T.Bepler, A.Morin, M.Rapp, J.Brasch, L.Shapiro, A. J.Noble, B.Berger, Positive-unlabeled convolutional neural networks for particle picking in cryo-electron micrographs, Automated comparative protein structure modeling with SWISS-MODEL and Swiss-PdbViewer: A historical perspective. Given that 5 leader mRNAs in Salmonella have an average length of 2065 nucleotides (nt)23, the presence of a >400 nt intergenic region upstream of the corA start codon (Fig. Epub 2012 Mar 22. 2015 Dec 4; 427(24): 38343849. Labeled primer was purified on a G-50 column (GE Healthcare) and 0.4 pmol was mixed with 8 g of total RNA extracted from Salmonella grown to mid-log phase in N-minimal media (10 mM MgCl2). 1 g of template DNA was used and reactions were incubated at 37C overnight. Epub 2021 Oct 27. Rho inactivation negatively affects biofilm. What are the two modes of Bacterial RNA polymerase termination Factor Independent and Factor Dependent 3. S4). It is likely that Salmonella shares this regulatory mechanism with other enteric bacteria because the corA leader sequence is highly conserved within this family (Fig. The particles from two datasets were then merged for further 3D classification, which revealed the classes of apo RNAP, EC, and RNAP-Rho complexes 1 and 2 (fig. Local CTF estimation and beam tilt refinement were performed to obtain a final 3.5- map. Together these results indicate that stem-loops C and D promote expression of the coding region by reducing the efficiency of Rho-dependent termination within the corA leader. 1A) raises the possibility that an unusually long mRNA leader precedes the corA coding region. 3A), the short ORF corL and the corA Shine-Dalgarno (SD) sequence are shown. 2022 Jun 21;204(6):e0008922. 8600 Rockville Pike The transcription elongation factor NusG facilitates this termination process by directly interacting with Rho. Accessibility 10. How does rho factor work? | Genes VII Rho dependent termination The three main stages of transcription are known as initiation, elongation and termination, the latter of which can be assisted by a protein that associates with the RNA polymerase complex, in Escherichia coli this known as a Rho () factor. Go to: Abstract The process of transcription termination is essential to proper expression of bacterial genes and, in many cases, to the regulation of bacterial gene expression. S5A). Rho factor - Wikipedia Unauthorized use of these marks is strictly prohibited. In this review, we discuss current models describing the mechanism of action of this hexameric transcription terminator, its regulation by different cis and trans factors, and the effects of the termination process on physiological processes in bacterial cells, particularly E. coli and Salmonella enterica Typhimurium. Proc Natl Acad Sci U S A. Federal government websites often end in .gov or .mil. 4A,B), indicating that both pyrimidine-rich tracts are required for efficient Rho-dependent termination. As a service to our customers we are providing this early version of the manuscript. The key difference between Rho dependent and Rho independent termination is that in Rho dependent termination, Rho factor binds with the transcript and terminates transcription by breaking hydrogen bonds between the template and the transcript, while Rho independent termination terminates the transcription by producing FOIA For plasmid maintenance, 20 g/ml chloramphenicol (Cm), 50 g/ml kanamycin (Kan) and/or 50 g/ml ampicillin (Ap) were used. Rho Protein: Roles and Mechanisms - PubMed 2006 Sep;152(Pt 9):2515-2528. doi: 10.1099/mic.0.28982-0. Similarly, the Salmonella sRNA ChiX can base pair with the chiPQ transcript at the chiP translation start site to uncouple transcription and translation, thus allowing Rho to abort transcription of chiQ18. J Biol Chem. We identified two regions at positions U87-U97 and C125-C140 that have high pyrimidine content and thus might contribute to recognition of the corA leader as a Rho substrate. Calculation of termination efficiency and generation of an RNA sequencing ladder were performed as described in Materials and Methods. Cumulatively, the in-line probing results indicate that the corA leader can adopt the mutually exclusive conformations consisting of stem-loops A+B versus C+D (Fig. However, this notion has recently been challenged by the observation that corA mRNA and protein levels do not correlate during the transition from exponential to stationary growth31. Suppose that there are Rho-dependent terminators within the transcription unit, that is, before the terminator that usually is used. S1). Cumulatively, these data indicate that stem-loop D is necessary to promote transcription into the associated coding region. The involvement of . Subsequent physiological analyses demonstrated that a considerable part of Rho-controlled transcription is connected to balanced regulation of three mutually exclusive differentiation programs: cell motility, biofilm formation, and sporulation. That is, the formation of RNA secondary structures controlling rut site availability is independently determined by direct binding of Mg2+ to the mgtA leader mRNA and by translation of mgtL, a short ORF in the mgtA leader20. Factor-stimulated intrinsic termination: getting by with a little help from some friends. S4C).Approximately 693,000 Rho-hexamer particles were selected by 2D classification (fig. When translation of corL is impaired (2), either by inefficient translation initiation or elongation, the kinetic and thermodynamic advantages of stem-loop C over stem-loop B lead to formation of the stem-loops C+D conformation, which sequesters the rut site and allows RNAP to continue into the corA coding region. (A) The accumulation of free phosphate was monitored to quantify Rhos ATPase activity. Regulation of mgtA and mgtB expression. An official website of the United States government. Plasmid pACYC-corL-lacZ was constructed by cloning a PCR product generated from 14028s genomic DNA with primers 11903 and 11905 between the XmaI and XbaI sites of plasmid pACYC-lacZ. Once the PBS binds an RNA substrate, transient opening of the ring-shaped protein allows a downstream RNA segment to thread into the central channel and contact a secondary binding site8; 12. Plasmid pYS1040FL, which contains the entire corA 5 intergenic region and lacks the plac16 promoter, was constructed by cloning a PCR fragment generated from 14028s genomic DNA using primers 11992 and 11987 between the SalI and XhoI sites of pYS1040. Incubation was continued at 37C with 250 rpm shaking with samples collected for OD600 measurement and Western blotting every 12 h. Western blot samples were prepared by re-suspending cell pellets in a volume of SDS-PAGE loading buffer (2.3% v/v sodium dodecyl sulfate, 22% v/v glycerol, 0.015 g/ml Tris, 0.05 mg/ml bromophenol blue, 87 L/ml -mercaptoethanol) determined by the formula: loading buffer (l) = OD600 x sample volume (l) x 100. Bogden CE, Fass D, Bergman N, Nichols MD, Berger JM. Termination factor Rho mediates transcriptional reprogramming of Bacillus subtilis stationary phase. Transcriptional pause extension benefits the stand-by rather than catch-up Rho-dependent termination. Babina AM, Surkov S, Ye W, Jerlstrm-Hultqvist J, Larsson M, Holmqvist E, Jemth P, Andersson DI, Knopp M. Elife. (BE) -galactosidase activity (Miller units) exhibited by wild-type Salmonella (14028s) harboring plasmid pYS1040 or derivatives containing mutations that alter the efficiency of corL translation initiation (M11-M14) or introduce a premature stop codon into corL (M15-M17). Science. 5E) suggests that some Rho-dependent termination still occurs when the RNA adopts the stem-loop C+D conformation. A.Waterhouse, M.Bertoni, S.Bienert, G.Studer, G.Tauriello, R.Gumienny, F. T.Heer, T.A.P.de Beer, C.Rempfer, L.Bordoli, R.Lepore, T.Schwede, SWISS-MODEL: Homology modelling of protein structures and complexes. By contrast, Rho displayed reduced affinity for an RNA harboring a mutation in the rut site (M9+M10, Fig. Rho inactivation accelerates sporulation of. rut Sites in the nascent transcript mediate Rho-dependent transcription termination in vivo. Subsequently, ~239,000 particles were selected for 3D refinement with C6 symmetry enforced and subsequent Bayesian polishing. Author contributions: Conceptualization: Y.M. 1997 Jul 1;36(26):7993-8004. doi: 10.1021/bi963180r. Rho-dependent transcription termination: more questions than answers Describe factor Indepennt termination Intrinsic termination 4. Rho factor: transcription termination in four steps. In Escherichia coli, about half of the transcription events are terminated by the Rho protein. By contrast, the downstream pyrimidine-rich tract in the corA leader is most important for Rho loading because mutation of this region (M9+M10) has larger effects than mutation of the upstream pyrimidine-rich tract (M8) on transcription in vivo (Fig. To determine the effect of the of corA leader mRNA conformation on transcription of the associated coding region, we measured the -galactosidase activity originating from wild-type Salmonella harboring plasmid pYS1040 or derivatives containing point mutations expected to favor or disrupt a particular RNA structure. If corL translation exerts its effect by promoting formation of particular secondary structures in the corA leader RNA, then mutations forcing the RNA to adopt a certain conformation should be epistatic over those affecting corL translation. Here, we summarize the current state of the ever-increasing knowledge about the various aspects of the physiological functions of Rho, such as limitation of deleterious foreign DNA expression, control of gene expression, suppression of pervasive transcription, prevention of R-loops and maintenance of chromosome integrity, focusing on similaritie. Bidnenko V, Nicolas P, Gurin C, Drozier S, Chastanet A, Dairou J, Redko-Hamel Y, Jules M, Bidnenko E. PLoS Genet. In WT, transcription terminates at Rho-controlled terminator (transcript is shown in black). The kinetic pathway of RNA binding to the Escherichia coli transcription termination factor Rho. The accessibility of rut sites within 5 mRNA leaders can be modulated by a variety of intracellular signals including ions16, small molecule metabolites16, amino acid availability17, sRNAs18 and proteins19. Following incubation at 37C for 4 h with 250 rpm shaking, 50 l chloroform and 20 l 0.1% SDS were added and cultures were vortexed vigorously for 1 min. Mutations used to disrupt base pairing (red) or Rho loading (blue) are denoted next to the mutated nucleotides. Biochemistry. Selective ribosome profiling reveals the cotranslational chaperone action of trigger factor in vivo. 2C). In rho-dependent termination in prokaryotes, how does RNA polymerase "know" that it has reached the end of a gene and that it has to stop so that the rho-factor can bind mRNA's rut site? The .gov means its official. 2023 Feb 3;19(2):e1010618. 2016 Aug;41(8):690-699. doi: 10.1016/j.tibs.2016.05.012. the contents by NLM or the National Institutes of Health. It is based on the elements of pervasive genome-wide transcription controlled by the termination factor Rho. FOIA Bidnenko V, Nicolas P, Gurin C, Drozier S, Chastanet A, Dairou J, Redko-Hamel Y, Jules M, Bidnenko E. PLoS Genet. Epub 2022 May 31. In the absence of Rho, several up-regulated sense and antisense transcripts affect key structural and regulatory elements of these differentiation programs, thereby suppressing motility and biofilm formation and stimulating sporulation. Transcription Part 3 Flashcards | Quizlet Gene. Transcription. In both the corA and mgtA leaders, the conformation that presents an accessible rut site contains an RNA hairpin (i.e., stem-loop B) that brings together two single-stranded pyrimidine-rich regions required for Rho loading. Prieto A, Bernabeu M, Snchez-Herrero JF, Prez-Bosque A, Mir L, Buerl C, Collado C, Httener M, Jurez A. Commun Biol. 6C was performed in an intermediate MgCl2 concentration of 1.5 mM to allow for resolution of mutants that both increase and decrease ATPase activity. We now report the identification of a Rho . Models of RNAP-Rho complexes were generated by fitting the coordinates of the EC-unbound Rho and the Rho-unbound EC/RNAP into the maps. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. The structure and activities of the archaeal transcription termination factor Eta detail vulnerabilities of the transcription elongation complex. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. The https:// ensures that you are connecting to the J. Y.Kang, R. A.Mooney, Y.Nedialkov, J.Saba, T. V.Mishanina, I.Artsimovitch, R.Landick, S. A.Darst, Structural basis for transcript elongation control by NusG family universal regulators. Careers. HHS Vulnerability Disclosure, Help One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Rho inactivation increases Spo0A phosphorylation. S3). Khudyakov YuE, Neplyueva VS, Kalinina TI, Smirnov VD. official website and that any information you provide is encrypted J Mol Biol. 2023 Mar 15;12:e78299. Accessibility J. The .gov means its official. The RNA conformation that promotes Rho-dependent termination is favored by efficient translation of corL, a short open reading frame located within the corA leader. Careers. In the absence of Rho, RNAP transcribed full-length RNA (Fig. Murayama Y, Ehara H, Aoki M, Goto M, Yokoyama T, Sekine SI. If the corA leader mRNA is required for normal expression of the corA coding region, then deletion of the corresponding DNA sequence from the Salmonella chromosome should alter the abundance of the CorA protein. An overview of the steps involved in the termination by Rho. Transcription. By contrast, in-line probing of an RNA harboring a mutation predicted to disrupt stem-loop C (M1 [G84C G85C C86G], Fig. This transcription start site, hereafter denoted nucleotide +1, has near-consensus 10 and 35 promoter elements (Fig. Models to explain Rhos access to RNA-DNA hybrid and release RNA. 2004 Dec;7(6):579-86. doi: 10.1016/j.mib.2004.10.001. Calculation of termination efficiency and generation of an RNA sequencing ladder were performed as described in Materials and Methods. 1994 Mar;11(6):983-90. doi: 10.1111/j.1365-2958.1994.tb00376.x. 6C). Mol Microbiol. 2007; 8: 413423. The effect of the M5 mutation is specifically due to disruption of stem-loop D and not B because other mutations predicted to prevent formation of stem-loop D without disrupting stem-loop B (M5* [G123A], M5+M7 [T97G C99G G121T G123T], Fig. Datsenko KA, Wanner BL. Keywords: This RNA conformation presents an accessible rut site (dark blue), allowing Rho (orange) to load onto the corA mRNA leader and trigger RNA release by RNAP (green) prior to transcription of the corA coding region (light blue). The site is secure. RNA randomly collides with the Rho molecule forming a dynamic Rho-RNA complex. The cryo-EM experiments were performed with the Krios G4 microscope at the RIKEN Yokohama Cryo-EM facility. Schematic representation of the conserved domains of. All experiments were carried out with wild-type Salmonella enterica serovar Typhimurium strain 14028s39 or its derivative MK112, in which has the sequence corresponding to the corA leader is replaced by an 85 nt scar sequence. Kim DE, Patel SS. For example, binding of the protein CsrA to the pgaA leader or the small molecule flavin mononucleotide to the ribB leader in Escherichia coli both unfold RNA secondary structures that normally sequester a rut site16; 19. For instance, excess tryptophan promotes ribosome stalling during translation of a short ORF in the leader region of the E. coli tnaA mRNA, thereby exposing a rut site that would normally be occluded by a ribosome17. FOIA C, Rho contact with elongation complex changes the conformation of RNA exit channel. Strains. If the effects of mutations in the pyrimidine-rich regions are due to rut site disruption, then the corresponding RNAs should exhibit a decreased ability to stimulate Rhos ATPase activity in vitro. However, the stem-loop A+B conformation has a kinetic advantage over the stem-loop C+D conformation because nucleotides G24-G30 and C80-C86 have the opportunity to base pair before nucleotides G101-G111 emerge from RNAP (Fig. Unauthorized use of these marks is strictly prohibited. We identified two possible RNA conformations whose regions of base pairing overlap such that they are mutually exclusive (Fig. Bacteria require phase separation for fitness in the mammalian gut. official website and that any information you provide is encrypted subtilis kinB gene contains intragenic Rho-dependent terminator. The mode of action of bicyclomycin was studied in the 1980s and early 1990s, and only information concerning its locus of action in E. coli, that is, the rho transcription termination factor (rho is a protein responsible for termination of RNA synthesis), was reported. Morgan WD, Bear DG, von Hippel PH. When a translocating Rho molecule catches up to a paused RNAP, it induces dissociation of the elongation complex2; 15. Mouse anti-DnaK (Enzo Life Sciences) was used at a dilution of 1:5000 as a loading control. Before Here, we report that the Mg2+ channel gene corA in Salmonella enterica serovar Typhimurium, which was previously thought to be constitutively expressed, is regulated by a Rho-dependent terminator located within its 5 leader region. 7A). Bossi L, Schwartz A, Guillemardet B, Boudvillain M, Figueroa-Bossi N. A role for Rho-dependent polarity in gene regulation by a noncoding small RNA. Removal of pyrimidines from positions 6668 or 7477 actually decreased expression in vivo (Fig. To clearly define the two identified conformations, we performed in-line probing of RNAs harboring mutations anticipated to favor formation of one conformation or the other (Fig. 2012 Jun;158(Pt 6):1468-1481. doi: 10.1099/mic.0.056804-0. Vmax was taken to be the slope during a 10 min window with the largest linear change in absorbance. The recently solved crystal structure could explain many of its physiological functions in terms of its structure. Tandem attenuators control expression of the Salmonella mgtCBR virulence operon. Termination factor - Wikipedia 3A). These results confirm that ribosome occupancy of the corL ribosome binding site is necessary for preferential formation of stem-loop C, likely because stem-loop A can compete with stem-loop C in the absence of a ribosome at the RBS. Normally these earlier terminators are not used, because the ribosome prevents Rho from reaching RNA polymerase. HHS Vulnerability Disclosure, Help MeSH 8). For both the corA and mgtA leader mRNAs, sequences within the right arm of stem-loop A and the left-arm of stem-loop B can alternatively base pair to form a structure (stem-loop C) that sequesters rut site nucleotides. Yeast RNA was added to 0.05 mg/ml to prevent non-specific binding. Genetic networks controlling the initiation of sporulation and the development of genetic competence in Bacillus subtilis. As hypothesized, RNAs harboring mutations that reduce the pyrimidine content of U87-U97 and C125-C140 stimulated Rhos ATPase activity to a much lesser extent than wild-type RNA (Fig. Epub 2022 Sep 12. J. M.Peters, A. D.Vangeloff, R.Landick, Bacterial transcription terminators: The RNA 3-end chronicles. HHS Vulnerability Disclosure, Help Many aspects of this process are still being studied. Kang W, Hwang S, Kang JY, Kang C, Hohng S. Int J Mol Sci. In accordance with this notion, the affinity of Rho for a mutant corA leader mRNA unable to form stem-loop C (M1) was significantly higher than that for the wild-type corA leader RNA (Fig. Accessibility Biochemistry. For example, Rho defines the 3' end of many operons, suppresses antisense transcription, and implements transcriptional polarity by triggering RNA release from RNA polymerase (RNAP) when the normal coupling between transcription and translation is disrupted 1; 2; 3. D. G.Vassylyev, M. N.Vassylyeva, A.Perederina, T. H.Tahirov, I.Artsimovitch, Structural basis for transcription elongation by bacterial RNA polymerase, V. Molodtsov, C.Wang, E.Firlar, J.T.Kaelber, R.H.Ebright, Structural basis of Rho-dependent transcription termination. Termination efficiency was calculated using the formula: 100 100* (runoff band intensity/total lane intensity). Epub 2022 Sep 25. [1] Rho factor binds to the transcription terminator pause site, an exposed region of single stranded RNA (a stretch of 72 nucleotides) after the open reading frame at C-rich/G-poor sequences that lack obvious secondary structure. The mixture was incubated at 65C for 5 min and cooled on ice for 1 min to promote annealing. Most bacteria possess termination factor Rho, which represses pervasive, mostly antisense, transcription. National Library of Medicine In vitro approaches to analysis of transcription termination. M.Boudvillain, N.Figueroa-Bossi, L.Bossi, Terminator still moving forward: Expanding roles for Rho factor. 5C). USA. USA. doi: 10.1073/pnas.2207581119. Factor-dependent termination of transcription in bacteria relies on the activity of a specific RNA helicase, the termination factor Rho. Kinetics of mRNA elongation during transcription from the bacteriophage lambda PR promoter. Rho Factor: Transcription Termination in Four Steps 3A) showed no expression (Fig. The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). Fig 7. FOIA Formation of stem-loop D alone is not sufficient to promote constitutive transcription elongation because a triple mutant that retains stem-loop D but cannot form stem-loop C (M1+M5+M6, Fig.