Understanding translation efficiency is crucial to pharmaceutical companies that have invested substantial time and effort in engineering bacteria to produce recombinant proteins. While translation initiation and elongation have been studied intensively, much remains obscure in the subprocess of translation termination. We aim to understand how stop codons and the first 3’ flanking (+4) base affect translation termination efficiency.
In chapter two, we hypothesized that stop codon usage of UAG and UGA is dependent on the abundance of their respective decoders, RF1 and RF2. We predicted and observed that bacterial species with high relative proportions of RF1 uses UAG more, and vice versa for UGA. In addition, the usage of UGA, not UAG, is always avoided in highly expressed genes. Thus, we argued against the claim made by a recent study that UAG is a minor stop codon in bacteria. The claim is incorrect because UAG does not meet the two criteria of a minor codon: i) it is most avoided in highly expressed genes, and ii) it corresponds to the least abundant decoder. Interestingly, we found that the proportion of RF2 decreases rapidly towards zero in species with high AT contents; this explains why UGA is reassigned to a sense codon in bacterial lineages with high AT content.
In chapter three, we examined the role of the first downstream (+4) base Uracil in bacterial translation termination. The +4U is associated with a decrease in stop codon read-through in bacteria and yeast. We hypothesized that i) +4U enhances the termination efficiency of stop signals, and ii) +4U may serve to prevent stop codon misreading by near cognate tRNAs (nc_tRNAs). We predicted that i) +4U is preferred in highly expressed genes (HEGs) than lowly expressed genes (LEGs), and ii) +4U usage increases with the frequency of stop codon nc_tRNAs. We found +4U consistently over-represented in HEGs in contrast to LEGs; however, +4U usage in HEGs decreases in GC-rich species where most stop codons are UGA and UAG. In addition, +4U usage increases significantly with UAA usage in the known highly expressed ribosomal protein genes. These results suggest that +4U is a strong stop signal enhancer for UAA, not UAG or UGA. Furthermore, in HEGs, +4U usage also increases significantly with the abundance of UAA nc_tRNAs, suggesting that +4U increases UAA termination efficiency presumably by reducing misreading of UAA by nc_tRNAs.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/35529 |
| Date | January 2016 |
| Creators | Wei, Yulong |
| Contributors | Xia, Xuhua |
| Publisher | Université d'Ottawa / University of Ottawa |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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