Transcription activation by a sliding clamp

AbstractTranscription activation of bacteriophage T4 late genes is accomplished by a transcription activation complex containing RNA polymerase (RNAP), the promoter specificity factor gp55, the coactivator gp33, and a universal component of cellular DNA replication, the sliding clamp gp45. Although genetic and biochemical studies have elucidated many aspects of T4 late gene transcription, no precise structure of the transcription machinery in the process is available. Here, we report the cryo-EM structures of a gp55-dependent RNAP-promoter open complex and an intact gp45-dependent transcription activation complex. The structures reveal the interactions between gp55 and the promoter DNA that mediate the recognition of T4 late promoters. In addition to the σR2 homology domain, gp55 has a helix-loop-helix motif that chaperons the template-strand single-stranded DNA of the transcription bubble. Gp33 contacts both RNAP and the upstream double-stranded DNA. Gp45 encircles the DNA and tethers RNAP to it, supporting the idea that gp45 switches the promoter search from three-dimensional diffusion mode to one-dimensional scanning mode.

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Structural origins of Escherichia coli RNA polymerase open promoter complex stability

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2112877118 ◽

2021 ◽

Vol 118 (40) ◽

pp. e2112877118

Author(s):

Ruth M. Saecker ◽

James Chen ◽

Courtney E. Chiu ◽

Brandon Malone ◽

Johanna Sotiris ◽

...

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

De Novo ◽

Promoter Strength ◽

Transcription Bubble ◽

Template Strand ◽

Solution Studies ◽

Active Site Cleft ◽

Promoter Dna

The first step in gene expression in all organisms requires opening the DNA duplex to expose one strand for templated RNA synthesis. In Escherichia coli, promoter DNA sequence fundamentally determines how fast the RNA polymerase (RNAP) forms “open” complexes (RPo), whether RPo persists for seconds or hours, and how quickly RNAP transitions from initiation to elongation. These rates control promoter strength in vivo, but their structural origins remain largely unknown. Here, we use cryoelectron microscopy to determine the structures of RPo formed de novo at three promoters with widely differing lifetimes at 37 °C: λPR (t1/2 ∼10 h), T7A1 (t1/2 ∼4 min), and a point mutant in λPR (λPR-5C) (t1/2 ∼2 h). Two distinct RPo conformers are populated at λPR, likely representing productive and unproductive forms of RPo observed in solution studies. We find that changes in the sequence and length of DNA in the transcription bubble just upstream of the start site (+1) globally alter the network of DNA–RNAP interactions, base stacking, and strand order in the single-stranded DNA of the transcription bubble; these differences propagate beyond the bubble to upstream and downstream DNA. After expanding the transcription bubble by one base (T7A1), the nontemplate strand “scrunches” inside the active site cleft; the template strand bulges outside the cleft at the upstream edge of the bubble. The structures illustrate how limited sequence changes trigger global alterations in the transcription bubble that modulate the RPo lifetime and affect the subsequent steps of the transcription cycle.

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Structural origins of Escherichia coli RNA polymerase open promoter complex stability

10.1101/2021.09.08.459427 ◽

2021 ◽

Author(s):

Ruth M. Saecker ◽

James Chen ◽

Courtney E. Chiu ◽

Brandon Malone ◽

Johanna Sotiris ◽

...

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

De Novo ◽

Promoter Strength ◽

Transcription Bubble ◽

Template Strand ◽

Solution Studies ◽

Active Site Cleft ◽

Promoter Dna

AbstractThe first step of gene expression in all organisms requires opening the DNA duplex to expose one strand for templated RNA synthesis. In Escherichia coli, promoter DNA sequence fundamentally determines how fast the RNA polymerase (RNAP) forms “open” complexes (RPo), whether RPo persists for seconds or hours, and how quickly RNAP transitions from initiation to elongation. These rates control promoter strength in vivo but their structural origins remain largely unknown. Here we use cryo-electron microscopy to determine structures of RPo formed de novo at three promoters with widely differing lifetimes at 37°C: λPR (t1/2 ∼ 10 hours), T7A1 (t1/2 ∼ 4 minutes), and a point mutant in λPR (λPR-5C) (t1/2 ∼ 2 hours). Two distinct RPo conformers are populated at λPR, likely representing productive and unproductive forms of RPo observed in solution studies. We find that changes in the sequence and length of DNA in the transcription bubble just upstream of the start site (+1) globally alter the network of DNA-RNAP interactions, base stacking, and strand order in the single-stranded DNA of the transcription bubble; these differences propagate beyond the bubble to upstream and downstream DNA. After expanding the transcription bubble by one base (T7A1), the nontemplate-strand “scrunches” inside the active site cleft; the template-strand bulges outside the cleft at the upstream edge of the bubble. The structures illustrate how limited sequence changes trigger global alterations in the transcription bubble that modulate RPo lifetime and affect the subsequent steps of the transcription cycle.

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Structure of a bacterial RNA polymerase holoenzyme open promoter complex

eLife ◽

10.7554/elife.08504 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 109

Author(s):

Brian Bae ◽

Andrey Feklistov ◽

Agnieszka Lass-Napiorkowska ◽

Robert Landick ◽

Seth A Darst

Keyword(s):

Rna Polymerase ◽

Bubble Formation ◽

Thermus Aquaticus ◽

Transcription Bubble ◽

Template Strand ◽

Bacterial Rna ◽

Promoter Complex ◽

Primary Means ◽

Promoter Dna ◽

Biochemical Analyses

Initiation of transcription is a primary means for controlling gene expression. In bacteria, the RNA polymerase (RNAP) holoenzyme binds and unwinds promoter DNA, forming the transcription bubble of the open promoter complex (RPo). We have determined crystal structures, refined to 4.14 Å-resolution, of RPo containing Thermus aquaticus RNAP holoenzyme and promoter DNA that includes the full transcription bubble. The structures, combined with biochemical analyses, reveal key features supporting the formation and maintenance of the double-strand/single-strand DNA junction at the upstream edge of the −10 element where bubble formation initiates. The results also reveal RNAP interactions with duplex DNA just upstream of the −10 element and potential protein/DNA interactions that direct the DNA template strand into the RNAP active site. Addition of an RNA primer to yield a 4 base-pair post-translocated RNA:DNA hybrid mimics an initially transcribing complex at the point where steric clash initiates abortive initiation and σA dissociation.

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Dimerization through the helix-loop-helix motif enhances phosphorylation of the transcription activation domains of myogenin

Molecular and Cellular Biology ◽

10.1128/mcb.14.9.6232-6243.1994 ◽

1994 ◽

Vol 14 (9) ◽

pp. 6232-6243

Author(s):

J Zhou ◽

E N Olson

Keyword(s):

Transcriptional Activity ◽

Transcription Activation ◽

Bhlh Protein ◽

Specific Gene ◽

Phosphorylation Sites ◽

Gene Products ◽

Activation Domains ◽

Helix Loop Helix ◽

Carboxyl Terminal ◽

Bhlh Proteins

The muscle-specific basic helix-loop-helix (bHLH) protein myogenin activates muscle transcription by binding to target sequences in muscle-specific promoters and enhancers as a heterodimer with ubiquitous bHLH proteins, such as the E2A gene products E12 and E47. We show that dimerization with E2A products potentiates phosphorylation of myogenin at sites within its amino- and carboxyl-terminal transcription activation domains. Phosphorylation of myogenin at these sites was mediated by the bHLH region of E2A products and was dependent on dimerization but not on DNA binding. Mutations of the dimerization-dependent phosphorylation sites resulted in enhanced transcriptional activity of myogenin, suggesting that their phosphorylation diminishes myogenin's transcriptional activity. The ability of E2A products to potentiate myogenin phosphorylation suggests that dimerization induces a conformational change in myogenin that unmasks otherwise cryptic phosphorylation sites or that E2A proteins recruit a kinase for which myogenin is a substrate. That phosphorylation of these dimerization-dependent sites diminished myogenin's transcriptional activity suggests that these sites are targets for a kinase that interferes with muscle-specific gene expression.

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Three-dimensional model and molecular dynamics simulation of the active site of the self-splicing intervening sequence of the bacteriophage T4 nrdB messenger RNA

Biochemistry ◽

10.1021/bi00497a005 ◽

1990 ◽

Vol 29 (45) ◽

pp. 10317-10322 ◽

Cited By ~ 5

Author(s):

Lennart Nilsson ◽

Agneta Aahgren-Staalhandske ◽

Ann Sofie Sjoegren ◽

Solveig Hahne ◽

Britt Marie Sjoeberg

Keyword(s):

Molecular Dynamics ◽

Active Site ◽

Messenger Rna ◽

Bacteriophage T4 ◽

Three Dimensional ◽

The Self ◽

Dynamics Simulation ◽

Dimensional Model ◽

Three Dimensional Model ◽

Self Splicing

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Tracking Sliding Clamp Opening and Closing during Bacteriophage T4 DNA Polymerase Holoenzyme Assembly†

Biochemistry ◽

10.1021/bi992377r ◽

2000 ◽

Vol 39 (11) ◽

pp. 3076-3090 ◽

Cited By ~ 44

Author(s):

Stephen C. Alley ◽

Ernesto Abel-Santos ◽

Stephen J. Benkovic

Keyword(s):

Dna Polymerase ◽

Bacteriophage T4 ◽

Sliding Clamp ◽

Opening And Closing

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The universally-conserved transcription factor RfaH is recruited to a hairpin structure of the non-template DNA strand

eLife ◽

10.7554/elife.36349 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 18

Author(s):

Philipp K Zuber ◽

Irina Artsimovitch ◽

Monali NandyMazumdar ◽

Zhaokun Liu ◽

Yuri Nedialkov ◽

...

Keyword(s):

Virulence Genes ◽

Complex Structure ◽

Transcription Regulator ◽

Transcription Bubble ◽

Template Strand ◽

Domains Of Life ◽

Dna Strand ◽

Functional Analyses ◽

Template Dna

RfaH, a transcription regulator of the universally conserved NusG/Spt5 family, utilizes a unique mode of recruitment to elongating RNA polymerase to activate virulence genes. RfaH function depends critically on an ops sequence, an exemplar of a consensus pause, in the non-template DNA strand of the transcription bubble. We used structural and functional analyses to elucidate the role of ops in RfaH recruitment. Our results demonstrate that ops induces pausing to facilitate RfaH binding and establishes direct contacts with RfaH. Strikingly, the non-template DNA forms a hairpin in the RfaH:ops complex structure, flipping out a conserved T residue that is specifically recognized by RfaH. Molecular modeling and genetic evidence support the notion that ops hairpin is required for RfaH recruitment. We argue that both the sequence and the structure of the non-template strand are read out by transcription factors, expanding the repertoire of transcriptional regulators in all domains of life.

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Studies on deoxyribonucleic acid-dependent ribonucleic acid polymerase from Escherichia coli. Variations of the enzyme activity during growth

Biochemical Journal ◽

10.1042/bj1290291 ◽

1972 ◽

Vol 129 (2) ◽

pp. 291-299 ◽

Cited By ~ 3

Author(s):

K. A. Abraham ◽

K. J. Andersen ◽

A. Rognes

Keyword(s):

Escherichia Coli ◽

Stationary Phase ◽

Stationary Phases ◽

Bacteriophage T4 ◽

Polymerase Activity ◽

Exponential Phase ◽

Enzyme Preparations ◽

Rna Polymerase Activity ◽

Enzyme Molecules ◽

Cellular Dna

1. RNA polymerase activity of Escherichia coli extracts prepared from cells in exponential and stationary phases of growth, when measured in the presence and absence of external template, showed significant qualitative differences. 2. In both extracts, polymerase activity was higher when assayed with external template, suggesting the presence of a pool of enzyme not bound to cellular DNA. 3. In the crude extract, the fraction of enzyme bound to cellular DNA is higher during the exponential phase of growth. 4. A method is described for the purification of enzyme molecules not tightly bound to cellular DNA from exponential- and stationary-phase cultures. 5. Purified enzyme preparations showed differences in template requirement and subunit composition. 6. On phosphocellulose chromatography of stationary-phase enzyme, a major portion of polymerase activity eluted from the column with 0.25m-KCl. In the case of exponential-phase enzyme, polymerase activity eluted from a phosphocellulose column mainly with 0.35m-KCl. 7. Enzyme assays done with excess of bacteriophage T4 DNA showed a strong inhibition of stationary-phase enzyme by this template. The exponential-phase enzyme was only slightly inhibited by excess of bacteriophage T4 DNA.

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Direct binding of TFEα opens DNA binding cleft of RNA polymerase

Nature Communications ◽

10.1038/s41467-020-19998-x ◽

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.

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The extruded non-template strand determines the architecture of R-loops

Nucleic Acids Research ◽

10.1093/nar/gkz341 ◽

2019 ◽

Vol 47 (13) ◽

pp. 6783-6795 ◽

Cited By ~ 8

Author(s):

Yeraldinne Carrasco-Salas ◽

Amélie Malapert ◽

Shaheen Sulthana ◽

Bastien Molcrette ◽

Léa Chazot-Franguiadakis ◽

...

Keyword(s):

Single Molecule ◽

Genome Stability ◽

Three Dimensional ◽

Dna Breaks ◽

Yeast Gene ◽

Physical Constraints ◽

Force Microscopy ◽

Template Strand ◽

Atomic Force

Abstract Three-stranded R-loop structures have been associated with genomic instability phenotypes. What underlies their wide-ranging effects on genome stability remains poorly understood. Here we combined biochemical and atomic force microscopy approaches with single molecule R-loop footprinting to demonstrate that R-loops formed at the model Airn locus in vitro adopt a defined set of three-dimensional conformations characterized by distinct shapes and volumes, which we call R-loop objects. Interestingly, we show that these R-loop objects impose specific physical constraints on the DNA, as revealed by the presence of stereotypical angles in the surrounding DNA. Biochemical probing and mutagenesis experiments revealed that the formation of R-loop objects at Airn is dictated by the extruded non-template strand, suggesting that R-loops possess intrinsic sequence-driven properties. Consistent with this, we show that R-loops formed at the fission yeast gene sum3 do not form detectable R-loop objects. Our results reveal that R-loops differ by their architectures and that the organization of the non-template strand is a fundamental characteristic of R-loops, which could explain that only a subset of R-loops is associated with replication-dependent DNA breaks.

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