scholarly journals How Single-Molecule Localization Microscopy Expanded Our Mechanistic Understanding of RNA Polymerase II Transcription

2021 ◽  
Vol 22 (13) ◽  
pp. 6694
Author(s):  
Peter Hoboth ◽  
Ondřej Šebesta ◽  
Pavel Hozák
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Spatial Organization ◽  
Super Resolution ◽  
Temporal Organization ◽  
Transcription Machinery ◽  
Mechanistic Understanding ◽  
Rnapii Transcription

Classical models of gene expression were built using genetics and biochemistry. Although these approaches are powerful, they have very limited consideration of the spatial and temporal organization of gene expression. Although the spatial organization and dynamics of RNA polymerase II (RNAPII) transcription machinery have fundamental functional consequences for gene expression, its detailed studies have been abrogated by the limits of classical light microscopy for a long time. The advent of super-resolution microscopy (SRM) techniques allowed for the visualization of the RNAPII transcription machinery with nanometer resolution and millisecond precision. In this review, we summarize the recent methodological advances in SRM, focus on its application for studies of the nanoscale organization in space and time of RNAPII transcription, and discuss its consequences for the mechanistic understanding of gene expression.

scholarly journals How Single-Molecule Localization Microscopy Expanded Our Mechanistic Understanding of RNA Polymerase II Transcription

2021 ◽  
Author(s):  
Peter Hoboth ◽  
Ondřej Šebesta ◽  
Pavel Hozak
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Spatial Organization ◽  
Super Resolution ◽  
Temporal Organization ◽  
Transcription Machinery ◽  
Mechanistic Understanding ◽  
Rnapii Transcription

Classical models of gene expression were built using genetics and biochemistry. Although these approaches are powerful, they have very limited consideration of the spatial and temporal organization of gene expression. Although the spatial organization and dynamics of RNA polymerase II (RNAPII) transcription machinery has fundamental functional consequences for gene expression, its detailed studies have been for long time abrogated by the limits of classical light microscopy. The advent of super-resolution microscopy (SRM) techniques allowed for the visualization of the RNAPII transcription machinery with nanometer resolution and millisecond precision. In this review, we summarize the recent methodological advances in SRM, focus on its application for studies of the nanoscale organization in space and time of RNAPII transcription, and discuss its consequences for the mechanistic understanding of gene expression.

scholarly journals A dual role for H2A.Z.1 in modulating the dynamics of RNA Polymerase II initiation and elongation

2020 ◽  
Author(s):  
Constantine Mylonas ◽  
Alexander L. Auld ◽  
Choongman Lee ◽  
Ibrahim I. Cisse ◽  
Laurie A. Boyer
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Mammalian Cells ◽  
Embryonic Stem ◽  
Super Resolution ◽  
Fine Tuning ◽  
Transcriptional Initiation ◽  
Single Molecule Level

AbstractRNAPII pausing immediately downstream of the transcription start site (TSS) is a critical rate limiting step at most metazoan genes that allows fine-tuning of gene expression in response to diverse signals1–5. During pause-release, RNA Polymerase II (RNAPII) encounters an H2A.Z.1 nucleosome6–8, yet how this variant contributes to transcription is poorly understood. Here, we use high resolution genomic approaches2,9 (NET-seq and ChIP-nexus) along with live cell super-resolution microscopy (tcPALM)10 to investigate the role of H2A.Z.1 on RNAPII dynamics in embryonic stem cells (ESCs). Using a rapid, inducible protein degron system11 combined with transcriptional initiation and elongation inhibitors, our quantitative analysis shows that H2A.Z.1 slows the release of RNAPII, impacting both RNAPII and NELF dynamics at a single molecule level. We also find that H2A.Z.1 loss has a dramatic impact on nascent transcription at stably paused, signal-dependent genes. Furthermore, we demonstrate that H2A.Z.1 inhibits re-assembly and re-initiation of the PIC to reinforce the paused state and acts as a strong additional pause signal at stably paused genes. Together, our study suggests that H2A.Z.1 fine-tunes gene expression by regulating RNAPII kinetics in mammalian cells.

scholarly journals Live-cell imaging reveals the spatiotemporal organization of endogenous RNA polymerase II phosphorylation at a single gene

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Linda S. Forero-Quintero ◽  
William Raymond ◽  
Tetsuya Handa ◽  
Matthew N. Saxton ◽  
Tatsuya Morisaki ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Computational Models ◽  
Spatial Organization ◽  
Fluorescent Antibody ◽  
Single Gene ◽  
Single Copy ◽  
Living Cells ◽  
Live Cell

AbstractThe carboxyl-terminal domain of RNA polymerase II (RNAP2) is phosphorylated during transcription in eukaryotic cells. While residue-specific phosphorylation has been mapped with exquisite spatial resolution along the 1D genome in a population of fixed cells using immunoprecipitation-based assays, the timing, kinetics, and spatial organization of phosphorylation along a single-copy gene have not yet been measured in living cells. Here, we achieve this by combining multi-color, single-molecule microscopy with fluorescent antibody-based probes that specifically bind to different phosphorylated forms of endogenous RNAP2 in living cells. Applying this methodology to a single-copy HIV-1 reporter gene provides live-cell evidence for heterogeneity in the distribution of RNAP2 along the length of the gene as well as Serine 5 phosphorylated RNAP2 clusters that remain separated in both space and time from nascent mRNA synthesis. Computational models determine that 5 to 40 RNAP2 cluster around the promoter during a typical transcriptional burst, with most phosphorylated at Serine 5 within 6 seconds of arrival and roughly half escaping the promoter in ~1.5 minutes. Taken together, our data provide live-cell support for the notion of efficient transcription clusters that transiently form around promoters and contain high concentrations of RNAP2 phosphorylated at Serine 5.

scholarly journals Spatial Organization of RNA Polymerase II Revealed by Super-Resolution Imaging of Mammalian Cell Nucleus

2013 ◽  
Vol 104 (2) ◽  
pp. 339a
Author(s):  
Ziqing Zhao ◽  
Rahul Roy ◽  
J. Christof M. Gebhardt ◽  
David M. Suter ◽  
Alec R. Chapman ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Mammalian Cell ◽  
Cell Nucleus ◽  
Spatial Organization ◽  
Super Resolution ◽  
Resolution Imaging

scholarly journals Live-cell imaging reveals the spatiotemporal organization of endogenous RNA polymerase II phosphorylation at a single gene

2020 ◽  
Author(s):  
Linda S. Forero-Quintero ◽  
William Raymond ◽  
Tetsuya Handa ◽  
Matthew Saxton ◽  
Tatsuya Morisaki ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Spatial Organization ◽  
Fluorescent Antibody ◽  
Single Gene ◽  
Single Copy ◽  
Living Cells ◽  
Live Cell ◽  
Highly Efficient

The carboxyl-terminal domain of RNA polymerase II is dynamically phosphorylated during transcription in eukaryotic cells. While residue-specific phosphorylation has been mapped with exquisite spatial resolution along the 1D genome in a population of fixed cells using immunoprecipitation-based assays, the timing, kinetics, and spatial organization of phosphorylation along a single-copy gene have not yet been measured in living cells. Here, we achieve this by combining multi-color, single-molecule microscopy with fluorescent antibody-based probes that specifically bind to unphosphorylated and phosphorylated forms of endogenous RNAP2 in living cells. Applying this methodology to a single-copy HIV-1 reporter gene provides live-cell evidence for heterogeneity in the distribution of RNAP2 along the length of the gene as well as clusters of Serine 5 phosphorylated RNAP2 that form around active genes and are separated in both space and time from nascent mRNA synthesis. Computational models fit to our data determine that 5 to 40 RNAP2 cluster around the promoter of a gene during typical transcriptional bursts. Nearly all RNAP2 either arrive with Serine 5 phosphorylation or acquire the modification within a minute. Transcription from the cluster appears to be highly efficient, with nearly half of the clustered RNAP2 ultimately escaping the promoter in a minute or so to elongate a full-length mRNA in approximately five minutes. The highly dynamic and spatially organized concentrations of RNAP2 we observe support the notion of highly efficient transcription clusters that form around promoters and contain high concentrations of RNAP2 phosphorylated at Serine 5.

scholarly journals Therapeutic Targeting of the General RNA Polymerase II Transcription Machinery

2020 ◽  
Vol 21 (9) ◽  
pp. 3354 ◽  
Author(s):  
Ryan D. Martin ◽  
Terence E. Hébert ◽  
Jason C. Tanny
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Complex Diseases ◽  
Cellular Models ◽  
Transcription Machinery ◽  
Transcription Inhibitors ◽  
Preclinical And Clinical Studies ◽  
Rna Polymerase Ii Transcription ◽  
Rnapii Transcription

Inhibitors targeting the general RNA polymerase II (RNAPII) transcription machinery are candidate therapeutics in cancer and other complex diseases. Here, we review the molecular targets and mechanisms of action of these compounds, framing them within the steps of RNAPII transcription. We discuss the effects of transcription inhibitors in vitro and in cellular models (with an emphasis on cancer), as well as their efficacy in preclinical and clinical studies. We also discuss the rationale for inhibiting broadly acting transcriptional regulators or RNAPII itself in complex diseases.

scholarly journals Nucleosomal arrangement affects single-molecule transcription dynamics

2016 ◽  
Vol 113 (45) ◽  
pp. 12733-12738 ◽  
Author(s):  
Veronika Fitz ◽  
Jaeoh Shin ◽  
Christoph Ehrlich ◽  
Lucas Farnung ◽  
Patrick Cramer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Optical Tweezers ◽  
Single Molecule ◽  
Pol Ii ◽  
Dna Molecule ◽  
Chromatin Arrangement ◽  
Dna Template ◽  
Drift Coefficient

In eukaryotes, gene expression depends on chromatin organization. However, how chromatin affects the transcription dynamics of individual RNA polymerases has remained elusive. Here, we use dual trap optical tweezers to study single yeast RNA polymerase II (Pol II) molecules transcribing along a DNA template with two nucleosomes. The slowdown and the changes in pausing behavior within the nucleosomal region allow us to determine a drift coefficient, χ, which characterizes the ability of the enzyme to recover from a nucleosomal backtrack. Notably, χ can be used to predict the probability to pass the first nucleosome. Importantly, the presence of a second nucleosome changes χ in a manner that depends on the spacing between the two nucleosomes, as well as on their rotational arrangement on the helical DNA molecule. Our results indicate that the ability of Pol II to pass the first nucleosome is increased when the next nucleosome is turned away from the first one to face the opposite side of the DNA template. These findings help to rationalize how chromatin arrangement affects Pol II transcription dynamics.

scholarly journals A Single-Molecule Surface-Based Platform to Detect the Assembly and Function of the Human RNA Polymerase II Transcription Machinery

Structure ◽  
2020 ◽  
Vol 28 (12) ◽  
pp. 1337-1343.e4 ◽  
Author(s):  
Sang Ryul Park ◽  
Jesse Hauver ◽  
Yunxiang Zhang ◽  
Andrey Revyakin ◽  
Robert A. Coleman ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Transcription Machinery ◽  
And Function ◽  
Rna Polymerase Ii Transcription ◽  
Molecule Surface

Nutrient-regulated gene expression in eukaryotes

2006 ◽  
Vol 73 ◽  
pp. 85-96 ◽  
Author(s):  
Richard J. Reece ◽  
Laila Beynon ◽  
Stacey Holden ◽  
Amanda D. Hughes ◽  
Karine Rébora ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Control ◽  
Direct Interaction ◽  
Signalling Pathways ◽  
A Cell ◽  
One Step ◽  
Higher Eukaryotes ◽  
Regulated Gene Expression

The recognition of changes in environmental conditions, and the ability to adapt to these changes, is essential for the viability of cells. There are numerous well characterized systems by which the presence or absence of an individual metabolite may be recognized by a cell. However, the recognition of a metabolite is just one step in a process that often results in changes in the expression of whole sets of genes required to respond to that metabolite. In higher eukaryotes, the signalling pathway between metabolite recognition and transcriptional control can be complex. Recent evidence from the relatively simple eukaryote yeast suggests that complex signalling pathways may be circumvented through the direct interaction between individual metabolites and regulators of RNA polymerase II-mediated transcription. Biochemical and structural analyses are beginning to unravel these elegant genetic control elements.

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