Mutations in and Monoclonal Antibody Binding to Evolutionary Hypervariable Region of Escherichia coli RNA Polymerase β′ Subunit Inhibit Transcript Cleavage and Transcript Elongation

Zakharova, Natalya; Bass, I. A.; Arsenieva, E. L.; Nikiforov, Vadim; Severinov, Konstantin

doi:10.1074/jbc.273.38.24912

Cited by 51 publications

(59 citation statements)

References 40 publications

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“…2). Other findings implicate segment G directly in transcript elongation, transcriptional arrest, and transcript cleavage (17,18,34,35). Interestingly, the location and nature of rpoC931 correspond exactly to the rpb1-502 mutation isolated by Hekmatpanah and Young (12) in the Saccharomyces cerevisiae RNAP II large-subunit gene RPB1.…”

Section: Discussionmentioning

confidence: 96%

Escherichia coli RNA Polymerase Is the Target of the Cyclopeptide Antibiotic Microcin J25

et al. 2001

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Section: Discussionmentioning

confidence: 96%

Escherichia coli RNA Polymerase Is the Target of the Cyclopeptide Antibiotic Microcin J25

et al. 2001

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“…To determine transcription elongation rates, elongation complexes stalled at position ϩ20 (EC 20 ) were prepared in 50-l reaction mixtures containing 40 mM Tris-HCl (pH 7.9), 40 mM KCl, 10 mM MgCl 2 , 20 nM DNA fragment containing the T7 A1 promoter fused to the tR2 terminator (15), 50 nM RNAP, 0.05 mM CpApUpC, 50 M (each) ATP and GTP, and 2.5 M [␣-…”

Section: Methodsmentioning

confidence: 99%

“…Having established conditions for promoter-specific transcription initiation by T. aquaticus RNAP, we wished to compare the transcription elongation, transcription termination, and transcript cleavage properties of T. aquaticus RNAP with those of the prototypic E. coli enzyme. Immobilized RNAP was used to obtain stalled elongation complexes containing 20-mer RNA on the T7 A1 promoter-containing DNA fragment (15). Purified elongation complexes were incubated in the presence of different concentrations of NTP, to monitor the transcription elongation rate and transcription termination on the rhoindependent tR2 terminator located downstream.…”

Section: Vol 183 2001mentioning

confidence: 99%

Recombinant Thermus aquaticus RNA Polymerase, a New Tool for Structure-Based Analysis of Transcription

et al. 2001

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The three-dimensional structure of DNA-dependent RNA polymerase (RNAP) from thermophilic Thermus aquaticus has recently been determined at 3.3 Å resolution. Currently, very little is known about T. aquaticus transcription and no genetic system to study T. aquaticus RNAP genes is available. To overcome these limitations, we cloned and overexpressed T. aquaticus RNAP genes in Escherichia coli. Overproduced T. aquaticus RNAP subunits assembled into functional RNAP in vitro and in vivo when coexpressed in E. coli. We used the recombinant T. aquaticus enzyme to demonstrate that transcription initiation, transcription termination, and transcription cleavage assays developed for E. coli RNAP can be adapted to study T. aquaticus transcription. However, T. aquaticus RNAP differs from the prototypical E. coli enzyme in several important ways: it terminates transcription less efficiently, has exceptionally high rate of intrinsic transcript cleavage, and is highly resistant to rifampin. Our results, together with the high-resolution structural information, should now allow a rational analysis of transcription mechanism by mutation.Most bacterial RNA polymerase (RNAP) core enzymes consist of four core subunits (␤Ј, ␤, and a dimer of identical ␣ subunits). Binding of one of the several factors converts the core into the holoenzyme, which is able to initiate transcription from promoters. In vitro activity of several bacterial RNAPs can be recovered after separation of individual subunits in the presence of denaturing agents and subsequent mixing of the subunits and dialysis under controlled conditions (19). In vitro reconstitution of Escherichia coli RNAP from cloned and individually overexpressed and purified subunits provides a means of obtaining RNAP harboring lethal mutations in quantities sufficient for biochemical analyses (16). Structure-function studies of reconstituted recombinant E. coli RNAP mutants have provided crucial insights into the transcription mechanism and regulation and are primarily responsible for our increased understanding of this enzyme (see, for example, references 6, 8, and 17).Recent structural analysis of RNAP from Thermus aquaticus provided the first tantalizing view of the bacterial RNAP core enzyme structure at 3.3 Å resolution (18). The availability of a relatively high-resolution structure qualitatively raises the importance of a rational structure-function analysis of RNAP. Despite obvious homology between RNAPs from T. aquaticus and E. coli, it would be highly desirable to perform mutational and structural studies using T. aquaticus RNAP rather than the better-studied E. coli counterpart. The important advantages of studying T. aquaticus RNAP include the ability to reduce assembly effects of mutations to a minimum and the ability to perform structural analysis of mutants. The disadvantages of the T. aquaticus system are the lack of functional assays, the general absence of data on gene transcription in this organism, and, more importantly, the inability to manipulate T. aquaticus RNAP subu...

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“…The sequence in this region folds into a threestranded antiparallel ␤-sheet that cross-links to the nontemplate DNA strand near ϩ15 (3), is variable in sequence among bacterial RNAPs (Fig. 1A), and lies near an insertion of 187 aa that is present only in some bacteria (44). Gly-1161, however, is invariant among bacterial RNAPs and occurs at a turn leading to the first ␤ strand.…”

Section: Jaw Mutant Rnaps Exhibit Lesser Effects On Tec Stability Andmentioning

confidence: 99%

The Downstream DNA Jaw of Bacterial RNA Polymerase Facilitates Both Transcriptional Initiation and Pausing

Ederth¹,

Artsimovitch²,

Isaksson³

et al. 2002

Journal of Biological Chemistry

116

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Regulation of RNA polymerase during initiation, elongation, and termination of transcription is mediated in part by interactions with intrinsic regulatory signals encoded in the RNA and DNA that contact the enzyme. These interactions include contacts to an 8 -9-bp RNA: DNA hybrid within the active-site cleft of the enzyme, contacts to the melted nontemplate DNA strand in the vicinity of the hybrid, contacts to exiting RNA upstream of the hybrid, and contacts to ϳ20 bp of duplex DNA downstream of the active site. Based on characterization of an amino acid substitution (G1161R) and a deletion (⌬1149 -1190) in the jaw domain of the bacterial RNA polymerase largest subunit (␤), we report here that contacts of the jaw domain to downstream DNA at the leading edge of the transcription complex contribute to regulation during all three phases of transcription. The results provide insight into the role of the jaw domain-downstream DNA contact in transcriptional initiation and pausing and suggest possible explanations for the previously reported isolation of the jaw mutants based on reduced ColEI plasmid replication. Cellular, multisubunit RNA polymerases (RNAPs) 1 participate in a complex cycle of conformational changes to initiate, elongate, and terminate RNA transcripts. Each step in this cycle is mediated by a network of protein-nucleic acid interactions composed of interconnected parts of RNAP that contact DNA and product RNA (1-9). During elongation, most nucleic acid contacts are made by two large subunits of similar structure and sequence in prokaryotic and eukaryotic RNAPs, called ␤Ј and ␤ in bacteria or RPB1 and RPB2 in eukaryotes. During initiation, these contacts are supplemented by sequence-specific DNA contacts made by auxiliary initiation factors ( in bacteria) that mediate promoter engagement.In both initiation and elongation complexes, a key component in this protein-nucleic acid interaction network occurs between ϳ20 bp of duplex DNA downstream of the polymerization site and a channel in RNAP composed of a trough formed mostly by ␤Ј(RPB1) and a cover formed by the lobe domain of ␤(RPB2). During promoter engagement, establishment of this contact is coupled to formation of the ϳ15 bp melted transcription bubble and insertion of the template DNA strand into the active site of RNAP (Refs. 1 and 10, and references therein). Upon promoter escape, when RNAP forms a transcription elongation complex (TEC), the downstream contact persists and participates in the response of RNAP to pause, arrest, and termination signals (11-16).The downstream DNA interaction, which stretches from the position of duplex melting 1-3 nt in front of the catalytic center to ϳ20 bp further downstream, can be subdivided into activesite proximal and active-site distal sets of contacts (Fig. 1, B and C). The active-site proximal set of contacts is made at ϩ5 to ϩ8 by the lobe and domain called the clamp (formed mostly by ␤Ј), both of which can move relative to the central core of the enzyme (2, 8, 9). The active-site distal set of contacts ...

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Mutations in and Monoclonal Antibody Binding to Evolutionary Hypervariable Region of Escherichia coli RNA Polymerase β′ Subunit Inhibit Transcript Cleavage and Transcript Elongation

Cited by 51 publications

References 40 publications

Escherichia coli RNA Polymerase Is the Target of the Cyclopeptide Antibiotic Microcin J25

Escherichia coli RNA Polymerase Is the Target of the Cyclopeptide Antibiotic Microcin J25

Recombinant Thermus aquaticus RNA Polymerase, a New Tool for Structure-Based Analysis of Transcription

The Downstream DNA Jaw of Bacterial RNA Polymerase Facilitates Both Transcriptional Initiation and Pausing

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