Stop codon recognition and interactions with peptide release factor RF3 of truncated and chimeric RF1 and RF2 from Escherichia coli.
Release factors RF1 and RF2 recognize stop codons present at the A-site of the ribosome and activate hydrolysis of peptidyl-tRNA to release the peptide chain. Interactions with RF3, a ribosome-dependent GTPase, then initiate a series of reactions that accelerate the dissociation of RF1 or RF2 and their recycling between ribosomes. Two regions of Escherichia coli RF1 and RF2 were identified previously as involved in stop codon recognition and peptidyl-tRNA hydrolysis. We show here that removing the N-terminal domain of RF1 or RF2 or exchanging this domain between the two factors does not affect RF specificity but has different effects on the activity of RF1 and RF2: truncated RF1 remains highly active and able to support rapid cell growth, whereas cells with truncated RF2 grow only poorly. Transplanting a loop of 13 amino acid residues from RF2 to RF1 switches the stop codon specificity. The interaction of the truncated factors with RF3 on the ribosome is defective: they fail to stimulate guanine nucleotide exchange on RF3, recycling is not stimulated by RF3, and nucleotide-free RF3 fails to stabilize the binding of RF1 or RF2 to the ribosome. However, the N-terminal domain seems not to be required for the expulsion of RF1 or RF2 by RF3:GTP.
Mora
Liliana
L
UPR9073 du CNRS, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, Paris 75005, France.
Zavialov
Andrei
A
Ehrenberg
Måns
M
Buckingham
Richard H
RH
D000595:Amino Acid Sequence; D018388:Codon, Terminator / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010454:Peptide Termination Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism
D018388:Codon, Terminator; D029968:Escherichia coli Proteins; D010454:Peptide Termination Factors; D012346:RNA, Transfer, Amino Acyl; D011993:Recombinant Fusion Proteins; C041460:peptide chain termination release factor 2; C087971:peptide-chain-release factor 3; C489005:prfA protein, E coli; C489006:prfB protein, E coli; C489007:prfC protein, E coli; C019037:tRNA, peptidyl-