Initiation is the rate-limiting step of translation and is an important regulatory step in gene expression. Translation initiation requires ribosome recognition of the mRNA’s start codon followed by formation of a stable ternary complex involving initiator tRNA. Studies have shown specific contacts between a AUG start codon and several ribosomal proteins (r-proteins). We employed short-leadered mRNAs in order to further characterize the path of an mRNA’s 5’-AUG from initial association with the ribosome to its final placement at the ribosome’s decoding site (P-site). Our results suggest that bacterial ribosomes can bind and inspect the 5’-terminus of all mRNAs for an AUG triplet, with close proximity of AUG to the 5’-terminus positively influencing ribosome binding and ternary initiation complex formation. Crosslinking studies showed that while 5’-AUG interactions with r-proteins S1, S3, S4, S7, S10 and S18 can occur in the absence of tRNA-fMet, movement of a 5’-AUG into the Psite...
