Do PhoH2 proteins regulate SigF in mycobacteria?
Rzoska-Smith, E. (2018). Do PhoH2 proteins regulate SigF in mycobacteria? (Thesis, Master of Science (Research) (MSc(Research))). The University of Waikato, Hamilton, New Zealand. Retrieved from https://hdl.handle.net/10289/12265
Permanent Research Commons link: https://hdl.handle.net/10289/12265
There is little known about the role of the PhoH2 proteins in biology. PhoH2 consists of two domains, an N-terminal PIN domain with RNase activity and a C-terminal PhoH domain shown to have ATP induced RNA helicase activity. PhoH2 proteins from Mycobacterium tuberculosis and Mycobacterium smegmatis show ATP and magnesium dependent, sequence-specific RNA unwinding and cleavage activity. It is hypothesised based on RNAseq data that PhoH2 from M. smegmatis may be acting as a potential regulator of sigma factor SigF, through its targeted RNA helicase and RNase activity. Regulation may occur directly on the sigF mRNA transcript, or indirectly through regulation of anti-sigma factor antagonists. This thesis used purified PhoH2 proteins to examine the activity of PhoH2 against mRNA substrates designed to span regions of the sigF gene and the genes co-expressed in the sigF operon; rsbW and chaB. Results show that PhoH2 co-purifies with E. coli nucleic acid and that despite this co-purification, activity was observed on sigF mRNA, that was improved with increasing amounts of ATP. qPCR experiments were designed to test expression levels of 12 genes, that showed significant up or down regulation expression in M. smegmatis mc²155 ΔphoH2 compared to wild-type M. smegmatis mc²155. qPCR analysis revealed a significant upregulation of the genes MSMEG_1804 (sigF) and MSMEG_0064 (PPE) in M. smegmatis mc²155 ΔphoH2 compared to M. smegmatis mc²155. These results differ from the gene expression results obtained from RNAseq data, yet results still point towards regulation of SigF by PhoH2. Further experiments using qPCR and activity assays with PhoH2 aim to verify the precise role of PhoH2 in the regulation of SigF.
The University of Waikato
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