Poster abstracts

Poster number 44 submitted by Daoming Qin

An in vitro mRNA competition assay to study translation initiation

Daoming Qin (Ohio State Biochemistry Program), Kurt Fredrick (Department of Microbiology, Ohio State Biochemistry Program)

Abstract:
Initiation is usually the rate-limiting step of protein synthesis and the step targeted for regulation. In bacterial, several cis-acting elements located in the translation initiation region (TIR) of the mRNA determine the efficiency of initiation. These include the Shine-Dalgarno sequence, the start codon, and the local secondary structure. Here, in order to study the contribution of different TIRs to initiation under various conditions, we set up an in vitro mRNA competition assay. In this assay, two different mRNAs (both in excess) were mixed with various components of the translational machinery (e. g. ribosomal subunits, initiation factors, initiator tRNA) and the relative efficiency of initiation complex formation was determined by toeprinting and dipeptide formation. In experiments where mRNA containing the canonical start codon AUG was competed with mRNA containing AUC, initiation factor IF3 increased mRNA selectivity from a factor of ~2 to ~100. Substantial effects of initiation factor IF1 on mRNA selectivity have also been observed and are being further characterized. This methodology should be generally useful for studying gene regulation at the level of translation initiation.

Keywords: translation initiation, ribosome, start codon