scholarly journals Mechanism of transcription initiation and promoter escape byE.coliRNA polymerase

2017 ◽  
Vol 114 (15) ◽  
pp. E3032-E3040 ◽  
Author(s):  
Kate L. Henderson ◽  
Lindsey C. Felth ◽  
Cristen M. Molzahn ◽  
Irina Shkel ◽  
Si Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Free Energy ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Initial Phase ◽  
Rna Synthesis ◽  
Quantitative Model ◽  
Ternary Complexes ◽  
Promoter Escape ◽  
Abortive Cycling

To investigate roles of the discriminator and open complex (OC) lifetime in transcription initiation byEscherichia coliRNA polymerase (RNAP; α2ββ’ωσ70), we compare productive and abortive initiation rates, short RNA distributions, and OC lifetime for the λPRand T7A1 promoters and variants with exchanged discriminators, all with the same transcribed region. The discriminator determines the OC lifetime of these promoters. Permanganate reactivity of thymines reveals that strand backbones in open regions of long-lived λPR-discriminator OCs are much more tightly held than for shorter-lived T7A1-discriminator OCs. Initiation from these OCs exhibits two kinetic phases and at least two subpopulations of ternary complexes. Long RNA synthesis (constrained to be single round) occurs only in the initial phase (<10 s), at similar rates for all promoters. Less than half of OCs synthesize a full-length RNA; the majority stall after synthesizing a short RNA. Most abortive cycling occurs in the slower phase (>10 s), when stalled complexes release their short RNA and make another without escaping. In both kinetic phases, significant amounts of 8-nt and 10-nt transcripts are produced by longer-lived, λPR-discriminator OCs, whereas no RNA longer than 7 nt is produced by shorter-lived T7A1-discriminator OCs. These observations and the lack of abortive RNA in initiation from short-lived ribosomal promoter OCs are well described by a quantitative model in which ∼1.0 kcal/mol of scrunching free energy is generated per translocation step of RNA synthesis to overcome OC stability and drive escape. The different length-distributions of abortive RNAs released from OCs with different lifetimes likely play regulatory roles.

scholarly journals Micro-analysis of pure deoxyribonucleic acid-dependent ribonucleic acid polymerase from Escherichia coli. Action of heparin and rifampicin on structure and function

1970 ◽  
Vol 117 (3) ◽  
pp. 623-631 ◽  
Author(s):  
Volker Neuhoff ◽  
Wolf-Bernhard Schill ◽  
Hans Sternbach
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
Deoxyribonucleic Acid ◽  
Structure And Function ◽  
Disc Electrophoresis ◽  
Inhibitory Mechanism ◽  
And Function ◽  
Micro Analysis ◽  
Template Complex

By using micro disc electrophoresis and micro-diffusion techniques, the interaction of pure DNA-dependent RNA polymerase (EC 2.7.7.6) from Escherichia coli with the template, the substrates and the inhibitors heparin and rifampicin was investigated. The following findings were obtained: (1) heparin converts the 24S and 18S particles of the polymerase into the 13S form; (2) heparin inhibits RNA synthesis by dissociating the enzyme–template complex; (3) rifampicin does not affect the attachment of heparin to the enzyme; (4) the substrates ATP and UTP are bound by enzyme loaded with rifampicin; (5) rifampicin is bound by an enzyme–template complex to the same extent as by an RNA-synthesizing enzyme–template complex. From this it is concluded that the mechanism of the inhibition of RNA synthesis by rifampicin is radically different from that by heparin. As a working hypothesis to explain the inhibitory mechanism of rifampicin, it is assumed that it becomes very firmly attached to a position close to the synthesizing site and only blocks this when no synthesis is in progress.

scholarly journals Interactions between the Escherichia coli cAMP receptor protein and the C-terminal domain of the α subunit of RNA polymerase at Class I promoters

1999 ◽  
Vol 337 (3) ◽  
pp. 415-423 ◽  
Author(s):  
Emma C. LAW ◽  
Nigel J. SAVERY ◽  
Stephen J. W. BUSBY
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Class I ◽  
Receptor Protein ◽  
Camp Receptor Protein ◽  
Α Subunit ◽  
Terminal Domain ◽  
Up Elements ◽  
Camp Receptor

The Escherichia coli cAMP receptor protein (CRP) is a factor that activates transcription at over 100 target promoters. At Class I CRP-dependent promoters, CRP binds immediately upstream of RNA polymerase and activates transcription by making direct contacts with the C-terminal domain of the RNA polymerase α subunit (αCTD). Since αCTD is also known to interact with DNA sequence elements (known as UP elements), we have constructed a series of semi-synthetic Class I CRP-dependent promoters, carrying both a consensus DNA-binding site for CRP and a UP element at different positions. We previously showed that, at these promoters, the CRP–αCTD interaction and the CRP–UP element interaction contribute independently and additively to transcription initiation. In this study, we show that the two halves of the UP element can function independently, and that, in the presence of the UP element, the best location for the DNA site for CRP is position -69.5. This suggests that, at Class I CRP-dependent promoters where the DNA site for CRP is located at position -61.5, the two αCTDs of RNA polymerase are not optimally positioned. Two experiments to test this hypothesis are presented.

scholarly journals Effects of distortions by A-tracts of promoter B-DNA spacer region on the kinetics of open complex formation by Escherichia coli RNA polymerase.

2003 ◽  
Vol 50 (4) ◽  
pp. 909-920 ◽  
Author(s):  
Iwona K Kolasa ◽  
Tomasz Łoziński ◽  
Kazimierz L Wierzchowski
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Specific Interaction ◽  
Structural Data ◽  
Structural Morphology ◽  
Promoter Dna ◽  
Sigma Subunit ◽  
Kinetics Of

A-tracts in DNA due to their structural morphology distinctly different from the canonical B-DNA form play an important role in specific recognition of bacterial upstream promoter elements by the carboxyl terminal domain of RNA polymerase alpha subunit and, in turn, in the process of transcription initiation. They are only rarely found in the spacer promoter regions separating the -35 and -10 recognition hexamers. At present, the nature of the protein-DNA contacts formed between RNA polymerase and promoter DNA in transcription initiation can only be inferred from low resolution structural data and mutational and crosslinking experiments. To probe these contacts further, we constructed derivatives of a model Pa promoter bearing in the spacer region one or two An (n = 5 or 6) tracts, in phase with the DNA helical repeat, and studied the effects of thereby induced perturbation of promoter DNA structure on the kinetics of open complex (RPo) formation in vitro by Escherichia coli RNA polymerase. We found that the overall second-order rate constant ka of RPo formation, relative to that at the control promoter, was strongly reduced by one to two orders of magnitude only when the A-tracts were located in the nontemplate strand. A particularly strong 30-fold down effect on ka was exerted by nontemplate A-tracts in the -10 extended promoter region, where an involvement of nontemplate TG (-14, -15) sequence in a specific interaction with region 3 of sigma-subunit is postulated. A-tracts in the latter location caused also 3-fold slower isomerization of the first closed transcription complex into the intermediate one that precedes formation of RPo, and led to two-fold faster dissociation of the latter. All these findings are discussed in relation to recent structural and kinetic models of RPo formation.

A mutation in the RNA polymerase β′ subunit causing depressed ribosomal RNA synthesis in Escherichia coli

1981 ◽  
Vol 183 (1) ◽  
pp. 54-58 ◽  
Author(s):  
Ben A. Oostra ◽  
Klaas Kok ◽  
Adri J. Van Vliet ◽  
AB Geert ◽  
Max Gruber
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Ribosomal Rna ◽  
Rna Synthesis ◽  
Β Subunit

scholarly journals Cryo-EM structures reveal transcription initiation steps by yeast mitochondrial RNA polymerase

2020 ◽  
Author(s):  
Brent De Wijngaert ◽  
Shemaila Sultana ◽  
Chhaya Dharia ◽  
Hans Vanbuel ◽  
Jiayu Shen ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Large Scale ◽  
Rna Synthesis ◽  
Mitochondrial Rna ◽  
Initiation Complex ◽  
Mitochondrial Rna Polymerase ◽  
Catalytically Active ◽  
Promoter Melting ◽  
Antiviral Nucleosides

Cryo-EM structures of transcription pre-initiation complex (PIC) and initiation complex (IC) of yeast mitochondrial RNA polymerase show fully resolved transcription bubbles and explain promoter melting, template alignment, DNA scrunching, transition into elongation, and abortive synthesis. Promoter melting initiates in PIC with MTF1 trapping the −4 to −2 non-template (NT) bases in its NT-groove. Transition to IC is marked by a large-scale movement that aligns the template with RNA at the active site. RNA synthesis scrunches the NT strand into an NT-loop, which interacts with centrally positioned MTF1 C-tail. Steric clashes of the C-tail with RNA:DNA and NT-loop, and dynamic scrunching-unscrunching of DNA explain abortive synthesis and transition into elongation. Capturing the catalytically active IC-state with UTPαS poised for incorporation enables modeling toxicity of antiviral nucleosides/nucleotides.

Action of Trifluralin on Chromatin Activity in Corn and Soybean

Weed Science ◽  
1972 ◽  
Vol 20 (4) ◽  
pp. 364-366 ◽  
Author(s):  
Donald Penner ◽  
Roy W. Early
Keyword(s):  
Escherichia Coli ◽  
Zea Mays ◽  
Glycine Max ◽  
Rna Polymerase ◽  
Melting Temperature ◽  
Ribonucleic Acid ◽  
Rna Synthesis ◽  
Subsequent Reduction

Trifluralin (α,α,α-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine) at 10−5M applied to etiolated corn(Zea maysL. ‘Michigan 500′) seedlings 6 or 12 hr before the isolation of chromatin from the roots markedly reduced ribonucleic acid (RNA) synthesis supported by the chromatin. The addition ofEscherichia coliRNA polymerase failed to overcome the inhibition. Trifluralin increased the melting temperature of the chromatin. The presence of trifluralin during the isolation and reaction procedure inhibited RNA synthesis indicating possible trifluralin binding to the chromatin with subsequent reduction of template availability for transcription. Trifluralin did not inhibit chromatin activity in soybean [Glycine max(L.) Merr. ‘Hark’] seedlings.

scholarly journals CarD and RbpA modify the kinetics of initial transcription and slow promoter escape of the Mycobacterium tuberculosis RNA polymerase

2019 ◽  
Vol 47 (13) ◽  
pp. 6685-6698 ◽  
Author(s):  
Drake Jensen ◽  
Ana Ruiz Manzano ◽  
Jayan Rammohan ◽  
Christina L Stallings ◽  
Eric A Galburt
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Complex Formation ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Promoter Escape ◽  
Rate Limiting Step ◽  
Nucleotide Incorporation ◽  
Transcriptional Regulatory ◽  
Open Complex Formation ◽  
Transcriptional Regulatory Mechanisms

Abstract The pathogen Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, enacts unique transcriptional regulatory mechanisms when subjected to host-derived stresses. Initiation of transcription by the Mycobacterial RNA polymerase (RNAP) has previously been shown to exhibit different open complex kinetics and stabilities relative to Escherichia coli (Eco) RNAP. However, transcription initiation rates also depend on the kinetics following open complex formation such as initial nucleotide incorporation and subsequent promoter escape. Here, using a real-time fluorescence assay, we present the first in-depth kinetic analysis of initial transcription and promoter escape for the Mtb RNAP. We show that in relation to Eco RNAP, Mtb displays slower initial nucleotide incorporation but faster overall promoter escape kinetics on the Mtb rrnAP3 promoter. Furthermore, in the context of the essential transcription factors CarD and RbpA, Mtb promoter escape is slowed via differential effects on initially transcribing complexes. Finally, based on their ability to increase the rate of open complex formation and decrease the rate of promoter escape, we suggest that CarD and RbpA are capable of activation or repression depending on the rate-limiting step of a given promoter's basal initiation kinetics.

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