Mutagenesis of Escherichia coli for sm-FRET and FIRMS study of domain IV EF-G studies.

Abstract

The translation of mRNA into protein is performed by a ribonucleoprotein complex which consists of two subunits which come together to form three tRNA binding sites; the A-, P-, and E-site. The complex is associated with several proteins, one of which is Elongation Factor G (EF-G) which advances the mRNA sequence through the ribosome in a process known as translocation. During translocation tRNAs move from the A- and P-sites to the P- and E-sites, respectively, and the ribosome slides the mRNA exactly three nucleotides. Two key residues (Q508 and H584) in the domain IV region of EF-G form interactions with nucleotides of the A-site tRNA during translocation. The primary goal of this project will be to reveal the function of EF-G mediated translocation via mutagenesis of these two residues. Single molecule fluorescent resonance energy transfer (sm-FRET) will be used to determine the confirmational change of mutant EF-G. Force production of these mutants will also be measured using force-induced remnant magnetization spectroscopy (FIRMS) to determine the overall effect the domain IV mutants have on power-stroke force. We expect that the EF-G mutants will only be able to complete partial translocation and will have a reduction in force exertion, while GTP hydrolysis will not compromised, which will imply an active role in the translocation mechanism by these residues. These findings together will visualize the ribosome translocation mechanism with sub-nucleotide resolution, which only the FIRMS method can do to our best knowledge.