The biological role and mechanism of action of rbpA and carD in Streptomyces coelicolor A3 (2) and Mycobacterium tuberculosis

Jagatia, Heena (2018) The biological role and mechanism of action of rbpA and carD in Streptomyces coelicolor A3 (2) and Mycobacterium tuberculosis. Doctoral thesis (PhD), University of Sussex.

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Abstract

RbpA is unique to and conserved across the Actinobacteria and binds to the group I sigma factors to form an RNA polymerase holoenzyme complex resulting in stabilisation of the transcription initiation bubble. RbpA is composed of an unstructured N-terminus, RbpA core domain (RCD), Basic linker (BL) and Sigma Interaction Domain (SID). The function of the RbpA-SID is the only well characterised domain. Therefore, this study focuses on elucidating the function of the RbpA BL. A recently published full length RbpA crystal structure has shown that the RbpA BL is positioned to interact with the extended -10 promoter DNA. Alanine substitutions of the RbpA BL did show a small colony phenotype with actinorhodin production in S. coelicolor. The equivalent RbpA BL residue substitutions with alanine in M. tuberculosis were constructed and tested in vivo. The RbpA BL mutant (3KRA) failed to culture on solid media, but no growth inhibitory phenotype was present in liquid media. Additionally, RNA-seq of the BL mutant in both organisms revealed major transcriptional changes. Together these studies, suggest that the RbpA BL does not contribute to the essential function of RbpA and therefore RbpA may fulfil other functions. To explore these alternative functions, we constructed mutations in HrdB, a principle sigma factor in S. coelicolor, which prevented RbpA binding to RNAP/σHrdB holoenzyme. Chromatin immunoprecipitation techniques followed by high-throughput ChIP sequencing of the HrdB mutant revealed that HrdB was capable of co-localising at all RbpA dependant promoters in the absence of RbpA binding.

CarD is another essential transcription factor in Mycobacterium spp which binds to RNAP/σA holoenzyme. Several structural models have suggested that RbpA and CarD bind to the extended -10 promoter DNA from opposite sides of the double helix DNA. Therefore, to explore a potential relationship between RbpA and CarD, we constructed a degradation tag system to enable co-depletion of RbpA and CarD. This provides a genetic tool for studying essential genes without the use of antibiotic dependant inducible systems.

Item Type: Thesis (Doctoral)
Schools and Departments: School of Life Sciences > Biochemistry
Subjects: Q Science > QR Microbiology > QR0075 Bacteria
Depositing User: Library Cataloguing
Date Deposited: 30 Nov 2018 15:44
Last Modified: 04 Jan 2021 07:09
URI: http://sro.sussex.ac.uk/id/eprint/80557

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