Leaderless mRNAs in bacteria: surprises in ribosomal recruitment and translational control trast, the mechanism(s) leading to translation of leaderless mRNA, in which the start codon is either preceded by only a few nucleotides or which starts directly with a 5¢-terminal AUG, has remained elusive. In this review, we summarize the current knowledge on translation of leaderless mRNAs in Escherichia coli and discuss their possible biological implication. Ribosomal recruitment signals on leaderless mRNAs downstream of the start codon: do they exist?The finding of a Watson and Crick complementarity between the initiation-proximal coding region (downstream box, DB) of several phage and bacterial mRNAs and bases 1469-1483 within helix 44 of 16S rRNA (anti-DB, aDB) was at the origin of the hypothesis that, similarly to the Shine-Dalgarno (SD)-anti-SD interaction, a DB-aDB pairing could enhance the translational efficiency (Sprengart et al., 1990). Soon afterwards, it was suggested that the DB could compensate for the absence of a SD sequence in leaderless mRNAs (Shean and Gottesman, 1992). However, evidence in support of the DB-aDB interaction from biochemistry, mRNA-rRNA co-variation and rRNA mutagenesis is invariably lacking. Chemical probing studies have failed to show protection of the putative DB of the leaderless lcI mRNA bound in 70S initiation complexes (Resch et al., 1996). Moreover, the 3D crystal structure of the Thermus thermophilus 30S subunit (Wimberley et al., 2000) revealed that the whole shoulder of the body of the ribosomal particle is situated between the putative DB of the mRNA and that part of helix 44 of 16S rRNA comprising the proposed aDB . This renders any model in which the start codon is placed in the ribosomal P-site whereas the adjacent DB interacts with the aDB (Sprengart and Porter, 1997) or in which the DB basepairs synergistically with the SD-anti-SD interaction (Sprengart et al., 1996) untenable. Likewise, there is no experimental support for the speculation that a DB-aDB basepairing could contribute to the initial 30S-mRNA interaction before translation initiation complex formation (Sprengart and Porter, 1997). Neither natural lcI mRNA nor a derivative of this leaderless mRNA with an optimized basepairing potential with the aDB formed a binary complex with ribosomes . This result was consistent with previous kinetic toeprint experiments (Resch et al., 1996), as well as with the findings that the aDB is not accessible to a Molecular Microbiology (2002) 43(1), 239

Translation initiation in bacteria involves a stochastic binding mechanism in which the 30S ribosomal subunit ®rst binds either to mRNA or to initiator tRNA, fMet-tRNA f Met . Leaderless l cI mRNA did not form a binary complex with 30S ribosomes, which argues against the view that ribosomal recruitment signals other than a 5¢-terminal start codon are essential for translation initiation of these mRNAs. We show that, in Escherichia coli, translation initiation factor 2 (IF2) selectively stimulates translation of l cI mRNA in vivo and in vitro. These experiments suggest that the start codon of leaderless mRNAs is recognized by a 30S±fMet-tRNA f Met ±IF2 complex, an intermediate equivalent to that obligatorily formed during translation initiation in eukaryotes. We further show that leaderless l cI mRNA is faithfully translated in vitro in both archaebacterial and eukaryotic translation systems. This suggests that translation of leaderless mRNAs re¯ects a fundamental capability of the translational apparatus of all three domains of life and lends support to the hypothesis that the translation initiation pathway is universally conserved.

Summary Leaderless mRNAs beginning with the AUG initiating codon occur in all kingdoms of life. It has been previously reported that translation of the leaderless λcI mRNA is stimulated in an Escherichia coli rpsB mutant deficient in ribosomal protein S2. Here, we have studied this phenomenon at the molecular level by making use of an E. coli rpsBts mutant. The analysis of the ribosomes isolated under the non‐permissive conditions revealed that in addition to ribosomal protein S2, ribosomal protein S1 was absent, demonstrating that S2 is essential for binding of S1 to the 30S ribosomal subunit. In vitro translation assays and the selective translation of a leaderless mRNA in vivo at the non‐permissive temperature corroborate and extend previous in vitro ribosome binding studies in that S1 is indeed dispensable for translation of leaderless mRNAs. The deaD/csdA gene, encoding the ‘DeaD/CsdA’ DEAD‐box helicase, has been isolated as a multicopy suppressor of rpsBts mutations. Here, we show that expression of a plasmid borne deaD/csdA gene restores both S1 and S2 on the ribosome at the non‐permissive temperature in the rpsBts strain, which in turn leads to suppression of the translational defect affecting canonical mRNAs. These data are discussed in terms of a model, wherein DeaD/CsdA is involved in ribosome biogenesis rather than acting directly on mRNA.

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.