Poster abstracts
Poster number 134 submitted by Tyler Smith
Mechanistic framework for understanding ribosome sliding on homopolymeric A stretches
Tyler J. Smith (Department of Chemistry - University of Michigan ), Kristin S. Koutmou (Department of Chemistry - University of Michigan )
Abstract:
Translation speed and fidelity can be modulated by a variety of factors including mRNA structure, codon usage, and peptide identity. The synthesis of positively charged peptides is thought to cause the ribosome to stall because of interactions between the cationic nascent peptide chain and the negatively charged ribosome exit tunnel. However, emerging evidence suggests that strong charge-charge interactions between the ribosome and nascent chain may be insufficient to explain reduced protein output during the translation of positively charged proteins. Recent work demonstrated that the ribosome translates mRNAs containing iterative lysine AAA and AAG codons differently, losing frame and undergoing non-canonical ribosome movement (sliding) on homopolymeric AAA, but not AAG, sequences. Here, we investigate the contributions of peptide charge, mRNA sequence, mRNA modification, and EF-G to ribosome sliding events using a reconstituted in vitro E. coli translation system. We demonstrate that the ribosome slides into the -2, -1 and +1 frames on poly(A) sequences, and are working to characterize the kinetics of each of these shifts. Our data indicate that ribosome sliding depends on EF-G, much as in -1 programmed frameshifting events. Furthermore, we find that codon modification status contributes to the degree of sliding; the incorporation of a single m6A into AAA codons modulates sliding. Moreover, neither ribosome stalling and sliding on homopolymeric (A) sequences appear to strictly require the translation of a positively charged peptide sequence – sliding and slow translation still occur when tRNALys is mischarged with Val (Val-tRNALys). Our data provide a mechanistic framework for understanding how protein synthesis is modulated by mRNA sequences encoding positively charged amino acids.
Keywords: Translation, Frame-loss, Sliding