scholarly journals NusG inhibits RNA polymerase backtracking by stabilizing the minimal transcription bubble

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Matti Turtola ◽  
Georgiy A Belogurov
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Transcription Elongation ◽  
Single Nucleotide ◽  
E Coli ◽  
Transcription Bubble ◽  
Nucleotide Addition ◽  
Nascent Rna ◽  
Comprehensive Mapping

Universally conserved factors from NusG family bind at the upstream fork junction of transcription elongation complexes and modulate RNA synthesis in response to translation, processing, and folding of the nascent RNA. Escherichia coli NusG enhances transcription elongation in vitro by a poorly understood mechanism. Here we report that E. coli NusG slows Gre factor-stimulated cleavage of the nascent RNA, but does not measurably change the rates of single nucleotide addition and translocation by a non-paused RNA polymerase. We demonstrate that NusG slows RNA cleavage by inhibiting backtracking. This activity is abolished by mismatches in the upstream DNA and is independent of the gate and rudder loops, but is partially dependent on the lid loop. Our comprehensive mapping of the upstream fork junction by base analogue fluorescence and nucleic acids crosslinking suggests that NusG inhibits backtracking by stabilizing the minimal transcription bubble.

scholarly journals Transcription of bacteriophage T4 deoxyribonucleic acid in vitro

1969 ◽  
Vol 115 (3) ◽  
pp. 353-361 ◽  
Author(s):  
John O. Bishop ◽  
Forbes W. Robertson
Keyword(s):  
Rna Polymerase ◽  
Messenger Rna ◽  
Rna Synthesis ◽  
Deoxyribonucleic Acid ◽  
Bacteriophage T4 ◽  
Rna Sequences ◽  
Experimental Conditions ◽  
E Coli ◽  
New Methods

1. RNA was synthesized in vitro from a template of bacteriophage T4 DNA, in the presence of Mn2+. A comparison was made of the RNA synthesized by purified RNA polymerase from two sources, Micrococcus lysodeikticus and Escherichia coli; these are referred to as Micrococcus cRNA and E. coli cRNA respectively (where cRNA indicates RNA synthesized in vitro by using purified RNA polymerase and a DNA primer). 2. Both types of RNA were self-complementary as judged by resistance to digestion with ribonuclease after self-annealing, Micrococcus cRNA being more self-complementary (40%) than was E. coli cRNA (30%). The cRNA was found to be much less self-complementary if Mg2+ was present during RNA synthesis instead of Mn2+. 3. Micrococcus cRNA hybridized with a larger part of bacteriophage T4 DNA than did E. coli cRNA. The E. coli cRNA competed with only part (70%) of the Micrococcus cRNA in hybridization-competition experiments. It is concluded that more sequences of bacteriophage T4 DNA are transcribed by Micrococcus polymerase than by E. coli polymerase. 4. The RNA sequences synthesized by Micrococcus RNA polymerase but not by E. coli RNA polymerase are shown by hybridization competition to compete with specifically late bacteriophage T4 messenger RNA sequences. The relevance of this finding to the control of transcription is discussed. 5. In an Appendix, new methods are described for the analysis of hybridization-saturation and -competition experiments. Particular attention is paid to the effects produced if different RNA sequences are present at different relative concentrations. 6. By using cRNA isolated from an enzymically synthesized DNA–RNA hybrid, it is estimated that, of the DNA that is complementary to cRNA, only about half can become hybridized with cRNA under the experimental conditions used.

scholarly journals Asymmetric RNA synthesis in vitro: heterologous DNA-enzyme systems; E. coli RNA polymerase.

1965 ◽  
Vol 53 (5) ◽  
pp. 1140-1147 ◽  
Author(s):  
A. J. Colvill ◽  
L. C. Kanner ◽  
G. P. Tocchini-Valentini ◽  
M. T. Sarnat ◽  
E. P. Geiduschek
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
E Coli ◽  
Enzyme Systems

scholarly journals Purification of synchronized E. coli transcription elongation complexes by reversible immobilization on magnetic beads

2022 ◽  
Author(s):  
Skyler L. Kelly ◽  
Courtney E. Szyjka ◽  
Eric J. Strobel
Keyword(s):  
Rna Polymerase ◽  
High Performance ◽  
Magnetic Beads ◽  
Solid Phase ◽  
Transcription Elongation ◽  
E Coli ◽  
First Occurrence ◽  
Transcription Antitermination ◽  
Dna Strand

Synchronized transcription elongation complexes (TECs) are a fundamental tool for in vitro studies of transcription and RNA folding. Transcription elongation can be synchronized by omitting one or more NTPs from an in vitro transcription reaction so that RNA polymerase can only transcribe to the first occurrence of the omitted nucleotide(s) in the coding DNA strand. This approach was developed over four decades ago and has been applied extensively in biochemical investigations of RNA polymerase enzymes, but has not been optimized for RNA-centric assays. In this work, we describe the development of a system for isolating synchronized TECs from an in vitro transcription reaction. Our approach uses a custom 5′ leader sequence, called C3-SC1, to reversibly capture synchronized TECs on magnetic beads. We first show that complexes isolated by this procedure, called C3-SC1TECs, are >95% pure, >98% active, highly synchronous (94% of complexes chase in <15s upon addition of saturating NTPs), and compatible with solid-phase transcription; the yield of this purification is ~8%. We then show that C3-SC1TECs perturb, but do not interfere with, the function of ZTP-sensing and ppGpp-sensing transcriptional riboswitches. For both riboswitches, transcription using C3-SC1TECs improved the efficiency of transcription termination in the absence of ligand but did not inhibit ligand-induced transcription antitermination. Given these properties, C3-SC1TECs will likely be useful for developing biochemical and biophysical RNA assays that require high-performance, quantitative bacterial in vitro transcription.

scholarly journals Mycobacterial HelD is a nucleic acids-clearing factor for RNA polymerase

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Tomáš Kouba ◽  
Tomáš Koval’ ◽  
Petra Sudzinová ◽  
Jiří Pospíšil ◽  
Barbora Brezovská ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Rna Polymerase ◽  
Active Site ◽  
Mycobacterium Smegmatis ◽  
Rna Synthesis ◽  
Environmental Changes ◽  
Transcription Elongation ◽  
Specific Interactions ◽  
Inactive State ◽  
Transcription Machinery

AbstractRNA synthesis is central to life, and RNA polymerase (RNAP) depends on accessory factors for recovery from stalled states and adaptation to environmental changes. Here, we investigated the mechanism by which a helicase-like factor HelD recycles RNAP. We report a cryo-EM structure of a complex between the Mycobacterium smegmatis RNAP and HelD. The crescent-shaped HelD simultaneously penetrates deep into two RNAP channels that are responsible for nucleic acids binding and substrate delivery to the active site, thereby locking RNAP in an inactive state. We show that HelD prevents non-specific interactions between RNAP and DNA and dissociates stalled transcription elongation complexes. The liberated RNAP can either stay dormant, sequestered by HelD, or upon HelD release, restart transcription. Our results provide insights into the architecture and regulation of the highly medically-relevant mycobacterial transcription machinery and define HelD as a clearing factor that releases RNAP from nonfunctional complexes with nucleic acids.

scholarly journals Single-nucleotide control of tRNA folding cooperativity under near-cellular conditions

2019 ◽  
Vol 116 (46) ◽  
pp. 23075-23082
Author(s):  
Kathleen A. Leamy ◽  
Ryota Yamagami ◽  
Neela H. Yennawar ◽  
Philip C. Bevilacqua
Keyword(s):  
Rna Folding ◽  
3D Structure ◽  
Single Nucleotide ◽  
Transfer Rnas ◽  
E Coli ◽  
Posttranscriptional Processing ◽  
Structural Levels ◽  
Trna Folding ◽  
Precursor Transcript

RNA folding is often studied by renaturing full-length RNA in vitro and tracking folding transitions. However, the intracellular transcript folds as it emerges from the RNA polymerase. Here, we investigate the folding pathways and stability of numerous late-transcriptional intermediates of yeast and Escherichia coli transfer RNAs (tRNAs). Transfer RNA is a highly regulated functional RNA that undergoes multiple steps of posttranscriptional processing and is found in very different lengths during its lifetime in the cell. The precursor transcript is extended on both the 5′ and 3′ ends of the cloverleaf core, and these extensions get trimmed before addition of the 3′-CCA and aminoacylation. We studied the thermodynamics and structures of the precursor tRNA and of late-transcriptional intermediates of the cloverleaf structure. We examined RNA folding at both the secondary and tertiary structural levels using multiple biochemical and biophysical approaches. Our findings suggest that perhaps nature has selected for a single-base addition to control folding to the functional 3D structure. In near-cellular conditions, yeast tRNAPhe and E. coli tRNAAla transcripts fold in a single, cooperative transition only when nearly all of the nucleotides in the cloverleaf are transcribed by indirectly enhancing folding cooperativity. Furthermore, native extensions on the 5′ and 3′ ends do not interfere with cooperative core folding. This highly controlled cooperative folding has implications for recognition of tRNA by processing and modification enzymes and quality control of tRNA in cells.

scholarly journals Role of the 3′ tRNA-Like Structure in Tobacco Mosaic Virus Minus-Strand RNA Synthesis by the Viral RNA-Dependent RNA Polymerase In Vitro

2000 ◽  
Vol 74 (24) ◽  
pp. 11671-11680 ◽  
Author(s):  
T. A. M. Osman ◽  
C. L. Hemenway ◽  
K. W. Buck
Keyword(s):  
Rna Polymerase ◽  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Untranslated Region ◽  
Rna Synthesis ◽  
Central Core ◽  
Minus Strand ◽  
Stem Loop ◽  
Polymerase Binding

ABSTRACT A template-dependent RNA polymerase has been used to determine the sequence elements in the 3′ untranslated region of tobacco mosaic virus RNA that are required for promotion of minus-strand RNA synthesis and binding to the RNA polymerase in vitro. Regions which were important for minus-strand synthesis were domain D1, which is equivalent to a tRNA acceptor arm; domain D2, which is similar to a tRNA anticodon arm; an upstream domain, D3; and a central core, C, which connects domains D1, D2, and D3 and determines their relative orientations. Mutational analysis of the 3′-terminal 4 nucleotides of domain D1 indicated the importance of the 3′-terminal CA sequence for minus-strand synthesis, with the sequence CCCA or GGCA giving the highest transcriptional efficiency. Several double-helical regions, but not their sequences, which are essential for forming pseudoknot and/or stem-loop structures in domains D1, D2, and D3 and the central core, C, were shown to be required for high template efficiency. Also important were a bulge sequence in the D2 stem-loop and, to a lesser extent, a loop sequence in a hairpin structure in domain D1. The sequence of the 3′ untranslated region upstream of domain D3 was not required for minus-strand synthesis. Template-RNA polymerase binding competition experiments showed that the highest-affinity RNA polymerase binding element region lay within a region comprising domain D2 and the central core, C, but domains D1 and D3 also bound to the RNA polymerase with lower affinity.

scholarly journals Interactions between RNA polymerase and the core recognition element are a determinant of transcription start site selection

2016 ◽  
Vol 113 (21) ◽  
pp. E2899-E2905 ◽  
Author(s):  
Irina O. Vvedenskaya ◽  
Hanif Vahedian-Movahed ◽  
Yuanchao Zhang ◽  
Deanne M. Taylor ◽  
Richard H. Ebright ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Transcription Start Site ◽  
Transcription Initiation ◽  
Specific Protein ◽  
Start Site ◽  
Transcription Start ◽  
Transcription Bubble ◽  
Recognition Element

During transcription initiation, RNA polymerase (RNAP) holoenzyme unwinds ∼13 bp of promoter DNA, forming an RNAP-promoter open complex (RPo) containing a single-stranded transcription bubble, and selects a template-strand nucleotide to serve as the transcription start site (TSS). In RPo, RNAP core enzyme makes sequence-specific protein–DNA interactions with the downstream part of the nontemplate strand of the transcription bubble (“core recognition element,” CRE). Here, we investigated whether sequence-specific RNAP–CRE interactions affect TSS selection. To do this, we used two next-generation sequencing-based approaches to compare the TSS profile of WT RNAP to that of an RNAP derivative defective in sequence-specific RNAP–CRE interactions. First, using massively systematic transcript end readout, MASTER, we assessed effects of RNAP–CRE interactions on TSS selection in vitro and in vivo for a library of 47 (∼16,000) consensus promoters containing different TSS region sequences, and we observed that the TSS profile of the RNAP derivative defective in RNAP–CRE interactions differed from that of WT RNAP, in a manner that correlated with the presence of consensus CRE sequences in the TSS region. Second, using 5′ merodiploid native-elongating-transcript sequencing, 5′ mNET-seq, we assessed effects of RNAP–CRE interactions at natural promoters in Escherichia coli, and we identified 39 promoters at which RNAP–CRE interactions determine TSS selection. Our findings establish RNAP–CRE interactions are a functional determinant of TSS selection. We propose that RNAP–CRE interactions modulate the position of the downstream end of the transcription bubble in RPo, and thereby modulate TSS selection, which involves transcription bubble expansion or transcription bubble contraction (scrunching or antiscrunching).

scholarly journals Factors Required for the Uridylylation of the Foot-and-Mouth Disease Virus 3B1, 3B2, and 3B3 Peptides by the RNA-Dependent RNA Polymerase (3Dpol) In Vitro

2005 ◽  
Vol 79 (12) ◽  
pp. 7698-7706 ◽  
Author(s):  
Arabinda Nayak ◽  
Ian G. Goodfellow ◽  
Graham J. Belsham
Keyword(s):  
Rna Polymerase ◽  
Disease Virus ◽  
Rna Synthesis ◽  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Coding Region ◽  
Mouth Disease Virus ◽  
Foot And Mouth ◽  
Positive Strand Rna

ABSTRACT The 5′ terminus of picornavirus genomic RNA is covalently linked to the virus-encoded peptide 3B (VPg). Foot-and-mouth disease virus (FMDV) is unique in encoding and using 3 distinct forms of this peptide. These peptides each act as primers for RNA synthesis by the virus-encoded RNA polymerase 3Dpol. To act as the primer for positive-strand RNA synthesis, the 3B peptides have to be uridylylated to form VPgpU(pU). For certain picornaviruses, it has been shown that this reaction is achieved by the 3Dpol in the presence of the 3CD precursor plus an internal RNA sequence termed a cis-acting replication element (cre). The FMDV cre has been identified previously to be within the 5′ untranslated region, whereas all other picornavirus cre structures are within the viral coding region. The requirements for the in vitro uridylylation of each of the FMDV 3B peptides has now been determined, and the role of the FMDV cre (also known as the 3B-uridylylation site, or bus) in this reaction has been analyzed. The poly(A) tail does not act as a significant template for FMDV 3B uridylylation.

Sign in / Sign up

Export Citation Format

Share Document