Quantitative analysis of transcriptional pausing by Escherichia coli RNA polymerase: his leader pause site as paradigm

Landick, Robert; Wang, Daguang; Chan, Cathleen L.

doi:10.1016/s0076-6879(96)74029-6

Cited by 119 publications

(124 citation statements)

References 57 publications

Supporting

Mentioning

119

Contrasting

Order By: Relevance

“…Thus, for the experiments with RfaH-NTD (Fig. 7), we calculated and reported pause strength, 0 , as E/k, where E is the pause efficiency when only one paused state existed, or as (f 1 /k 1 ) ϩ (f 2 /k 2 ) when two paused states existed (35). f 1 and f 2 are the fractions of paused RNAPs that enter the paused states with escape rates k 1 and k 2 (see Fig.…”

Section: Methodsmentioning

confidence: 99%

Antisense Oligonucleotide-stimulated Transcriptional Pausing Reveals RNA Exit Channel Specificity of RNA Polymerase and Mechanistic Contributions of NusA and RfaH

Kolb

Hein

Landick

2014

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Background:In bacterial transcription complexes, nascent RNA structures increase pause duration. Results: Antisense RNA and to a lesser extent DNA oligonucleotides mimic effects of RNA structures synergistically with NusA and competitively with RfaH. Conclusion: Exit channel duplexes interact specifically with RNA polymerase and NusA and compete with RfaH to control clamp position. Significance: Insights into conformational dynamics of RNA polymerase improve mechanistic understanding of transcriptional regulation.

show abstract

Section: Methodsmentioning

confidence: 99%

Antisense Oligonucleotide-stimulated Transcriptional Pausing Reveals RNA Exit Channel Specificity of RNA Polymerase and Mechanistic Contributions of NusA and RfaH

Kolb

Hein

Landick

2014

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…Luo et al (18) similarly failed to demonstrate tRNA Thr -directed antitermination of the B. subtilis thrS gene in vitro. To increase the range of experimental conditions that could be tested, we moved to halted complex assays (15) so that transcription initiation and elongation could be carried out separately under different sets of conditions. Because correct folding of the nascent transcript was likely to be essential to antitermination, we tested the B. subtilis glyQS leader, which (like all glycyl leaders) lacks the complex stem II and stem IIA͞B pseudoknot elements present in most other leaders, including B. subtilis tyrS ( Fig.…”

Section: Methodsmentioning

confidence: 99%

tRNA-mediated transcription antitermination in vitro : Codon–anticodon pairing independent of the ribosome

Grundy

Winkler²,

Henkin³

2002

Proc. Natl. Acad. Sci. U.S.A.

110

138

View full text Add to dashboard Cite

Uncharged tRNA acts as the effector for transcription antitermination of genes in the T box family in Bacillus subtilis and other Gram-positive bacteria. Genetic studies suggested that expression of these genes is induced by stabilization of an antiterminator element in the leader RNA of each target gene by the cognate uncharged tRNA. The specificity of the tRNA response is dependent on a single codon in the leader, which was postulated to pair with the anticodon of the corresponding tRNA. It was not known whether the leader RNA-tRNA interaction requires additional factors. We show here that tRNA-dependent antitermination occurs in vitro in a purified transcription system, in the absence of ribosomes or accessory factors, demonstrating that the RNA-RNA interaction is sufficient to control gene expression by antitermination. The tRNA response exhibits similar specificity in vivo and in vitro, and the antitermination reaction in vitro is independent of NusA and functions with either B. subtilis or Escherichia coli RNA polymerase.T he T box transcription termination control system is widely used in Gram-positive bacteria to regulate expression of aminoacyl-tRNA synthetase, amino acid biosynthesis, and transporter genes (1). The transcripts of genes regulated by this mechanism contain a 200-to 300-nt untranslated leader that includes a factor-independent (intrinsic) transcription termination signal and a competing antiterminator structure (2). Readthrough of the leader region terminator occurs when the charged to uncharged ratio of the corresponding tRNA isoacceptor is decreased, signaling a deficiency in that aminoacyl-tRNA synthetase or amino acid. Sequence comparisons and mutational studies identified a single codon, displayed at a precise position within the leader RNA structure, that determines the specificity of the amino acid response, presumably by pairing with the anticodon of the cognate tRNA (2). The acceptor end of uncharged tRNA apparently makes a second interaction with a bulged region in the antiterminator, based on genetic analyses as well as gel-shift assays using a model 39-nt antiterminator RNA (3, 4); this interaction is proposed to stabilize the antiterminator, preventing the formation of a competing terminator helix (Fig. 1). Mutational studies of the Bacillus subtilis tyrS leader and tRNA Tyr indicated that read-through requires additional conserved features of both RNAs (5-7).Although it has been established that a tRNA is essential for antitermination, it was not known whether other factors are also required to mediate the tRNA-leader interaction. There was no evidence of translation of the leader RNA (2, 3, 8), but in vivo assays could not rule out the possibility that the tRNA-leader interaction occurs within the context of a ribosome. In this study, we demonstrate specific tRNA Gly -dependent antitermination of the Bacillus subtilis glyQS leader in a purified in vitro transcription system. Materials and MethodsBacterial Strains and Growth Conditions. B. subtilis strain BR151MA (lys-3 trp...

show abstract

“…Prominent transcripts are opsP (pause RNA transcript; 62 and 64 nt) and RO (run-off RNA transcript). The fractions of paused RNA were plotted against time; pause half-lives and pause efficiencies for the major pause site (64 nt) were determined as described under "Experimental Procedures" (25).…”

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

confidence: 99%

“…Gels were dried, and the RNA products were visualized and quantified using PhosphorImager screens and ImageQuant Software (Amersham Biosciences). Pause half-life (time during which half of the complexes re-enter the elongation pathway) and pause efficiency (fraction of transcribing RNAP molecules that pause) were determined by nonlinear regression analysis as described (25). Termination efficiencies were calculated as described by Reynolds et al (15).…”

mentioning

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

Self Cite

116

View full text Add to dashboard Cite

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 ...

show abstract

Quantitative analysis of transcriptional pausing by Escherichia coli RNA polymerase: his leader pause site as paradigm

Cited by 119 publications

References 57 publications

Antisense Oligonucleotide-stimulated Transcriptional Pausing Reveals RNA Exit Channel Specificity of RNA Polymerase and Mechanistic Contributions of NusA and RfaH

Antisense Oligonucleotide-stimulated Transcriptional Pausing Reveals RNA Exit Channel Specificity of RNA Polymerase and Mechanistic Contributions of NusA and RfaH

tRNA-mediated transcription antitermination in vitro : Codon–anticodon pairing independent of the ribosome

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

Contact Info

Product

Resources

About