Single-Molecule Studies of Human RNA Polymerase Ii Transcription Initiation: Core-Promoter Recognition

Gene transcription by RNA polymerase II (Pol II) is the first step in the expression of the eukaryotic genome and a focal point for cellular regulation during development, differentiation, and responses to the environment. Two decades after the determination of the structure of Pol II, the mechanisms of transcription have been elucidated with studies of Pol II complexes with nucleic acids and associated proteins. Here we provide an overview of the nearly 200 available Pol II complex structures and summarize how these structures have elucidated promoter-dependent transcription initiation, promoter-proximal pausing and release of Pol II into active elongation, and the mechanisms that Pol II uses to navigate obstacles such as nucleosomes and DNA lesions. We predict that future studies will focus on how Pol II transcription is interconnected with chromatin transitions, RNA processing, and DNA repair.

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Bacterial RNA polymerase can retain σ70 throughout transcription

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1513899113 ◽

2016 ◽

Vol 113 (3) ◽

pp. 602-607 ◽

Cited By ~ 35

Author(s):

Timothy T. Harden ◽

Christopher D. Wells ◽

Larry J. Friedman ◽

Robert Landick ◽

Ann Hochschild ◽

...

Keyword(s):

Rna Polymerase ◽

Single Molecule ◽

Transcription Initiation ◽

Messenger Rna ◽

Initiation Factors ◽

Bacterial Rna ◽

Direct Measurements ◽

Promoter Recognition ◽

Σ Factor ◽

Transcript Elongation

Production of a messenger RNA proceeds through sequential stages of transcription initiation and transcript elongation and termination. During each of these stages, RNA polymerase (RNAP) function is regulated by RNAP-associated protein factors. In bacteria, RNAP-associated σ factors are strictly required for promoter recognition and have historically been regarded as dedicated initiation factors. However, the primary σ factor in Escherichia coli, σ70, can remain associated with RNAP during the transition from initiation to elongation, influencing events that occur after initiation. Quantitative studies on the extent of σ70 retention have been limited to complexes halted during early elongation. Here, we used multiwavelength single-molecule fluorescence-colocalization microscopy to observe the σ70–RNAP complex during initiation from the λ PR′ promoter and throughout the elongation of a long (>2,000-nt) transcript. Our results provide direct measurements of the fraction of actively transcribing complexes with bound σ70 and the kinetics of σ70 release from actively transcribing complexes. σ70 release from mature elongation complexes was slow (0.0038 s−1); a substantial subpopulation of elongation complexes retained σ70 throughout transcript elongation, and this fraction depended on the sequence of the initially transcribed region. We also show that elongation complexes containing σ70 manifest enhanced recognition of a promoter-like pause element positioned hundreds of nucleotides downstream of the promoter. Together, the results provide a quantitative framework for understanding the postinitiation roles of σ70 during transcription.

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

Molecular and Cellular Biology ◽

10.1128/mcb.19.3.2130 ◽

1999 ◽

Vol 19 (3) ◽

pp. 2130-2141 ◽

Cited By ~ 39

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.

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Single-Molecule Insights on Human RNA Polymerase II Transcription Regulation: Assembly of the Pre-Initiation Complex and the Roles of Activators

Biophysical Journal ◽

10.1016/j.bpj.2012.11.2029 ◽

2013 ◽

Vol 104 (2) ◽

pp. 365a-366a

Author(s):

Zhengjian Zhang ◽

Andrey Revyakin ◽

Robert Coleman ◽

Steve Chu ◽

Robert Tjian

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription Regulation ◽

Single Molecule ◽

Initiation Complex ◽

Rna Polymerase Ii Transcription

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Live-cell single particle imaging reveals the role of RNA polymerase II in histone H2A.Z eviction

eLife ◽

10.7554/elife.55667 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 5

Author(s):

Anand Ranjan ◽

Vu Q Nguyen ◽

Sheng Liu ◽

Jan Wisniewski ◽

Jee Min Kim ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Single Molecule ◽

Transcription Initiation ◽

General Mechanism ◽

Pol Ii ◽

Promoter Escape ◽

Genome Wide ◽

A Genome ◽

Particle Imaging

The H2A.Z histone variant, a genome-wide hallmark of permissive chromatin, is enriched near transcription start sites in all eukaryotes. H2A.Z is deposited by the SWR1 chromatin remodeler and evicted by unclear mechanisms. We tracked H2A.Z in living yeast at single-molecule resolution, and found that H2A.Z eviction is dependent on RNA Polymerase II (Pol II) and the Kin28/Cdk7 kinase, which phosphorylates Serine 5 of heptapeptide repeats on the carboxy-terminal domain of the largest Pol II subunit Rpb1. These findings link H2A.Z eviction to transcription initiation, promoter escape and early elongation activities of Pol II. Because passage of Pol II through +1 nucleosomes genome-wide would obligate H2A.Z turnover, we propose that global transcription at yeast promoters is responsible for eviction of H2A.Z. Such usage of yeast Pol II suggests a general mechanism coupling eukaryotic transcription to erasure of the H2A.Z epigenetic signal.

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In vitro reconstitution of yeast RNA polymerase II transcription initiation with high efficiency

Methods ◽

10.1016/j.ymeth.2019.03.016 ◽

2019 ◽

Vol 159-160 ◽

pp. 82-89 ◽

Cited By ~ 2

Author(s):

Rina Fujiwara ◽

Kenji Murakami

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription Initiation ◽

High Efficiency ◽

In Vitro Reconstitution ◽

Rna Polymerase Ii Transcription

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Core promoter-selective RNA polymerase II transcription

Biochemical Society Symposium ◽

10.1042/bss0730225 ◽

2006 ◽

Vol 73 ◽

pp. 225-236 ◽

Cited By ~ 29

Author(s):

Petra Gross ◽

Thomas Oelgeschläger

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription Initiation ◽

Core Promoter ◽

Promoter Sequence ◽

Tata Box ◽

Promoter Elements ◽

The Core ◽

Core Promoter Sequence ◽

Rnap Ii

The initiation of mRNA synthesis in eukaryotic cells is a complex and highly regulated process that requires the assembly of general transcription factors and RNAP II (RNA polymerase II; also abbreviated as Pol II) into a pre-initiation complex at the core promoter. The core promoter is defined as the minimal DNA region that is sufficient to direct low levels of activator-independent (basal) transcription by RNAP II in vitro. The core promoter typically extends approx. 40 bp up- and down-stream of the start site of transcription and can contain several distinct core promoter sequence elements. Core promoters in higher eukaryotes are highly diverse in structure, and each core promoter sequence element is only found in a subset of genes. So far, only TATA box and INR (initiator) element have been shown to be capable of directing accurate RNAP II transcription initiation independent of other core promoter elements. Computational analysis of metazoan genomes suggests that the prevalence of the TATA box has been overestimated in the past and that the majority of human genes are TATA-less. While TATA-mediated transcription initiation has been studied in great detail and is very well understood, very little is known about the factors and mechanisms involved in the function of the INR and other core promoter elements. Here we summarize our current understanding of the factors and mechanisms involved in core promoter-selective transcription and discuss possible pathways through which diversity in core promoter architecture might contribute to combinatorial gene regulation in metazoan cells.

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