how does rho factor termination transcription

3A) suggests that the RNA conformation adopted by the corA leader might control Rhos ability to terminate transcription before the corA coding region. Sci Adv. Rho-dependent transcription terminators participate in sophisticated genetic regulatory mechanisms, in both bacteria and phages; they occur in regulatory regions preceding the coding sequences of genes and within coding sequences, as well as at the end of transcriptional units, to prevent readthrough transcription. For this classification, a multitemplate reference including type 1 and type 2 complexes, EC, and apo RNAP templates, created using maps obtained from preliminary classifications, was used for better discrimination between the complexes. Sci. is an Investigator of the Howard Hughes Medical Institute. In molecular biology, a termination factor is a protein that mediates the termination of RNA transcription by recognizing a transcription terminator and causing the release of the newly made mRNA.This is part of the process that regulates the transcription of RNA to preserve gene expression integrity and are present in both eukaryotes and prokaryotes, although the process in bacteria is more . 4B) and Rho ATPase activity in vitro (Fig. Recent studies performed in different species favour the view that Rho-dependent termination plays a significant role even in bacteria where Rho is not essential. Epub 2022 Oct 3. Data shown correspond to mean values from two independent experiments and error bars indicate standard deviation. EMBO Rep. 2009; 10: 973982. 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. The mutually exclusive RNA conformations adopted by the corA leader (Fig. The structure and activities of the archaeal transcription termination factor Eta detail vulnerabilities of the transcription elongation complex. 2013;110:258263. S4B). 0.8 ml aliquots of LB/Cm were inoculated with single wild-type Salmonella (14028s) colonies harboring plasmid pYS1040 or mutant derivatives and incubated overnight. and transmitted securely. 4AC). The pyrimidine-rich tract downstream of stem-loop B appears to play a larger role than the upstream one because mutation M9+M10 had a larger effect than M8, both in vitro and in vivo (Fig. By contrast, Rho displayed reduced affinity for an RNA harboring a mutation in the rut site (M9+M10, Fig. Two datasets of ~13,000 and ~20,000 micrographs were collected (table S1). J. Y.Kang, P. D.Olinares, J.Chen, E. A.Campbell, A.Mustaev, B. T.Chait, M. E.Gottesman, S. A.Darst, Structural basis of transcription arrest by coliphage HK022 Nun in an. S2C) and subjected to 3D classification to discard particles without RNAP. Rho Protein: Roles and Mechanisms | Annual Review of Microbiology Accessibility Whereas Rho executes these homeostatic functions genome-wide, it can also regulate expression of specific genes by controlling whether RNAP terminates transcription within a 5 leader region or continues into the associated coding region4. This result indicates that the corA leader represses expression of the corA coding region. 3A) had no effect on corA-lacZ transcription (Fig. doi: 10.1126/sciadv.ade7093. II. BeF3, in 400 l of buffer C.The sample was cross-linked with 3 mM BS3 (Thermo Fisher Scientific) for 30 min at 30C. RNA randomly collides with the Rho molecule forming a dynamic Rho-RNA complex. 4A) and decreased Rho-dependent termination in vitro (Fig. Rho factor is an essential protein in E. coli. D.Liebschner, P. V.Afonine, M. L.Baker, G.Bunkczi, V. B.Chen, T. I.Croll, B.Hintze, L. W.Hung, S.Jain, A. J.McCoy, N. W.Moriarty, R. D.Oeffner, B. K.Poon, M. G.Prisant, R. J. PMC The https:// ensures that you are connecting to the Epshtein V, Dutta D, Wade J, Nudler E. An allosteric mechanism of Rho-dependent transcription termination. Most of our current knowledge concerning the mechanism of Rho's activity and its physiological roles comes from the model micro-organism Escherichia coli, where Rho is essential and involved in the control of several important biological processes. 1B). The ribosome covers 1215 nt of mRNA on both sides of its P site30. 2019R1A2C2005209/National Research Foundation of Korea (NRF), 2018R1A2B2004602/National Research Foundation of Korea (NRF), 2019M3E5D6066058/National Research Foundation of Korea (NRF), Roberts JW. 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 similarities and differences of the activities of Rho in various bacterial species. Factor-____ termination: We will only discuss the Rho-dependent termination. official website and that any information you provide is encrypted Single round, synchronized in vitro transcription reactions were carried out as described43. doi: 10.1371/journal.pgen.1010618. 3A). National Library of Medicine Wei RR, Richardson JP. Rho is the main factor required for termination; it is a ring-shaped hexameric protein with ATPase and helicase activities. RNA secondary structures regulate three steps of Rho-dependent transcription termination within a bacterial mRNA leader. Unauthorized use of these marks is strictly prohibited. 3A). Reverse transcription was carried out using Superscript II (Life Technologies) according to the manufacturers instructions. Epub 2017 Jul 21. Epub 2023 Jan 25. Termination efficiency was calculated using the formula: 100 100* (runoff band intensity/total lane intensity). Bacterial Transcription Terminators: The RNA 3-End Chronicles Wang B, Pei H, Lu Z, Xu Y, Han S, Jia Z, Zheng J. iScience. Specific nucleotides required for termination have also been identified at Rho sites. Our results indicate that three kinetically distinct routes take place: (1) the catch-up mode leads first to RNA shearing for RNAP recycling on DNA, and (2) later to RNAP displacement for decomposition of the transcriptional complex; (3) the last termination usually follows the stand-by mode with displacing for decomposing. INTRODUCTION. Schematic representation of the conserved domains of. government site. 2 publications 2023 Jul 1;14(1):3891. doi: 10.1038/s41467-023-39553-8. The transcriptional landscape and small RNAs of Salmonella enterica serovar Typhimurium. Peters JM, Mooney RA, Kuan PF, Rowland JL, Keles S, Landick R. Rho directs widespread termination of intragenic and stable RNA transcription. doi: 10.1371/journal.pgen.1010618. 3A). Invivo regulation of bacterial Rho-dependent transcription termination by the nascent RNA. S2A), which is inconsistent with participation of these nucleotides in Rho loading. Rho factor is shown as an open ring, but it may be in equilibrium between open and closed states at this step. Analysis of a Cell Wall Mutant Highlights Rho-Dependent Genome Amplification Events in Staphylococcus aureus. Bookshelf 2. Rho's ATPase is activated by Rho-mRNA binding, and provides the energy for Rho translocation along the mRNA; translocation requires sliding of the message into the central hole of the hexamer. Before Federal government websites often end in .gov or .mil. Stages of transcription: initiation, elongation & termination (article Science. 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. Data shown correspond to mean values from at least three independent experiments performed in duplicate, and error bars indicate standard deviation. 5D). Regulation of CorA Mg2+ channel function affects the virulence of Salmonella enterica serovar typhimurium. Control RNase T1 and alkaline hydrolysis reactions were carried out using the RNase T1 (1U/l) kit (Ambion) according to the manufacturers instructions. Purified Rho protein was incubated with RNA corresponding to wild-type corA leader mRNA or variants harboring mutations in stem-loops A-C. (E) -galactosidase activity (Miller units) exhibited by wild-type Salmonella (14028s) harboring plasmid pYS1040 or derivatives harboring mutations in stem-loops A, C and/or D, in the presence or absence of the Rho-specific inhibitor bicyclomycin (BCM). Rho-dependent transcription termination is a multi-step process. Band intensities were measured using ImageQuant software (GE Healthcare) and normalized by subtracting the intensity of a blank region of the same area. Mandal M, Breaker RR. Babina AM, Surkov S, Ye W, Jerlstrm-Hultqvist J, Larsson M, Holmqvist E, Jemth P, Andersson DI, Knopp M. Elife. (AD) -galactosidase activity (Miller units) exhibited by wild-type Salmonella (14028s) harboring plasmid pYS1040 or derivatives containing mutations that disrupt base pairing in stem-loops A, B, C and/or D. Data shown correspond to mean values from at least three independent experiments performed in duplicate, and error bars indicate standard deviation. These experiments indicate that the stem-loop C+D conformation inhibits Rho binding to the corA leader independently of other effects that this conformation may have on Rho translocation and associated ATP hydrolysis. Epub 2023 Mar 16. Science. Unauthorized use of these marks is strictly prohibited. government site. Therefore, a ribosome that stalls early on during translation of corL might not occlude the left arm of stem-loop C (Fig. The corL SD sequence, start codon and stop codon are shown in purple. Calculation of termination efficiency and generation of an RNA sequencing ladder were performed as described in Materials and Methods. Effects of Rho over-expression in B . It is a homohexameric protein that recognizes and binds preferably to C-rich sites in the transcribed RNA. E. coli DH541 or XL-1 blue (Agilent) were used for cloning and plasmid DNA preparation. Interaction with the nascent RNA is a prerequisite for the recruitment of Rho to the transcription elongation complex in vitro. It is based on the elements of pervasive genome-wide transcription controlled by the termination factor Rho. So, even though the RNA polymerase is about 40 nt per second faster than Rho, it does not pose a problem for the Rho termination mechanism as the RNA polymerase allows Rho factor to catch up. In-line probing analysis of riboswitches. Epub 2016 Jun 17. 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. The recently solved crystal structure could explain many of its physiological functions in terms of its structure. In addition to these genome-wide activities, Rho implements regulation of specific genes by dictating whether RNA polymerase terminates transcription within the 5 leader region or continues into the downstream coding region. See this image and copyright information in PMC. In accordance with this notion, replacement of the corL start codon with the UUA stop codon increased expression from a corA-lacZ transcriptional fusion (M12 [G29A], Fig. The involvement of . sharing sensitive information, make sure youre on a federal Ciampi MS. Rho-dependent terminators and transcription termination. A, it spools RNA from a distance without making contact with elongation complex. What are the two modes of Bacterial RNA polymerase termination Factor Independent and Factor Dependent 3. The leader mRNA of the Salmonella Mg2+ transporter gene mgtA utilizes both of the above mechanisms to regulate Rho-dependent transcription termination. By contrast, in-line probing of an RNA harboring a mutation predicted to disrupt stem-loop C (M1 [G84C G85C C86G], Fig. Epub 2023 Feb 8. bioRxiv. Plasmid pcorL was constructed by cloning a PCR product generated from 14028s genomic DNA with primers 11903 and W574 into the HindIII site of pBAD1844. FOIA The cryo-EM experiments were performed with the Krios G4 microscope at the RIKEN Yokohama Cryo-EM facility. Models to explain Rhos access to RNA-DNA hybrid and release RNA. G. E.Johnson, J. Song E, Hwang S, Munasingha PR, Seo YS, Kang JY, Kang C, Hohng S. Nucleic Acids Res. Dual role of boxB RNA motif in the mechanisms of termination/antitermination at the lambda tR1 terminator revealed in vivo. Epub 2021 Oct 27. Purified Rho protein was incubated with RNA corresponding to the wild-type corA leader mRNA or variants harboring mutations in the pyrimidine-rich regions. S1). 8600 Rockville Pike This site needs JavaScript to work properly. 1A), we predicted that it might harbor a Rho-dependent transcription terminator. 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. 2016 Jun 2;85:319-47. doi: 10.1146/annurev-biochem-060815-014844. Federal government websites often end in .gov or .mil. The bacterial RNA helicase, Rho, is a transcription termination protein that dislodges the elongation complexes. 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). Datsenko KA, Wanner BL. Biochemistry. official website and that any information you provide is encrypted Together, these results imply that corA transcription is inversely correlated with the efficiency of corL translation initiation. Raising the MgCl2 concentration to 3 mM reduced ATPase activity stimulated by all RNAs and returned the M1 and M5 mutants to an asymptotic curve, allowing for better resolution of differences between mutants. subtilis cells. 5A); (ii) a double mutation that restores stem-loop A base pairing potential while disrupting stem-loop C (M1+M1*, Fig. A.McSwiggen, D. G.Bear, P. H.von Hippel. Murayama Y, Ehara H, Aoki M, Goto M, Yokoyama T, Sekine SI. National Library of Medicine 2023 Feb 10;9(6):eade7093. Suppose that there are Rho-dependent terminators within the transcription unit, that is, before the terminator that usually is used. A 5.8- map was reconstructed using ~147,000 particles. Nat Rev Genet. Biochemistry. To initiate synchronized transcription elongation, a 5X chase +/ Rho was added (100 mM Tris HCl (pH 8.0), 100 mM KCl, 0.1mg/ml rifampicin, 1mM NTPs, with 600 nM Rho or an equal volume of Rho buffer). sharing sensitive information, make sure youre on a federal 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. Fig 4. Careers. S3). The transcription start site (TSS) mapped in (B) is denoted with a black arrow. [3] In Escherichia coli, it is a ~274.6 kD hexamer of identical subunits. 10 nM to 10 M dilutions of Rho were prepared in Rho dilution buffer (10 mM Tris-HCL pH 7.9, 100mM KCL, 0.1 mM DTT, 0.1 mM EDTA, 50% v/v glycerol). Data shown correspond to mean values from two independent experiments, and error bars indicate standard deviation. Notably, the architecture of the mgtA leader region creates a situation opposite to canonical polarity; whereas Rho generally aborts transcription of incompletely translated messages, it instead attenuates transcription of mgtA when translation of mgtL is efficient20. Mol Microbiol. In step 1, the amino-terminal domain of Rho factor (blue), which contains the 1 mRNA binding site, binds to the ' rut' sequence of the mRNA transcript. and S.S. Writingreview and editing: Y.M., H.E., and S.S. (D) Western blot of cell lysates prepared from wild-type (14028s) Salmonella or a mutant strain in which the corA leader was replaced by a scar sequence from plasmid pKD4 (MK112). Unauthorized use of these marks is strictly prohibited. the contents by NLM or the National Institutes of Health. 2. Mutation of the corL SD sequence de-repressed corA-lacZ transcription only 25% (M11, Fig. In agreement with this notion, RNAs harboring mutations that disrupt stem-loops C or D (M1 or M5, respectively) stimulated Rhos ATPase activity to a much greater extent than wild-type RNA (Fig. NusA; NusG; Psu; RNA polymerase; Rho; transcription termination. The experiment shown was performed in 1.5 mM MgCl2. 2C). The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. C.Bardeleben, G. A.Kassavetis, E. P.Geiduschek. 2023 Jul 4;14(1):3931. doi: 10.1038/s41467-023-39576-1. (D) Percentage of 5 radiolabeled wild-type corA leader RNA (black) or mutant derivatives (M1, red and M9+M10, blue) bound by Rho and retained following vacuum filtration. MeSH Four sequencing reactions were performed, each in the presence of one ddNTP, using Vent (exo-) DNA polymerase (NEB) according to the manufacturers instructions with plasmid pYS1040FL as the template. sharing sensitive information, make sure youre on a federal Rho utilizes its RNA-dependent ATPase activities to translocate along the mRNA and eventually dislodges the RNAP via an unknown mechanism. R.Kohler, R. A.Mooney, D. J.

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