Published online 18 August 2004
Nucleic Acids Research, Vol. 32 No. 15 © Oxford University Press 2004; all rights reserved
Ribosome stalling and peptidyl-tRNA drop-off during translational delay at AGA codons
Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados del IPN, Apartado Postal 14-740, 07000 México D.F.
* To whom correspondence should be addressed. Tel: +52 55 5061 3338; Fax: +52 55 5747 7100; Email: gguarner{at}enigma.red.cinvestav.mx
Present address: Luis Rogelio Cruz-Vera, Department of Biological Sciences, Stanford University, Stanford CA 94305-5020, USA
Received May 19, 2004; Revised and Accepted August 3, 2004
Minigenes encoding the peptide Met–Arg–Arg have been used to study the mechanism of toxicity of AGA codons proximal to the start codon or prior to the termination codon in bacteria. The codon sequences of the ‘mini-ORFs’ employed were initiator, combinations of AGA and CGA, and terminator. Both, AGA and CGA are low-usage Arg codons in ORFs of Escherichia coli but, whilst AGA is translated by the scarce tRNAArg4, CGA is recognized by the abundant tRNAArg2. Overexpression of minigenes harbouring AGA in the third position, next to a termination codon, was deleterious to the cell and led to the accumulation of peptidyl-tRNAArg4 and of the peptidyl-tRNA cognate to the preceding CGA or AGA Arg triplet. The minigenes carrying CGA in the third position were not toxic. Minigene-mediated toxicity and peptidyl-tRNA accumulation were suppressed by overproduction of tRNAArg4 but not by overproduction of peptidyl-tRNA hydrolase, an enzyme that is only active on substrates that have been released from the ribosome. Consistent with these findings, peptidyl-tRNAArg4 was identified to be mainly associated with ribosomes in a stand-by complex. These and previous results support the hypothesis that the primary mechanism of inhibition of protein synthesis by AGA triplets in pth+ cells involves sequestration of tRNAs as peptidyl-tRNA on the stalled ribosome.
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