Regulation of 6S RNA biogenesis and ygfA transcription in ssrS transcription unit

Abstract

The ssrS transcription unit interestingly can produce an RNA product, 6S RNA, as well as a protein product, YgfA. The ssrS-encoded 6S RNA is an abundant noncoding RNA that binds to $\sigma ^{70}$ -RNA polymerase and regulates expression at a subset of promoters in Escherichia coli. It is transcribed from two tandem promoters, ssrS P1 ($\sigma ^{70}$) and ssrS P2 ($\sigma ^{70/S}$). In the first part of the thesis, regulation of transcription from two ssrS promoters for 6S RNA biogenesis was examined. Both P1 and P2 were growth phase-dependently regulated. Depletion of 6S RNA had no effect on growth-phase-dependent transcription from either promoter, whereas overexpression of 6S RNA increased P1 transcription and decreased P2 transcription, suggesting that transcription from P1 and P2 is subject to feedback activation and feedback inhibition, respectively. This feedback regulation disappeared in Δfis strains, supporting involvement of Fis in this process. The differential feedback regulation may provide a means for maintaining appropriate cellular concentrations of 6S RNA. YgfA, also called as Fau, is an uncharacterized protein that belongs to the 5-formyltetrahydrofolate cyclo-ligase family. It is known that the ygfA expression may be related to persister cell formation, which contributes to the antibiotic resistance of bacterial biofilm. The ygfA mRNA co-transcribed with 6S RNA from two ssrS promoters can be prematurely terminated by action of Rho factor at near site +279 (named $t_1$ in this thesis), relative to the 5’ end of 6S RNA. This transcription termination site, named $t_1$ in this thesis, is located within the ygfA open reading frame (ORF). Therefore, ygfA expression can be regulated by two ssrS promoters as well as Rho-dependent termination that occurs at $t_1$. In the second part of thesis, a pBR322-based E. coli genomic library was screened to identify factors that could affect the $t_1$ termination. Rof as a protein repressing the t1 termination was identified in this screening. Rof is known to modulate the activity of Rho factor by directly interacting with it. The increase of ygfA mRNA levels was found when Rof was overexpressed because Rof could inhibit termination at $t_1$. However, deletion experiments showed that there is an extra Rho-dependent termination site upstream of $t_1$ between +185 and +238. This site, named $t_2$, was more sensitive to Rof than $t_1$, suggesting that termination at $t_2$ requires lower concentration of Rho. This was further supported by the result that overexpression of Rho increased termination at $t_1$ more efficiently than at $t_2$. Therefore, this result showed that, besides transcriptional regulation by P1 and P2, both Rho and Rof function differentially at $t_1$ and $t_2$ depending on their cellular levels so that ygfA gene expression can be sophisticatedly controlled. Furthermore, this study suggests that Rho and Rof can orchestrate gene expression through regulating transcription termination according to their cellular levels.