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Title: Investigations into an archaeal RNA polymerase : structure to function analysis
Author: Mogni, Maria Elena
ISNI:       0000 0004 2745 7211
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2012
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The archaeal RNA polymerase (RNAP) is similar to the eukaryotic RNAP-II in terms of subunit composition and overall protein structure. Despite its similarity, a new archaeal-specific Rpo13 subunit has been identified. Rpo13 occupies a position in the enzyme which, in RNAP-II, is filled by the eukaryotic-specific Rpb5 jaw domain. It has therefore been proposed to contact DNA, where the positively charged C-terminal tail might mediate protein-DNA interactions. Furthermore, analysis of archaeal genomes has identified a homologue of the eukaryotic RNAP-III-specific RPC34 subunit. RPC34 may associate with the single archaeal RNAP, modulating the specificity of the archaeal RNAP and re-directing it to a subset of genes such as non-coding genes, in analogy to the RNAP-III/RPC34 eukaryotic system involved in the transcription of 5S rRNA, tRNAs and other small RNAs. More importantly, the association of RPC34 with the single archaeal RNAP would define an archaeal enzyme which acts as a precursor of the eukaryotic RNAP-III. Electrophoretic mobility shift assay (EMSA) analysis of purified Rpo13 protein by recombinant means subsequently incubated with a double-stranded (ds)DNA sequence reveals the formation of protein-DNA complexes, where Rpo13’s binding to DNA is non-sequence specific but discriminatory to dsDNA, as no binding is observed in the presence of single-stranded (ss)DNA. Also, it is found that the ma jor determinant of DNA binding is the Rpo13’s positively charged C-terminal tail, since DNA binding is abolished with a Rpo13 mutant deficient in this tail. Furthermore, neither ma jor groove nor minor groove interacting compounds have a major impact on Rpo13’s binding to DNA, suggesting that Rpo13 may associate with the negatively charged DNA phosphate backbone. Moreover, in vitro transcription assays indicate that a transcription product is observed upon RNAP incubation with a bubble DNA oligo shown to make Rpo13 contacts in the RNAP-DNA crystal structure. In addition, while a GST-pulldown experiment suggests the existence of an interaction between the archaeal RNAP and RCP34 in vitro, co-immunoprecipitation assays argue against the existence of such interaction from an in vivo point of view. Finally, a chromatin immunoprecipitation (ChIP)-sequencing approach to analyse Rpo13’s genomic distribution versus the one of the bulk RNAP was undertaken. While the gel filtration elution profile analysis of Rpo13 in the S. acidocaldarius cell extract versus the one of recombinant Rpo13 suggests that there is a free Rpo13 pool in the cell extract, indicating that Rpo13 may be acting as a transiently-associated RNAP subunit displaying a factor-like function, the ChIP-sequencing approach reveals that Rpo13 is a bona fide RNAP subunit since it co-localises from a genomic point of view with the bulk RNAP.
Supervisor: Bell, Stephen Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Biochemistry ; archaea ; RNA polymerase ; Rpo13