site, whereas the aminoacyl-tRNA is located in the A-site. After peptide transfer, the A-site carries the peptidyl-tRNA, whereas the P-site holds the deacylated tRNA. This is the state that should be recognized by EF-G. It has been reported that deacylated tRNA in the P-site stimulates multiple turnover GTPase activity of EF-G, but that peptidyl-tRNA does not (1, 2). This stimulation is attributed to the enhanced binding of EF-G to the ribosome (2).The P-site is located more than 70 Å away from the binding site of the elongation factors. Thus, an intriguing question is how can information from the P-site be transmitted across half the width of the ribosome? We believe that to answer this question one must seek for elements that are located between the peptidyltransferase center and the elongation factor binding site, the latter consisting of the GTPase-associated center (GAC) and the sarcin-ricin loop (SRL) (3). One can expect that some of the relevant elements should be mobile, as they have to transfer an allosteric signal by its movement relative to the rest of the large subunit. The SRL interacts with the switch regions of the elongation factors close to the GTP binding site (4), and it is likely that it is involved in the activation of GTP hydrolysis. However, in all available atomic structures of large ribosomal subunits from various organisms, as well as in cryoelectron microscop...

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