Published online 20 October 2005
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Function of the ribosomal E-site: a mutagenesis study
Department of Chemistry and A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University Moscow, 119899, Russia 1Department of Bioinformatics and Bioengineering, Moscow State University Moscow, 119899, Russia 2Max Planck Institut fur Molekulare Genetik Berlin, D-14195, Germany
*To whom correspondence should be addressed. Tel: +7 095 9328824; Fax: +7 095 9393181; Email: dontsova{at}genebee.msu.su
Received June 14, 2005. Revised September 21, 2005. Accepted October 1, 2005.
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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