Initiation factors IF1 and IF2 synergistically remove peptidyl-tRNAs with short polypeptides from the P-site of translating Escherichia coli ribosomes.
A novel function of initiation factors IF1 and IF2 in Escherichia coli translation has been identified. It is shown that these factors efficiently catalyse dissociation of peptidyl-tRNAs with polypeptides of different length from the P-site of E. coli ribosomes, and that the simultaneous presence of both factors is required for induction of drop-off. The factor-induced drop-off occurs with both sense and stop codons in the A-site and competes with peptide elongation or termination. The efficiency with which IF1 and IF2 catalyse drop-off decreases with increasing length of the nascent polypeptide, but is quite significant for hepta-peptidyl-tRNAs, the longest polypeptide chains studied. In the absence of IF1 and IF2 the rate of drop-off varies considerably for different peptidyl-tRNAs, and depends both on the length and sequence of the nascent peptide. Efficient factor-catalysed drop-off requires GTP but not GTP hydrolysis, as shown in experiments without guanine nucleotides, with GDP or with the non-cleavable analogue GMP-PNP.Simultaneous overexpression of IF1 and IF2 in vivo inhibits cell growth specifically in some peptidyl-tRNA hydrolase deficient mutants, suggesting that initiation factor-catalysed drop-off of peptidyl-tRNA can occur on a significant scale in the bacterial cell. Consequences for the bacterial physiology of this previously unknown function of IF1 and IF2 are discussed.
Karimi
R
R
Department of Molecular Biology, BMC, Uppsala, S-75124, Sweden.
Pavlov
M Y
MY
Heurgué-Hamard
V
V
Buckingham
R H
RH
Ehrenberg
M
M
D002265:Carboxylic Ester Hydrolases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D006160:Guanosine Triphosphate / Q000502:physiology; D009842:Oligopeptides / Q000378:metabolism*; D010448:Peptide Initiation Factors / Q000378:metabolism* / Q000494:pharmacology; D039881:Prokaryotic Initiation Factor-2; D014176:Protein Biosynthesis / Q000502:physiology*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012358:RNA, Transfer, Met / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*
D009842:Oligopeptides; D010448:Peptide Initiation Factors; D039881:Prokaryotic Initiation Factor-2; D012346:RNA, Transfer, Amino Acyl; D012358:RNA, Transfer, Met; C019037:tRNA, peptidyl-; D006160:Guanosine Triphosphate; D002265:Carboxylic Ester Hydrolases; C019674:aminoacyl-tRNA hydrolase