Interactions between mRNA and Escherichia coli ribosomes that contribute to the formation of translation initiation complexes

by Brock, Jay Edward.

Interactions Between mRNA and Escherichia coli Ribosomes that Contribute to the Formation of Translation Initiation Complexes By Jay Edward Brock Initiation is the rate-limiting step of translation involving the assembly of stable ternary complexes (mRNA-tRNA-ribosomes) on the translation initiation region (TIR) of the mRNA. Evidence for many non-Shine-Dalgarno (SD)-led genes, including genes encoding leaderless mRNA, provides motive for studying mRNA features within the coding sequence that contribute to the formation of these complexes. Binding and expression assays reveal here that adenines downstream from a mRNA’s start codon contribute significantly to the rate and amount of ternary complex formed as well as the level of in vivo expression, even in the presence of a canonical SD sequence. Leaderless mRNA, which lack signals upstream of the start codon, requires a 5’-terminal triphosphate for efficient translation. A mutagenesis approach was developed to search for ribosome sites involved in recognizing a leaderless mRNA’s 5’-terminal triphosphate. Binding assays demonstrated tRNA-dependent ribosome binding to leaderless mRNA; however, site-specific crosslinks between a leaderless mRNA’s start codon, containing a 4-thiouridine at +2 position, and components of the ribosome formed independent of initiator tRNA. Several leaderless mRNA-r-protein contacts were dependent on the presence of a 5’-terminal AUG, suggesting that specific r-proteins might facilitate recognition of a leaderless mRNA’s start codon. In addition, initiation factors did not influence the initial interactions between leaderless mRNA and ribosomes identified in site-specific crosslinking assays. This work provides the first evidence that recognition and ribosomal positioning of a leaderless mRNA’s 5’-AUG is a function of the ribosome. Interactions Between mRNA and Escherichia coli Ribosomes that Contribute to the Formation of Translation Initiation Complexes A Dissertation Submitted to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Microbiology by Jay Edward Brock Miami University Oxford, Ohio 2006 Dissertation Director: Gary R. Janssen, Ph.D. © Jay Edward Brock 2006