An AUG initiation codon, not codon–anticodon complementarity, is required for the translation of unleadered mRNA in Escherichia coli

Abstract

SummaryWe determined the in vivo translational efficiency of 'unleadered' lacZ compared with a conventionally leadered lacZ with and without a Shine-Dalgarno (SD) sequence in Escherichia coli and found that changing the SD sequence of leadered lacZ from the consensus 5Ј-AGGA-3Ј to 5Ј-UUUU-3Ј results in a 15-fold reduction in translational efficiency; however, removing the leader altogether results in only a twofold reduction. An increase in translation coincident with the removal of the leader lacking a SD sequence suggests the existence of stronger or novel translational signals within the coding sequence in the absence of the leader. We examined, therefore, a change in the translational signals provided by altering the AUG initiation codon to other naturally occurring initiation codons (GUG, UUG, CUG) in the presence and absence of a leader and find that mRNAs lacking leader sequences are dependent upon an AUG initiation codon, whereas leadered mRNAs are not. This suggests that mRNAs lacking leader sequences are either more dependent on perfect codon-anticodon complementarity or require an AUG initiation codon in a sequence-specific manner to form productive initiation complexes. A mutant initiator tRNA with compensating anticodon mutations restored expression of leadered, but not unleadered, mRNAs with UAG start codons, indicating that codon-anticodon complementarity was insufficient for the translation of mRNA lacking leader sequences. These data suggest that a cognate AUG initiation codon specifically serves as a stronger and different translational signal in the absence of an untranslated leader.

Ribosomes bind leaderless mRNA in Escherichia coli through recognition of their 5′-terminal AUG

“…Although gel shift and toeprint assays have demonstrated the importance of initiator tRNA in ternary complex stabilization, cross-linking experiments revealed that the initial contact and interaction between the ribosome and a leaderless mRNA's 59-AUG occurs through the 30S subunit and is independent of initiator tRNA. Together with earlier reports that codon-anticodon pairing is insufficient for expression (Van Etten and Janssen 1998) and that non-AUG start codons (i.e., GUG, UUG, CUG) do not support efficient ribosome binding and expression (O'Donnell and Janssen 2001), our results provide additional support that a 59 AUG is specifically required for efficient expression of leaderless mRNA in E. coli.…”

“…lanes 3 and 6). Taken together, these results suggest that a 59-terminal AUG is sufficient for an RNA molecule to form a ternary complex with tRNA and ribosomes and are consistent with a previous report demonstrating the importance of a 59-AUG for ribosome binding and translation of leaderless mRNA (Van Etten and Janssen 1998;O'Donnell and Janssen 2001).…”

“…Leaderless mRNAs have been reported to follow a novel pathway of translation initiation involving 70S ribosomes (Balakin et al 1992;O'Donnell and Janssen 2002;Moll et al 2004;Udagawa et al 2004) or 30S subunits free of IF3, which is reported to discriminate against a 59-terminal start codon (Moll et al 1998;Tedin et al 1999;Grill et al 2000Grill et al , 2001. Toeprint assays have demonstrated tRNA-dependent ribosome binding to leaderless mRNA, and an AUG start codon is required for efficient ribosome binding and translation in Escherichia coli (Van Etten and Janssen 1998;O'Donnell and Janssen 2001). The mRNA features or signals present in non-SDled genes that are important for ribosome association and P-site placement of the start codon are not well understood.…”

A 5′-terminal phosphate is required for stable ternary complex formation and translation of leaderless mRNA in Escherichia coli

“…The results presented here and elsewhere (Van Etten and Janssen 1998;O'Donnell and Janssen 2001;Brock et al 2008;Krishnan et al 2010) suggest that the combination of a 59-phosphate and 59-terminal AUG triplet represent a novel translation signal that uniquely identifies the 59 end of a leaderless mRNA for ribosome binding and translation initiation. Leaderless mRNAs provide a model system for studies of novel mRNA-ribosome interactions that may be different from those that occur with SDL mRNAs.…”

“…Also, the presence of a 59AUG, not codon-anticodon pairing alone, is required for the efficient translation of LL mRNAs in E. coli (Van Etten and Janssen 1998).…”