Rho-dependent transcription termination is essential to prevent excessive genome-wide R-loops in Escherichia coli

Abstract

Two pathways of transcription termination, factor-independent and -dependent, exist in bacteria. The latter pathway operates on nascent transcripts that are not simultaneously translated and requires factors Rho, NusG, and NusA, each of which is essential for viability of WT Escherichia coli. NusG and NusA are also involved in antitermination of transcription at the ribosomal RNA operons, as well as in regulating the rates of transcription elongation of all genes. We have used a bisulfite-sensitivity assay to demonstrate genome-wide increase in the occurrence of RNA-DNA hybrids (R-loops), including from antisense and read-through transcripts, in a nusG missense mutant defective for Rho-dependent termination. Lethality associated with complete deficiency of Rho and NusG (but not NusA) was rescued by ectopic expression of an R-loop-helicase UvsW, especially so on defined growth media. Our results suggest that factor-dependent transcription termination subserves a surveillance function to prevent translationuncoupled transcription from generating R-loops, which would block replication fork progression and therefore be lethal, and that NusA performs additional essential functions as well in E. coli. Prevention of R-loop-mediated transcription-replication conflicts by cotranscriptional protein engagement of nascent RNA is emerging as a unifying theme among both prokaryotes and eukaryotes.A ll bacterial transcription termination occurs by one of two pathways that are referred to as factor-independent (or intrinsic) and factor-dependent (or Rho-dependent), respectively (1). The latter pathway, which requires the action of factors Rho, NusG, and NusA in Escherichia coli, serves to terminate synthesis of transcripts that are not being simultaneously translated, for example, at the ends of various genes and operons (1, 2). The same mechanism is also responsible for the classic phenomenon of nonsense polarity (3), by which a stop codon mutation within the proximal gene of an operon results in absence of transcription of the distal genes; in this manner, Rho-dependent termination provides a back-up to other mechanisms (4, 5) that act to ensure the coupling of transcription with translation in bacteria. NusG and NusA are also involved in transcription antitermination during lytic growth of the lambdoid prophages and at the ribosomal RNA operons, as well as in regulating the rates of transcription elongation of all genes (6, 7).Rho, NusG, and NusA are each essential for viability of the prototypic WT E. coli strain MG1655. Cardinale et al. (8) have reported that NusG and NusA are dispensable in strain MDS42 [which is an engineered MG1655 derivative with 14% reduced genome content because of deletions of insertion elements and cryptic prophages (9)], based on which they have proposed that the essentiality of Rho-dependent termination stems from its need for silencing of horizontally acquired genes in bacteria. Even so, the Δrho mutation in MDS42, as in MG1655, is lethal (8, 10).Several phenotypes in nusG, rho, and nusA missen...

NusG-Spt5 Proteins—Universal Tools for Transcription Modification and Communication

“…The first antitermination mechanism, mediated by the N protein, was discovered in bacteriophage λ. Roberts proposed that λ N instructs RNAP to ignore Rho-dependent terminators in the early genes 97 , enabling transcription of the entire phage genome. Genetic screens for host proteins involved in λ N antitermination identified several N-utilization substances, NusA, NusB, NusE (the ribosomal protein S10) and NusG, that, together with N, assemble into a complex modifying RNAP to a termination-resistant form 98 .…”

Pervasive transcription: detecting functional RNAs in bacteria

“…11,12 Nevertheless, the average intensity of selection against such elements is weak, and, consequently, many spurious promoterlike sequences persist within populations. 13 Because uncontrolled transcription from genome-wide promoter-like sequences are potentially dangerous, bacteria have several systems in place to control the generation of spurious transcripts: (i) The histone-like nucleoid structuring protein (H-NS) suppresses transcription initiation from intragenic promoters, 14 (ii) the termination factor Rho and its cofactor NusG function in the termination of asRNA transcription, 15,16 and (iii) multiple RNases degrade aberrant RNAs. 17 A lack of asRNA conservation among closely related bacteria might not necessarily indicate lack of function because, as shown recently in Drosophila, functional genes can arise rapidly in a lineage-specific manner.…”

Decreased Expression of Stable RNA Can Alleviate the Lethality Associated with RNase E Deficiency in Escherichia coli

“…Several plasmids used in this study are derivatives of pMU575, which is an unstable single-copynumber plasmid with an IncW replicon and Tp r antibiotic marker: pHYD1613 (rne ϩ ) (41), pHYD2411 (Salmonella enterica serovar Typhimurium rho ϩ gene) (55), and pHYD2385 (Salmonella Typhimurium nusG ϩ ) (J. Krishna Leela, unpublished data). Other plasmids (with salient features in parentheses) employed include pHYD2373 (pWSK129 derivative carrying rne-R169Q,ΔCTH [encoding RNase E-R169Q,ΔCTH], the pSC101 replicon, Kan r , and Cm r ) (41), pRne-SG21 (pWSK129 derivative carrying rne-R169Q,529Δ [encoding RNase E-R169Q,529Δ], the pSC101 replicon, and Kan r ) (40), pCP20 (pSC101-based Ts replicon encoding Flp recombinase, Cm r , and Amp r ) (82), pJK537 (1.8-kb fragment carrying dksA cloned in pBR322, pMB1, and Amp r ) (83), and pHR53 (pJK537 derivative carrying truncated dksA gene, pMB1, and Amp r ) (84).…”

“…These findings were interpreted in the context of a model in which formation of excessive RNA-DNA hybrids (R-loops) in the rho and nusG mutants (55,56) provides an alternative means of mRNA degradation in the RNase E-deficient strains.…”