Mutagenesis of Escherichia coli Elongation Factor G for Domain IV sm-FRET and Force Studies

The translation of mRNA into protein is performed by the ribosome. This ribonucleoprotein complex 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. Previous mutation attempts using Q5 hot-start polymerase for two substitutions at this site (Q508A and H584A) resulted in failure. Low plasmid yield, undesired off-site mutations, and high polymerase chain reaction (PCR) failure rates were some of the primary issues faced when using this enzyme. In this experiment, a new polymerase enzyme was tested to determine if the desired mutations can be successfully amplified via PCR. Gel electrophoresis was used to confirm the presence of recombinant plasmid following PCR. Plasmid was isolated via midi-prep, and sequencing for the H584A mutant was performed using Sanger sequencing. These findings suggest that the H584A substitution mutation was not successfully inserted into Escherichia coli pDNA due to a deletion of G1742, causing a -1 frameshift. This project will be re-attempted using PAGE purified primers and a new PCR kit with fresh polymerase.