scholarly journals Kinetic-Mechanistic Evidence for Which E. coli RNA Polymerase-λPR Open Promoter Complex Initiates and for Stepwise Disruption of Contacts in Bubble Collapse

2020 ◽  
Author(s):  
Dylan Plaskon ◽  
Kate Henderson ◽  
Lindsey Felth ◽  
Cristen Molzahn ◽  
Claire Evensen ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rate Constants ◽  
Transcription Initiation ◽  
Bubble Collapse ◽  
Mechanism Analysis ◽  
Initiation Complex ◽  
Kinetic Measurements ◽  
Activation Barriers ◽  
Promoter Dna ◽  
Lower Temperature

AbstractIn transcription initiation, specific contacts between RNA polymerase (RNAP) and promoter DNA are disrupted as the RNA-DNA hybrid advances into the cleft, resulting in escape of RNAP. From the pattern of large and small rate constants for steps of initiation at λPR promoter at 19°C, we proposed that in-cleft interactions are disrupted in extending 3-mer to 5-mer RNA, −10 interactions are disrupted in extending 6-mer to 9-mer, and −35 interactions are disrupted in extending 10-mer to 11-mer, allowing RNAP to escape. Here we test this mechanism and determine enthalpic and entropic activation barriers of all steps from kinetic measurements at 25°C and 37°C. Initiation at 37°C differs significantly from expectations based on lower-temperature results. At low concentration of the second iNTP (UTP), synthesis of full-length RNA at 37°C is slower than at 25°C and no transient short RNA intermediates are observed, indicating a UTP-dependent bottleneck step early in the 37°C mechanism. Analysis reveals that the 37°C λPR OC (RPO) cannot initiate and must change conformation to a less-stable initiation complex (IC) capable of binding the iNTP. We find that IC is the primary λPR OC species below 25°C, and therefore conclude that IC must be the I3 intermediate in RPO formation. Surprisingly, Arrhenius activation energy barriers to five steps where RNAP-promoter in-cleft and −10 contacts are disrupted are much smaller than for other steps, including a negative barrier for the last of these steps. We interpret these striking effects as enthalpically-favorable, entropically-unfavorable, stepwise bubble collapse accompanying disruption of RNAP contacts.SignificanceTranscription initiation is highly regulated. To understand regulation, mechanisms of initiation and escape of RNA polymerase (RNAP) from the promoter must be understood. RNAP forms a highly-stable open complex (RPO) with λPR promoter at 37°C. From experiments determining effects of temperature on rate constants for each step of RNA synthesis, we find that RPO cannot bind the initiating nucleotides, that the I3 intermediate and not RPO is the initiation complex, and that contacts of RNAP with single-stranded DNA of the discriminator and −10 region and with −35 duplex DNA are disrupted stepwise as the RNA-DNA hybrid moves into the cleft. Evidence is obtained for stepwise bubble collapse and base stacking accompanying disruption of interactions of the single-stranded discriminator and −10 regions with RNAP.

scholarly journals Temperature effects on RNA polymerase initiation kinetics reveal which open complex initiates and that bubble collapse is stepwise

2021 ◽  
Vol 118 (30) ◽  
pp. e2021941118
Author(s):  
Dylan M. Plaskon ◽  
Kate L. Henderson ◽  
Lindsey C. Felth ◽  
Cristen M. Molzahn ◽  
Claire Evensen ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rate Constants ◽  
Transcription Initiation ◽  
Promoter Sequence ◽  
Bubble Collapse ◽  
Published Data ◽  
Transcription Inhibitors ◽  
Nucleotide Addition ◽  
Mechanisms Of Formation ◽  
Promoter Dna

Transcription initiation is highly regulated by promoter sequence, transcription factors, and ligands. All known transcription inhibitors, an important class of antibiotics, act in initiation. To understand regulation and inhibition, the biophysical mechanisms of formation and stabilization of the “open” promoter complex (OC), of synthesis of a short RNA–DNA hybrid upon nucleotide addition, and of escape of RNA polymerase (RNAP) from the promoter must be understood. We previously found that RNAP forms three different OC with λPR promoter DNA. The 37 °C RNAP-λPR OC (RPO) is very stable. At lower temperatures, RPO is less stable and in equilibrium with an intermediate OC (I3). Here, we report step-by-step rapid quench-flow kinetic data for initiation and growth of the RNA–DNA hybrid at 25 and 37 °C that yield rate constants for each step of productive nucleotide addition. Analyzed together, with previously published data at 19 °C, our results reveal that I3 and not RPO is the productive initiation complex at all temperatures. From the strong variations of rate constants and activation energies and entropies for individual steps of hybrid extension, we deduce that contacts of RNAP with the bubble strands are disrupted stepwise as the hybrid grows and translocates. Stepwise disruption of RNAP-strand contacts is accompanied by stepwise bubble collapse, base stacking, and duplex formation, as the hybrid extends to a 9-mer prior to disruption of upstream DNA–RNAP contacts and escape of RNAP from the promoter.

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.

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.

scholarly journals Direct binding of TFEα opens DNA binding cleft of RNA polymerase

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Sung-Hoon Jun ◽  
Jaekyung Hyun ◽  
Jeong Seok Cha ◽  
Hoyoung Kim ◽  
Michael S. Bartlett ◽  
...  
Keyword(s):  
Dna Binding ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Structural Basis ◽  
Transcription Bubble ◽  
Cryo Electron Microscopy ◽  
Direct Binding ◽  
Binding Cleft ◽  
Promoter Dna ◽  
Winged Helix Domain

AbstractOpening of the DNA binding cleft of cellular RNA polymerase (RNAP) is necessary for transcription initiation but the underlying molecular mechanism is not known. Here, we report on the cryo-electron microscopy structures of the RNAP, RNAP-TFEα binary, and RNAP-TFEα-promoter DNA ternary complexes from archaea, Thermococcus kodakarensis (Tko). The structures reveal that TFEα bridges the RNAP clamp and stalk domains to open the DNA binding cleft. Positioning of promoter DNA into the cleft closes it while maintaining the TFEα interactions with the RNAP mobile modules. The structures and photo-crosslinking results also suggest that the conserved aromatic residue in the extended winged-helix domain of TFEα interacts with promoter DNA to stabilize the transcription bubble. This study provides a structural basis for the functions of TFEα and elucidates the mechanism by which the DNA binding cleft is opened during transcription initiation in the stalk-containing RNAPs, including archaeal and eukaryotic RNAPs.

scholarly journals RNA polymerase and an activator form discrete subcomplexes in a transcription initiation complex

2006 ◽  
Vol 25 (16) ◽  
pp. 3784-3790 ◽  
Author(s):  
Sebastian Maurer ◽  
Jürgen Fritz ◽  
Georgi Muskhelishvili ◽  
Andrew Travers
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Initiation Complex ◽  
Transcription Initiation Complex

scholarly journals The General Transcription Factors IIA, IIB, IIF, and IIE Are Required for RNA Polymerase II Transcription from the Human U1 Small Nuclear RNA Promoter

1999 ◽  
Vol 19 (3) ◽  
pp. 2130-2141 ◽  
Author(s):  
T. C. Kuhlman ◽  
H. Cho ◽  
D. Reinberg ◽  
N. Hernandez
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Small Nuclear Rna ◽  
Initiation Complex ◽  
General Transcription Factors ◽  
Nuclear Rna ◽  
Transcription Initiation Complex ◽  
Rna Polymerase Ii Transcription

ABSTRACT RNA polymerase II transcribes the mRNA-encoding genes and the majority of the small nuclear RNA (snRNA) genes. The formation of a minimal functional transcription initiation complex on a TATA-box-containing mRNA promoter has been well characterized and involves the ordered assembly of a number of general transcription factors (GTFs), all of which have been either cloned or purified to near homogeneity. In the human RNA polymerase II snRNA promoters, a single element, the proximal sequence element (PSE), is sufficient to direct basal levels of transcription in vitro. The PSE is recognized by the basal transcription complex SNAPc. SNAPc, which is not required for transcription from mRNA-type RNA polymerase II promoters such as the adenovirus type 2 major late (Ad2ML) promoter, is thought to recruit TATA binding protein (TBP) and nucleate the assembly of the snRNA transcription initiation complex, but little is known about which GTFs other than TBP are required. Here we show that the GTFs IIA, IIB, IIF, and IIE are required for efficient RNA polymerase II transcription from snRNA promoters. Thus, although the factors that recognize the core elements of RNA polymerase II mRNA and snRNA-type promoters differ, they mediate the recruitment of many common GTFs.

scholarly journals E. coli TraR allosterically regulates transcription initiation by altering RNA polymerase conformation and dynamics

2019 ◽  
Author(s):  
James Chen ◽  
Saumya Gopalkrishnan ◽  
Courtney Chiu ◽  
Albert Y. Chen ◽  
Elizabeth A. Campbell ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Conformational Changes ◽  
Transcription Initiation ◽  
Structural Changes ◽  
E Coli ◽  
Bacterial Proteins ◽  
Cryo Electron Microscopy ◽  
Promoter Complex ◽  
Promoter Dna ◽  
Induced Changes

AbstractTraR and its homolog DksA are bacterial proteins that regulate transcription initiation by binding directly to RNA polymerase (RNAP) rather than to promoter DNA. Effects of TraR mimic the combined effects of DksA and its cofactor ppGpp. How TraR and its homologs regulate transcription is unclear. Here, we use cryo-electron microscopy to determine structures of Escherichia coli RNAP, with or without TraR, and of an RNAP-promoter complex. TraR binding induced RNAP conformational changes not seen in previous crystallographic analyses, and a quantitative analysis of RNAP conformational heterogeneity revealed TraR-induced changes in RNAP dynamics. These changes involve mobile regions of RNAP affecting promoter DNA interactions, including the βlobe, the clamp, the bridge helix, and several lineage-specific insertions. Using mutational approaches, we show that these structural changes, as well as effects on σ70 region 1.1, are critical for transcription activation or inhibition, depending on the kinetic features of regulated promoters.

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