UC Santa Cruz

The nature of the genetic code, where mRNA is decoded as a series of nonoverlapping triplets, is inherently prone to frameshifting errors that occur when the mRNA register is shifted into one of two improper reading frames. While a typical protein is several hundred amino acids in length, a single frameshifting error can produce a product that is not only non-functional, but that is also toxic to the cell. Frameshifting errors can occur through multiple potential routes, either by tRNA binding out-of-frame, mis-translocation, or by spontaneous slippage of the ribosome along the mRNA. The mechanisms by which cells achieve such remarkable accuracy throughout the dynamic process of protein synthesis are still not well understood. The work within this thesis aims to elucidate the mechanisms of translational reading frame maintenance and the pathways that result in frameshifting in the context of programmed frameshifting sequences. We measure frameshifting efficiency on these sequences using an in vitro protein synthesis assay reconstituted with mutant components. We first show that domain IV of elongation factor EF-G, a translational GTPase that catalyzes ribosomal translocation, protects against -1 frameshifting. We suggest domain IV of EF-G stabilizes the codon-anticodon duplex on its transit from the A site to the P site and restricts the duplex from moving too far. We then demonstrate that two of the few residues in 16S rRNA that contact the mRNA, A1503 and G926, help to prevent frameshifting, likely by restricting unregulated movement of the mRNA. We also compare two different types of programmed frameshifting motifs: ‘simultaneous slippage’ using the dnaX gene and ‘hungry frameshifting’ using the prfB gene. We demonstrate that the former stimulates frameshifting during translocation, while the latter proceeds via a different pathway. These results are consistent with the popular model for the prfB +1 frameshifting mechanism, which presumes that the frameshifting occurs on ribosomes with a vacant A site.