No abstract

SummaryIn this study, we have examined the influence of initiation factors on translation initiation of leaderless mRNAs whose 5Ј-terminal residues are the A of the AUG initiating codon. A 1:1 ratio of initiation factors to ribosomes abolished ternary complex formation at the authentic start codon of different leaderless mRNAs. Supporting this observation, in vitro translation assays using limiting ribosome concentrations with competing leaderless cI and Escherichia coli ompA mRNAs, the latter containing a canonical ribosome binding site, revealed reduced cI synthesis relative to OmpA in the presence of added initiation factors. Using in vitro toeprinting and in vitro translation assays, we show that this effect can be attributed to IF3. Moreover, in vivo studies revealed that the translational efficiency of a leaderless reporter gene is decreased with increased IF3 levels. These studies are corroborated by the observed increased translational efficiency of a leaderless reporter construct in an infC mutant strain unable to discriminate against non-standard start codons. These results suggest that, in the absence of a leader or a Shine-Dalgarno sequence, the function(s) of IF3 limits stable 30S ternary complex formation.

SummaryThe l S gene represents the prototype of holin genes with a dual-start motif, which leads to the synthesis of two polypeptides, S105 and S107. They differ at their N-terminus by only two amino acids, Met-1 and Lys-2, at the beginning of the longer product. Despite the minor difference, the two proteins have opposing functions in lysis, with protein S107 being an inhibitor and protein S105 being an effector of`hole formation' in the inner membrane. Here, we have studied the molecular mechanism underlying the`lysis clock' contributed by the dual-start motif. We have used protein fusions in which the secretory signal sequence of the M13 procoat protein VIII has been abutted to the Nterminal Met residues of S105 and S107 respectively. S-dependent`hole formation' required removal of the signal sequence in both fusion proteins, as both the VIII±S105 and the VIII±S107 fusion proteins were nonfunctional when leader peptidase cleavage was inhibited. These results strongly supported the hypothesis that functional assembly of S proteins requires translocation of their N-terminus to the periplasm. Using signal sequence cleavage as a measure of translocation, we observed that the translocation kinetics of the N-terminus of the S107 moiety was reduced about threefold when compared with the N-terminus of the S105 moiety. Moreover, depolarization of the membrane resulted in an immediate cleavage of the signal sequence and`hole formation' exerted by the S107 moiety of the VIII±S107 fusion protein. A model is presented in which S107 with a reversed topology of its N-terminus interacts with S105 and poisons`hole formation'. Upon depolarization of the membrane, translocation of the N-terminus of S107 to the periplasm results in the functional assembly of S proteins, i.e. hole formation'.