Bacterial translation of mRNA into protein requires a specific association of mRNA with the ribosome before initiation of protein synthesis can occur. While many bacterial mRNAs bind ribosomes through a complementary mRNA:rRNA interaction, a growing body of evidence suggests that many mRNAs lack this interaction but still bind ribosomes and are translated. Working with leaderless mRNA that lack any untranslated leader sequences upstream to the start codon, we have identified contacts between the start codon and specific ribosomal proteins of the 30S ribosomal subunit. Contacts to the leaderless mRNA's start codon have been localized to proteins found at the upstream and downstream ends of the mRNA binding channel of the 30S subunit. Our proposed experiments utilize a crosslinking approach, together with high resolution mass spectroscopy, to identify the specific sites within the r-proteins that contact the leaderless mRNA's start codon. The specific sites of contact will be identified within 30S subunits and 70S ribosomes. Reconstitution experiments will be used to reassemble 30S subunits from purified rRNA and purified r-proteins. The subunits will be assembled with wild type r-proteins, or proteins containing mutations in amino acids identified by mass spectroscopy as being in close contact with the start codon, or with complete absence of specific proteins implicated to contact the leaderless mRNA's start codon. A series of ribosome antibiotics will be used, as well as specific RNA competitors, to evaluate the observed crosslinking pattern and whether the crosslinks occur in random order or proceed through an ordered step-wise sequence of interactions. The results of this work will identify and define ribosome components (rRNA and r-proteins) that recognize and bind the AUG start codon at the 5'-terminus of a leaderless mRNA.
Translation initiation requires a specific recognition between mRNA and ribosomes for placement of the start codon into the decoding site. Translation of many bacterial mRNAs involves a specific interaction between a sequence on the mRNA and a complementary sequence in the ribosomal RNA. However, a number of mRNAs lack this complementary interaction but are still translated. Understanding the mechanism by which ribosomes recognize and bind these mRNAs provides potential targets for development of novel inhibitors and antibiotics. ? ? ?
