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J. Bacteriol. doi:10.1128/JB.01868-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

RRF and RF3 action promotes TnaC-peptidyl-tRNA drop-off and relieves ribosome stalling during tryptophan induction of tna operon expression in E. coli

Department of Biological Sciences, Stanford University, Stanford, California

^* To whom correspondence should be addressed. Email: yanofsky{at}cmgm.stanford.edu.

	Abstract

Upon tryptophan induction of tna operon expression in E. coli, the leader peptidyl-tRNA, TnaC-tRNA₂^Pro, resists cleavage, resulting in ribosome stalling at the tnaC stop codon. This stalled ribosome blocks Rho factor binding and action, preventing transcription termination in the tna operon's leader region. Plasmid-mediated overexpression of tnaC was previously shown to inhibit cell growth by reducing uncharged tRNA₂^Pro availability. Which factors relieve ribosome stalling, facilitate TnaC-tRNA₂^Pro cleavage, and relieve growth inhibition, were addressed in the current study. In strains containing the chromosomal tna operon and lacking a tnaC plasmid, overproduction of RRF and RF3 reduced tna operon expression. Their overproduction in vivo also increased the rate of cleavage of TnaC-tRNA₂^Pro, relieving the growth inhibition associated with plasmid-mediated tnaC overexpression. Overproduction of EF-G or IF3 did not have comparable effects, and tmRNA was incapable of attacking TnaC-tRNA₂^Pro in stalled ribosome complexes. The stability of TnaC-tRNA₂^Pro was increased appreciably in strains deficient in RRF and RF3, or deficient in peptidyl-tRNA hydrolase. These findings reveal the existence of a natural mechanism whereby an amino acid, tryptophan, binds to ribosomes that have just completed synthesis of TnaC-tRNA₂^Pro. Bound tryptophan inhibits RF2-mediated cleavage of TnaC-tRNA₂^Pro, resulting in stalling of the ribosome translating tnaC mRNA. This stalling results in increased transcription of the structural genes of the tna operon. RRF and RF3 then bind to this stalled ribosome complex and slowly release TnaC-tRNA₂^Pro. This release allows ribosome recycling, and permits cleavage of TnaC-tRNA₂^Pro by peptidyl-tRNA hydrolase.

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