Talk abstracts

Talk on Friday 07:30-07:50pm submitted by Sarah Walker

Role of the P/E state in ribosomal translocation

Sarah E. Walker (Department of Microbiology, The Ohio State University), Kurt Fredrick (Department of Microbiology and Ohio State Biochemistry Program, The Ohio State University)

Abstract:
During translation, tRNAs must move rapidly to their adjacent sites in the ribosome while maintaining precise pairing with the mRNA. A growing body of evidence suggests that this movement (translocation) occurs in a stepwise manner with hybrid-state intermediates, but it is unclear how these intermediate binding states relate to the kinetically defined events of translocation. Here we employ ribosomes harboring mutations of E-site rRNA nucleotide C2394 in a pre-steady-state kinetic analysis of translocation. These mutations are predicted to inhibit P/E state formation based on structural studies. Each substitution at position 2394 decreases growth rate, the maximal rate of translocation (ktrans), and the apparent affinity of EF-G for the pretranslocation complex (K1/2). The magnitude of these defects follows the trend (A > G > U > WT). The most deleterious mutation, C2394A, does not affect the rate of GTP hydrolysis. Thus, the substantial (>20-fold) increase in K1/2 cannot be attributed to either the initial binding of EF-G or the subsequent GTP hydrolysis step. We propose that P/E state formation occurs subsequent to GTP hydrolysis and contributes to K1/2 because each of the first three steps of translocation is readily reversible. Substitution of the peptidyl group for an aminoacyl group, which is predicted to inhibit A/P state formation, has a different effect on translocation. The aminoacyl group decreases ktrans but has no effect on K1/2. These data suggest that movement of tRNA into the P/E and A/P states are separable events and the latter is effectively irreversible in the presence of EF-G.

Keywords: ribosome, translocation, tRNA