An AUG start codon is an important determinant of ribosome binding and expression of leaderless mRNAs in Escherichia coli. Using reporter constructs encoding mRNAs where the AUG start codon is preceded by untranslated leaders of various length and sequence, we find that close proximity of the start codon to the 5 terminus and the leader sequence are strong determinants of both ribosome binding and expression.Ribosome recognition and binding to mRNA is a rate-limiting step of translation and a major determinant of gene expression. Although a Shine-Dalgarno (SD) sequence (25) is known to speed the formation of initiation complexes and result in higher expression, prokaryotes also contain noncanonical mRNAs, many of which lack a SD-type sequence but are highly expressed (6,9,24,26). Leaderless mRNAs carrying a start codon at their 5Ј terminus are a group of noncanonical mRNAs found in all domains of life (5,8,15,23). Much of our understanding about ribosome binding to a 5Ј-terminal AUG comes from studies of Escherichia coli with leaderless cI mRNA (1,3,(17)(18)(19)29), whereby in vitro ribosome binding and translation assays have successfully demonstrated the 70S ribosome's ability to bind and initiate translation of leaderless mRNA (16,17,28). A mutant initiator tRNA with compensating anticodon mutations restored expression of leadered, but not leaderless, mRNAs with UAG start codons, indicating that codon-anticodon complementarity was insufficient for the translation of leaderless mRNA (29). 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. In this report, we examine the influence of AUG start codon proximity to the 5Ј terminus and the influence of upstream nucleotides, if present, on 70S ribosome binding and on expression in E. coli.In order to determine if efficient ribosome recognition of leaderless mRNA requires the AUG to be at the 5Ј terminus, we prepared cI-lacZ fusions in which the distance from the transcriptional start site to the start codon was increased by the addition of "TC" multimers. Transcription in vivo results in mRNA containing a "UC" repeat sequence upstream (5Ј) to the start codon and can be used to assess the effects of the short leaders and recessed AUGs on expression. Transcription in vitro provides mRNAs with short leaders and recessed AUGs for use in ribosome binding studies; transcription with T7 RNA polymerase results in the addition of a G as the initiating nucleotide to all in vitro-synthesized mRNAs. The choice of "UC" for the untranslated region was made to avoid resemblance to a typical purine-rich Shine-Dalgarno-type sequence. ␤-Galactosidase assays (14) were performed using E. coli RFS859 (21) as a host. Ribosome purification (E. coli MRE600) (30), primer extension inhibition (toeprint) assays, and in vitro synthesis and purification of mRNAs were performed as described previously (2, 13); toeprint reaction products were separated on denaturing polyacrylamide gel...