Promoter-proximal elongation regulates transcription in archaea

Recruitment of RNA polymerase and initiation factors to the promoter is the only known mechanisms for transcription activation and repression in archaea. Whether any of the subsequent steps towards productive transcription elongation is involved in regulation is not known. We characterised how the basal transcription machinery is distributed along genes in the archaeon Sulfolobus solfataricus. We discovered a distinct early elongation phase where RNA polymerases sequentially recruit the elongation factors Spt4/5 and Elf1 to form the transcription elongation complex (TEC) before the TEC escapes into productive transcription. TEC escape is rate-limiting for transcription output during exponential growth. Oxidative stress causes changes in TEC escape that correlate with changes in the transcriptome. Our results thus establish that TEC escape contributes to the basal promoter strength and facilitates transcription regulation. Impaired TEC escape coincides with the accumulation of initiation factors at the promoter and recruitment of termination factor aCPSF1 to the early TEC. This suggests two possible mechanisms for how TEC escape limits transcription, physically blocking upstream RNA polymerases during transcription initiation and premature termination of early TECs.

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Mechanism of initiator-mediated transcription: evidence for a functional interaction between the TATA-binding protein and DNA in the absence of a specific recognition sequence

Molecular and Cellular Biology ◽

10.1128/mcb.13.7.3841-3849.1993 ◽

1993 ◽

Vol 13 (7) ◽

pp. 3841-3849

Author(s):

B Zenzie-Gregory ◽

A Khachi ◽

I P Garraway ◽

S T Smale

Keyword(s):

Transcription Initiation ◽

Binding Protein ◽

Tata Binding Protein ◽

Upstream Region ◽

Promoter Strength ◽

Recognition Sequence ◽

Specific Recognition ◽

Rate Limiting

Promoters containing Sp1 binding sites and an initiator element but lacking a TATA box direct high levels of accurate transcription initiation by using a mechanism that requires the TATA-binding protein (TBP). We have begun to address the role of TBP during transcription from Sp1-initiator promoters by varying the nucleotide sequence between -14 and -33 relative to the start site. With each of several promoters containing different upstream sequences, we detected accurate transcription both in vitro and in vivo, but the promoter strengths varied widely, particularly with the in vitro assay. The variable promoter activities correlated with, but were not proportional to, the abilities of the upstream sequences to function as TATA boxes, as assessed by multiple criteria. These results confirm that accurate transcription can proceed in the presence of an initiator, regardless of the sequence present in the -30 region. However, the results reveal a role for this upstream region, most consistent with a model in which initiator-mediated transcription requires binding of TBP to the upstream DNA in the absence of a specific recognition sequence. Moreover, in vivo it appears that the promoter strength is modulated less severely by altering the -30 sequence, consistent with a previous suggestion that TBP is not rate limiting in vivo for TATA-less promoters. Taken together, these results suggest that variations in the structure of a core promoter might alter the rate-limiting step for transcription initiation and thereby alter the potential modes of transcriptional regulation, without severely changing the pathway used to assemble a functional preinitiation complex.

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Distinct Mechanisms of Transcription Initiation by RNA Polymerases I and II

Annual Review of Biophysics ◽

10.1146/annurev-biophys-070317-033058 ◽

2018 ◽

Vol 47 (1) ◽

pp. 425-446 ◽

Cited By ~ 26

Author(s):

Christoph Engel ◽

Simon Neyer ◽

Patrick Cramer

Keyword(s):

Transcription Initiation ◽

Messenger Rna ◽

Rna Polymerases ◽

Chain Elongation ◽

Structural Studies ◽

Pol Ii ◽

Initiation Factors ◽

Initiation System ◽

Pol I ◽

Promoter Dna

RNA polymerases I and II (Pol I and Pol II) are the eukaryotic enzymes that catalyze DNA-dependent synthesis of ribosomal RNA and messenger RNA, respectively. Recent work shows that the transcribing forms of both enzymes are similar and the fundamental mechanisms of RNA chain elongation are conserved. However, the mechanisms of transcription initiation and its regulation differ between Pol I and Pol II. Recent structural studies of Pol I complexes with transcription initiation factors provided insights into how the polymerase recognizes its specific promoter DNA, how it may open DNA, and how initiation may be regulated. Comparison with the well-studied Pol II initiation system reveals a distinct architecture of the initiation complex and visualizes promoter- and gene-class-specific aspects of transcription initiation. On the basis of new structural studies, we derive a model of the Pol I transcription cycle and provide a molecular movie of Pol I transcription that can be used for teaching.

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Mechanism of initiator-mediated transcription: evidence for a functional interaction between the TATA-binding protein and DNA in the absence of a specific recognition sequence.

Molecular and Cellular Biology ◽

10.1128/mcb.13.7.3841 ◽

1993 ◽

Vol 13 (7) ◽

pp. 3841-3849 ◽

Cited By ~ 57

Author(s):

B Zenzie-Gregory ◽

A Khachi ◽

I P Garraway ◽

S T Smale

Keyword(s):

Transcription Initiation ◽

Binding Protein ◽

Tata Binding Protein ◽

Upstream Region ◽

Promoter Strength ◽

Recognition Sequence ◽

Specific Recognition ◽

Rate Limiting

Promoters containing Sp1 binding sites and an initiator element but lacking a TATA box direct high levels of accurate transcription initiation by using a mechanism that requires the TATA-binding protein (TBP). We have begun to address the role of TBP during transcription from Sp1-initiator promoters by varying the nucleotide sequence between -14 and -33 relative to the start site. With each of several promoters containing different upstream sequences, we detected accurate transcription both in vitro and in vivo, but the promoter strengths varied widely, particularly with the in vitro assay. The variable promoter activities correlated with, but were not proportional to, the abilities of the upstream sequences to function as TATA boxes, as assessed by multiple criteria. These results confirm that accurate transcription can proceed in the presence of an initiator, regardless of the sequence present in the -30 region. However, the results reveal a role for this upstream region, most consistent with a model in which initiator-mediated transcription requires binding of TBP to the upstream DNA in the absence of a specific recognition sequence. Moreover, in vivo it appears that the promoter strength is modulated less severely by altering the -30 sequence, consistent with a previous suggestion that TBP is not rate limiting in vivo for TATA-less promoters. Taken together, these results suggest that variations in the structure of a core promoter might alter the rate-limiting step for transcription initiation and thereby alter the potential modes of transcriptional regulation, without severely changing the pathway used to assemble a functional preinitiation complex.

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ChIP-exo interrogation of Crp, DNA, and RNAP holoenzyme interactions

10.1101/069021 ◽

2016 ◽

Cited By ~ 1

Author(s):

Haythem Latif ◽

Stephen Federowicz ◽

Ali Ebrahim ◽

Janna Tarasova ◽

Richard Szubin ◽

...

Keyword(s):

Transcription Initiation ◽

Receptor Protein ◽

Elongation Complex ◽

Template Strand ◽

Rate Limiting Step ◽

Dna Motif ◽

Genome Scale ◽

Rate Limiting

ABSTRACTNumerous in vitro studies have yielded a refined picture of the structural and molecular associations between Cyclic-AMP receptor protein (Crp), the DNA motif, and RNA polymerase (RNAP) holoenzyme. In this study, high-resolution ChIP-exonuclease (ChIP-exo) was applied to study Crp binding in vivo and at genome-scale. Surprisingly, Crp was found to provide little to no protection of the DNA motif under activating conditions. Instead, Crp demonstrated binding patterns that closely resembled those generated by σ70. The binding patterns of both Crp and σ70 are indicative of RNAP holoenzyme DNA footprinting profiles associated with stages during transcription initiation that occur post-recruitment. This is marked by a pronounced advancement of the template strand footprint profile to the +20 position relative to the transcription start site and a multimodal distribution on the nontemplate strand. This trend was also observed in the familial transcription factor, Fnr, but full protection of the motif was seen in the repressor ArcA. Given the time-scale of ChIP studies and that the rate-limiting step in transcription initiation is typically post recruitment, we propose a hypothesis where Crp is absent from the DNA motif but remains associated with RNAP holoenzyme post-recruitment during transcription initiation. The release of Crp from the DNA motif may be a result of energetic changes that occur as RNAP holoenzyme traverses the various stable intermediates towards elongation complex formation.

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DNA supercoiling-mediated collective behavior of co-transcribing RNA polymerases

10.1101/2021.03.04.433986 ◽

2021 ◽

Author(s):

Shubham Tripathi ◽

Sumitabha Brahmachari ◽

José Nelson Onuchic ◽

Herbert Levine

Keyword(s):

Transcription Initiation ◽

Transcription Elongation ◽

Group Behavior ◽

Dna Supercoiling ◽

Elongation Rate ◽

Rna Polymerases ◽

Stochastic Simulations ◽

Physical Interaction ◽

Mechanical Coupling ◽

Torsional Response

Multiple RNA polymerases (RNAPs) transcribing a gene have been known to exhibit collective group behavior, causing the transcription elongation rate to increase with the rate of transcription initiation. Such behavior has long been believed to be driven by a physical interaction or "push" between closely spaced RNAPs. However, recent studies have posited that RNAPs separated by longer distances may cooperate via the DNA segment under transcription. Here, we present a theoretical model incorporating the mechanical coupling between RNAP translocation and the torsional response of supercoiled DNA. Using stochastic simulations, we demonstrate long-range cooperation between co-transcribing RNAPs mediated by DNA supercoiling. We find that inhibiting transcription initiation can slow down the already recruited RNAPs, in agreement with recent experimental observations, and predict that the average transcription elongation rate varies non-monotonically with the rate of transcription initiation. We further show that while RNAPs transcribing neighboring genes oriented in tandem can cooperate, those transcribing genes in divergent or convergent orientations can act antagonistically, and that such behavior holds over a large range of intergenic separations. Our model makes testable predictions, revealing how the mechanical interplay between RNAPs and the DNA they transcribe can govern a key cellular process.

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The primary σ factor in Escherichia coli can access the transcription elongation complex from solution in vivo

eLife ◽

10.7554/elife.10514 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 12

Author(s):

Seth R Goldman ◽

Nikhil U Nair ◽

Christopher D Wells ◽

Bryce E Nickels ◽

Ann Hochschild

Keyword(s):

Escherichia Coli ◽

Transcription Elongation ◽

Elongation Factor ◽

Direct Effects ◽

Elongation Complex ◽

Initiation Factors ◽

Bacterial Rna ◽

Σ Factor ◽

In Trans

The σ subunit of bacterial RNA polymerase (RNAP) confers on the enzyme the ability to initiate promoter-specific transcription. Although σ factors are generally classified as initiation factors, σ can also remain associated with, and modulate the behavior of, RNAP during elongation. Here we establish that the primary σ factor in Escherichia coli, σ70, can function as an elongation factor in vivo by loading directly onto the transcription elongation complex (TEC) in trans. We demonstrate that σ70 can bind in trans to TECs that emanate from either a σ70-dependent promoter or a promoter that is controlled by an alternative σ factor. We further demonstrate that binding of σ70 to the TEC in trans can have a particularly large impact on the dynamics of transcription elongation during stationary phase. Our findings establish a mechanism whereby the primary σ factor can exert direct effects on the composition of the entire transcriptome, not just that portion that is produced under the control of σ70-dependent promoters.

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Opposing functions of the Hda1 complex and histone H2B mono-ubiquitylation in regulating cryptic transcription initiation in Saccharomyces cerevisiae

10.1101/2020.10.12.336081 ◽

2020 ◽

Author(s):

Margaret K. Shirra ◽

Rachel A. Kocik ◽

Mitchell A. Ellison ◽

Karen M. Arndt

Keyword(s):

Saccharomyces Cerevisiae ◽

Histone Deacetylase ◽

Chromatin Structure ◽

Histone Deacetylases ◽

Transcription Initiation ◽

Transcription Elongation ◽

Chromatin Accessibility ◽

Elongation Complex ◽

Gene Encoding ◽

Cryptic Transcription

ABSTRACTMaintenance of chromatin structure under the disruptive force of transcription requires cooperation among numerous chromatin regulatory factors. Histone post-translational modifications can regulate nucleosome stability and influence the disassembly and reassembly of nucleosomes during transcription elongation. The Paf1 transcription elongation complex, Paf1C, is required for several transcription-coupled histone modifications, including the mono-ubiquitylation of H2B. In Saccharomyces cerevisiae, amino acid substitutions in the Rtf1 subunit of Paf1C greatly diminish H2B ubiquitylation and cause transcription to initiate at a cryptic promoter within a coding gene, an indicator of chromatin disruption. In a genetic screen to identify factors that functionally interact with Paf1C, we identified a mutation in HDA3, a gene encoding a subunit of the Hda1C histone deacetylase, as a suppressor of an rtf1 mutation. Absence of Hda1C also suppresses the cryptic initiation phenotype of other mutants defective in H2B ubiquitylation. The genetic interactions between Hda1C and the H2B ubiquitylation pathway appear specific: loss of Hda1C does not suppress the cryptic initiation phenotypes of other chromatin mutants and absence of other histone deacetylases does not suppress the absence of H2B ubiquitylation. Providing further support for an appropriate balance of histone acetylation in regulating cryptic initiation, we find that deletion of the Sas3 histone acetyltransferase elevates cryptic initiation in rtf1 mutants. Our data suggest a coordination between two epigenetic modifiers, the H2B ubiquitylation pathway and Hda1C, in regulating chromatin structure during transcription elongation and reveal an unexpected role for a histone deacetylase in supporting chromatin accessibility.

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Faculty Opinions recommendation of Visualizing RNA extrusion and DNA wrapping in transcription elongation complexes of bacterial and eukaryotic RNA polymerases.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1012321.189106 ◽

2003 ◽

Author(s):

Daniel Reines

Keyword(s):

Transcription Elongation ◽

Rna Polymerases ◽

Dna Wrapping

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DIPG-03. THERAPEUTIC TARGETING OF TRANSCRIPTIONAL ELONGATION IN DIFFUSE INTRINSIC PONTINE GLIOMA

Neuro-Oncology ◽

10.1093/neuonc/noaa222.055 ◽

2020 ◽

Vol 22 (Supplement_3) ◽

pp. iii287-iii287

Author(s):

Hiroaki Katagi ◽

Nozomu Takata ◽

Yuki Aoi ◽

Yongzhan Zhang ◽

Emily J Rendleman ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Transcription Elongation ◽

Xenograft Model ◽

Diffuse Intrinsic Pontine Glioma ◽

Transcriptional Elongation ◽

Pontine Glioma ◽

Elongation Complex ◽

Pol Ii ◽

Repair Genes ◽

Novel Therapeutic

Abstract Diffuse intrinsic pontine glioma (DIPG) is highly aggressive brain stem tumor and needed to develop novel therapeutic agents for the treatment. The super elongation complex (SEC) is essential for transcription elongation through release of RNA polymerase II (Pol II). We found that AFF4, a scaffold protein of the SEC, is required for the growth of H3K27M-mutant DIPG cells. In addition, the small molecule SEC inhibitor, KL-1, increased promoter-proximal pausing of Pol II, and reduced transcription elongation, resulting in down-regulate cell cycle, transcription and DNA repair genes. KL-1 treatment decreased cell growth and increased apoptosis in H3K27M-mutant DIPG cells, and prolonged animal survival in our human H3K27M-mutant DIPG xenograft model. Our results demonstrate that the SEC disruption by KL-1 is a novel therapeutic strategy for H3K27M-mutant DIPG.

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