The ssrA gene encoded tmRNA acts as both a tRNA carrying an Ala to enter the A site of stalled ribosomes and as an mRNA allowing trans-translation to continue until ribosomes reach the stop codon of the tmRNA tag to help release the stalled ribosome, label the truncated peptide for degradation, and also facilitate degradation of the ribosome-stalling mRNAs. Functions of tmRNA rely on its binding to an essential protein factor SmpB that is encoded by the smpB gene. The mycelial bacteria streptomycetes have a well-defined growth and developmental cycle culminating at sporulation and provide a good model to study tmRNA function in bacteria growth and development. During different developmental stages, expression of some critical molecules are increased or decreased to control the developing procedures including a bldA-encoded tRNA that decodes the rare codon UUA. Translation elongation of genes containing UUA rare codons may be stalled and elicits tmRNA tagging, suggesting that tmRNA the tagging system may be important for Streptomyces growth and development. We use the most well studied strain, S. coelicolor whose genome sequence was the first sequenced, as our model organism. Here I report my ssrA knockout study with two different strategies. Using a temperature sensitive replicon, I found that the ssrA gene could be disrupted only in cells with an extra ssrA gene but not in wild type cells or cells with an extra-copy of tmRNA variant--tmRNADD that encodes a degradation-resistant tag. These results imply that ssrA is an essential gene and that degradation of truncated proteins is also an essential function for S. coelicolor. On the contrary, with the second method that does not need high temperature screening steps I was able to disrupt both the ssrA and smpB genes separately and at the same time, suggesting that the tmRNA tagging system may be required for cell survival under high temperature. Further characterization of mutant cells revealed that the tmRNA tagging system is important for cell growth and development at both high temperature and optimal growth conditions as well as under stress conditions that affect the translation elongation process. The second part of my thesis documents analyses of the expression, regulation and stability of S. coelicolor tmRNA. My results suggested that the well known metabolic stability of bacterial tmRNA might be due to its tight binding to the ribosome. Finally, I report my investigation of the tagging activity and the importance of some structural elements of S. coelicolor tmRNA. Particularly, I demonstrated that pseudoknot 4 is important for tmRNA tagging activity and mutations to some structural elements lead to a decrease of not only the mutant tmRNA but also the wild type tmRNA when expressed together in vivo.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/19113 |
| Date | 23 February 2010 |
| Creators | Yang, Chunzhong |
| Contributors | Glover, John R. |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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