Transcription termination in Mycobacteria

Abstract

Transcription termination is one of the key events of gene expression that decides the boundary of the gene. Termination either before the stop codon or transcriptional read-through into the downstream gene both have unfavourable consequences. The absence of efficient and accurate transcription termination can be deleterious for cell survival and can cause genome instability. In bacteria, transcription can be terminated by either of the two modes – intrinsic or Rho-dependent termination (RDT). Our understanding of bacterial transcription and the associated molecular machinery comes majorly from Escherichia coli (E. coli). Hence the presence of species-specific features with respect to transcription and its termination in distinct phyla across bacterial domain needs to be explored. The success of the pathogen Mycobacterium tuberculosis (Mtb) is credited to its efficient gene expression. However, very little is known about their transcription termination which is an essential part of gene expression. The work presented in the thesis attempts to uncover the unique features associated with both intrinsic termination and RDT in mycobacteria. Chapter 1 of the thesis provides a general introduction on transcription, associated molecular machinery especially RNA polymerase (RNAP), and the process of transcription termination in bacteria. The introduction emphasis more on the distinct features associated with intrinsic terminators and Rho with a brief compilation of what is already known about them. It also discusses the recently elucidated mechanism of their action and the impact of elongation factors like NusA and NusG on transcription termination. The introduction ends with summarizing briefly about transcription termination in mycobacteria and how this study contributes to the understanding of transcription termination in mycobacteria. In Chapter 2 the study is directed to understand the importance of U-tract downstream of an intrinsic terminator in transcription termination. A canonical intrinsic terminator has a G/C-rich hairpin structure followed by a 7-9 Us located at the 3’ region of the nascent RNA and are crucial determinants for intrinsic termination. In mycobacteria, there is a scarcity of such intrinsic terminators. However, secondary structures having G/C-rich stem devoid of any U’s or with suboptimal U-tracts were identified earlier as terminators and found to be functional both in vitro and in vivo. Two different observations - that a mycobacterial RNAP does not function at intrinsic terminators devoid of U-tracts and the identification of an altogether new motif for termination in mycobacteria necessitated re-examining a number of putative terminators for their function as terminators. In this chapter, in silico identified non-canonical terminators were subjected to experimental validation, they were found to dissociate RNA from the elongating RNAP. Termination was observed when the U-tracts were reduced, or totally absent both in vitro and in vivo. Our results, thus indicate that the presence of U-tract following the G/C-rich stem in an intrinsic terminator may not be an essential determinant for transcription termination in mycobacteria. Chapter 3 is focused on MtbRho and RDT in mycobacteria. Although the two transcription termination mechanisms contribute to nearly in equal measures to terminate transcription in E. coli, understanding their role in mycobacterial gene expression is at the early phase. In this chapter, we highlight the dominance of RDT in mycobacteria and pose Rho as a key regulatory factor. The lower abundance of intrinsic terminators, high cellular levels of Rho, and its genome-wide association with a majority of transcriptionally active genes indicate the predominance of RDT in Mtb. Unlike in other well-studied systems, Rho modulates the termination of RNA synthesis for both protein-coding and stable RNA genes in Mtb. Concordantly, the depletion of Rho in mycobacteria enhances the transcription read-through of transcription units. These properties suggest an evolutionary adaptation of Rho as the efficient and predominant mode of transcription termination in mycobacteria. Chapter 4 attempts to draw a connection between intrinsic termination and RDT. Generally, the two mechanisms have been illustrated as two independent pathways with contrasting requirements to halt RNA synthesis. However, a majority of the intrinsic terminators terminate transcription inefficiently leading to transcriptional read-through. The unwanted transcription in the downstream region beyond the terminator would have undesired consequences. To prevent such transcriptional read-through, bacteria must have evolved ways to terminate transcription more efficiently at or near the termination sites. This chapter describes the collaboration between the two mechanisms, where intrinsic terminator and Rho factor functionally interact to prevent transcriptional read-through. Contribution from both the termination processes is demonstrated at the downstream regions of the genes both in vitro and in vivo in mycobacteria. Distinct patterns of cooperation between the two modes of termination were observed at the 3’ untranslated regions of the genes to ensure efficient termination. A similar interaction between the two termination processes was observed in E. coli as well suggesting a likely prevalence of this functional cooperation across bacteria. The reporter system developed to assess the Rho – intrinsic termination collaboration in vivo for mycobacteria and E. coli can readily be applied to other bacteria.