Protein synthesis occurs in ribosomes, megadalton RNA-protein machines that use aminoacyl-tRNA (aa-tRNA) molecules to translate messenger RNA (mRNA) with high fidelity. During translation elongation, the ribosome orchestrates 3 major events: decoding, peptidyl transfer and translocation. The process of proteins synthesis is also one of the major targets of antibiotics and hence understanding the basics of ribosome function should provide insight for the development of novel drugs.
Genomes are maintained and expressed with remarkable fidelity and the accuracy of each process involved represents a compromise that optimizes the evolutionary fitness of the organism. The process of translation elongation is a complex one, and therefore there are potentially many ways the process can go awry. Chapter 1 introduces translation elongation errors and discusses the differences between missense, nonsense and frameshift errors. Mutations in the ribosome and other translation factors that affect the fidelity of translation elongation are also discussed.
Chapter 2 is focused on the ribosomal exit (E) site and its role in maintaining the translational reading frame. It has been proposed that a critical role for the E site is in maintenance of translational reading frame, dependent on codon-anticodon pairing (191). Though several studies support the idea that codon-anticodon interaction in the E site contributes to frame maintenance (167), direct in vivo evidence for this hypothesis has been scant. In chapter 2, we investigated this fundamental question and found that the E site helps to maintain the reading frame, but does not contribute to the accuracy of decoding, as has been suggested (chapter 2, 204). We also showed that the mutation of the 30S E site does not inhibit EF-G-catalyzed translocation, in sharp contrast to the effects of mutations in 50S E site. These data provided evidence that the function of the E site in translocation is largely confined to the 50S subunit.
One of the earliest identified examples of translational frameshifting occurs in the prfB gene of E. coli, encoding the peptide release factor 2 (RF2). While the genetic studies have identified the determinants of prfB programmed frameshifting and their relative importance, how these determinants act to promote frameshifting has remained unclear. In chapter 3, we compared ribosomal complexes with various spacer lengths between the SD sequence and P codon. We found that a close juxtaposition of the SD–ASD helix and P codon strongly destabilized P-site tRNA but had little or no effect on RF2-dependent termination or EF-Tu-dependent decoding. These data suggested that the intragenic SD of prfB destabilizes pairing of peptidyl-tRNALeu to the zero-frame CUU and promotes directional movement of the mRNA template with respect to the bound tRNA.
In chapter 4, we have isolated 16S rRNA mutations that could suppress a +1 frameshift mutation in E. coli. In one of the screens (where the slippery sequence in the frameshift window had a stop codon), 31 independent mutations were identified and mapped to four different positions, of which C1054U was isolated 28 times. The C1054U mutation has also been isolated previously as a nonsense suppressor. Purine substitutions at this position also increased UGA readthrough and miscoding. While the C1054U mutation significantly increased nonsense readthrough and frameshift errors, the mutation had a hyperaccurate phenotype with respect to decoding (i.e., reduced misreading). Other substitutions at this position also had differential effects on the three reporters (missense, nonsense and frameshift).
These interesting observations prompted us to characterize these A-site mutations as well as others in 16S rRNA (C1200U, G1491A and G299A) in vitro to get a better understanding of how the ribosome maintains its high fidelity (chapter 5). We investigated the effect of these mutations on RF2 function and found that all of the mutations tested had a defect in RF2-dependent termination. We directly tested the effect of these mutations on decoding by measuring the rate of GTP hydrolysis in both cognate and near-cognate mRNA. We found that all of the mutations tested (C1200U, G1491A, C1054U, C1054A, and G299A) had a substantial defect in initial selection, increasing the rate of GTP hydrolysis particularly on near-cognate mRNA. We also investigated the effect of these mutations on the stability of various tRNAs in the A site. Of the mutations analyzed, C1054U and G1491A seemed to differentially affect tRNA stability, suggesting that these mutations may stimulate GTP hydrolysis in a different way than the others.