Hill, Stuart A.
Ph.D. (Doctor of Philosophy)
Department of Biological Sciences
Neisseria gonorrhoeae--Molecular aspects
Initially, regulation of pilE expression was simplified into a model where integration host factor (IHF) activates a single -35/-10 promoter upon binding to an upstream region of the promoter. In this study, we show that pilE is potentially modulated at a transcriptional as well as a post-transcriptional level by transcription of cis-acting antisense RNAs within the pilE coding sequence and through formation of RNA secondary structures within its 5' untranslated region (5' UTR). Using a combination of in vitro transcription, site-directed mutagenesis, Northern blot, and quantitative real-time PCR (qRT-PCR) analysis, we have identified three unconventional promoter elements, in addition to the cognate promoter, within the pilE gene; two are located within the midgene region (one sense and one antisense), with the third, an antisense promoter, located immediately downstream of the pilE open reading frame. Through a strand-specific qRT-PCR analysis, an inverse correlation exists between the level of antisense expression and the amount of sense message. Moreover, transcription of the antisense RNAs appeared to be controlled by expression of H-NS, a DNA-binding protein that preferably binds to A/T-rich regions and inhibits intragenic transcription. Deleting the hns gene in N. gonorrhoeae resulted in a twofold increase of pilE antisense transcription, with a concomitant decrease in sense transcript levels. However, the most noticeable effect of H-NS observed in the hns mutant is suppressing pilE/pilS recombination. Mutational analysis, in conjunction with qRT-PCRs, also revealed that transcription of pilE is enhanced by the transcription cleavage factor GreB, but only in the absence of the GreA protein. More pilE transcrips were evident in a greA- greB+ gonococcal mutant compared to the wild type cells, whereas inactivating greB had no significant effect on the pilE mRNA level. In addition to antisense-mediated regulation, we have also demonstrated formation of three stem loops within the pilE 5' UTR in vivo and that, the presence of all these three RNA structures is required for mRNA protection from degradative activities. mRNA turnover studies also showed that pilE transcript is modulated by the enzymes RppH and RNase E, and that RNase III does not engage in RNA processing of the pilE mRNA. Consequently, this study reveals a multi-layered complexity in pilE expression that is controlled through both transcriptional and post-transcriptional regulations.
Masters, Thao Le, "Characterizing molecular mechanisms modulating pilE expression in Neisseria gonorrhoeae" (2016). Graduate Research Theses & Dissertations. 1764.
x, 165 pages
Northern Illinois University
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