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|Title:||Non-canonical RNA capping by DNA-dependent RNA polymerases & screen for bacteriophage regulators of mycobacterium smegmatis transcription|
|Abstract:||In the process of gene expression, all living cells synthesise RNA. RNA is polymerised from nucleotide monomers in dependence of a complementary sequence on a DNA template, a gene. This is done by DNA-dependent RNA Polymerase (RNAP) and is called transcription. Previously, it was believed that only eukaryotic RNA can be 5’-“capped”. The 5’-cap in eukaryotes is attached post-transcriptionally and serves several functions in RNA metabolism and in translation (process of protein synthesis from an RNA template). Recently, a cap was found on bacterial RNA. The bacterial cap is not a modified canonical nucleotide (like the methylated guanosine cap of eukaryotes), but consists of small metabolites or cell wall precursors which are covalently bound to 5’-RNA. This thesis investigates how bacterial RNAP incorporates the non-canonical initiating nucleotide (NCIN)- cap and discusses structural determinants of NCIN-capping of both RNAP enzyme and template DNA. RNAP accepts the cofactor and cell wall precursor as initiating substrate due to the free 3’-hydroyl group of the nucleotide moiety of the molecule. Functional moieties such as nicotinamide ribose or riboflavin on the 5’-end of the nucleotide ribose are bound as substrate by wiltype RNAP catalytic centre and RNA exit channel, and do not interact with upstream promoter regions. However, larger nucleotide-containing molecules such as uridine diphosphate N-acetylmuramic acid pentapeptide are inefficiently incorporated due to sigma subunit of RNAP. We further show that primase, RNA-polymerase of replication, incorporates NCIN into primer RNA and explore potential downstream effects on replication. DNA Polymerase I which replaces primer RNA with DNA, is differentially affected by 5’-cofactors on primer RNA. In the bigger picture, NCIN-caps as extensions of RNA primary structure might have a variety of physiological consequences, which are as of yet uncertain, but extensively discussed in this thesis. We argue that RNA stability, translation, localisation, and activity of regulatory RNA might be affected. RNA synthesis regulation in bacteria makes a fantastic target for drug development, as the cellular machineries of prokaryotes and eukaryotes differ. Also bacteria-infecting viruses, or bacteriophages, are often reported to interfere with host transcription in order to monopolise cellular resources in favour of their own gene expression and replication. We propose that studying the mechanisms by which bacteriophages hijack host gene expression could lead to the discovery or development of new antimicrobials. In order to investigate novel aspects of transcription regulation, we outline a methodology to study how bacteriophages regulate host transcription in mycobacteria. This bacterial family includes today’s most deadly bacterial pathogen, Mycobacterium tuberculosis. At the example of mycobacteriophage D29 gene product Gp53, we show that the protocol can be used to find new inhibitors of bacterial transcription.|
|Appears in Collections:||Institute for Cell and Molecular Biosciences|
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