Do PhoH2 proteins regulate SigF in mycobacteria?

Rzoska-Smith, Elizabeth

Do PhoH2 proteins regulate SigF in mycobacteria?

dc.contributor.advisor	Arcus, Vickery L.
dc.contributor.advisor	Andrews, Emma Sophie Vout
dc.contributor.author	Rzoska-Smith, Elizabeth
dc.date.accessioned	2019-01-15T02:01:55Z
dc.date.available	2019-01-15T02:01:55Z
dc.date.issued	2018
dc.date.updated	2018-12-17T22:45:36Z
dc.description.abstract	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.
dc.format.mimetype	application/pdf
dc.identifier.citation	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	en
dc.identifier.uri	https://hdl.handle.net/10289/12265
dc.language.iso	en
dc.publisher	The University of Waikato
dc.rights	All items in Research Commons are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.
dc.title	Do PhoH2 proteins regulate SigF in mycobacteria?
dc.type	Thesis
pubs.place-of-publication	Hamilton, New Zealand	en_NZ
thesis.degree.grantor	The University of Waikato
thesis.degree.level	Masters
thesis.degree.name	Master of Science (Research) (MSc(Research))