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.