scholarly journals Disruption of Higher-Order Folding by Core Histone Acetylation Dramatically Enhances Transcription of Nucleosomal Arrays by RNA Polymerase III

1998 ◽  
Vol 18 (8) ◽  
pp. 4629-4638 ◽  
Author(s):  
Christin Tse ◽  
Takashi Sera ◽  
Alan P. Wolffe ◽  
Jeffrey C. Hansen
Keyword(s):  
Rna Polymerase ◽  
Histone Acetylation ◽  
Molecular Mechanisms ◽  
Chromatin Condensation ◽  
Rna Polymerase Iii ◽  
Higher Order ◽  
Core Histone ◽  
Rrna Gene ◽  
Histone Octamer ◽  
Polymerase Iii

ABSTRACT We have examined the effects of core histone acetylation on the transcriptional activity and higher-order folding of defined 12-mer nucleosomal arrays. Purified HeLa core histone octamers containing an average of 2, 6, or 12 acetates per octamer (8, 23, or 46% maximal site occupancy, respectively) were assembled onto a DNA template consisting of 12 tandem repeats of a 208-bp Lytechinus 5S rRNA gene fragment. Reconstituted nucleosomal arrays were transcribed in a Xenopus oocyte nuclear extract and analyzed by analytical hydrodynamic and electrophoretic approaches to determine the extent of array compaction. Results indicated that in buffer containing 5 mM free Mg2+ and 50 mM KCl, high levels of acetylation (12 acetates/octamer) completely inhibited higher-order folding and concurrently led to a 15-fold enhancement of transcription by RNA polymerase III. The molecular mechanisms underlying the acetylation effects on chromatin condensation were investigated by analyzing the ability of differentially acetylated nucleosomal arrays to fold and oligomerize. In MgCl2-containing buffer the folding of 12-mer nucleosomal arrays containing an average of two or six acetates per histone octamer was indistinguishable, while a level of 12 acetates per octamer completely disrupted the ability of nucleosomal arrays to form higher-order folded structures at all ionic conditions tested. In contrast, there was a linear relationship between the extent of histone octamer acetylation and the extent of disruption of Mg2+-dependent oligomerization. These results have yielded new insight into the molecular basis of acetylation effects on both transcription and higher-order compaction of nucleosomal arrays.

scholarly journals Mitotic repression of transcription in vitro.

1993 ◽  
Vol 120 (3) ◽  
pp. 613-624 ◽  
Author(s):  
P Hartl ◽  
J Gottesfeld ◽  
D J Forbes
Keyword(s):  
Protein Kinase ◽  
Rna Polymerase ◽  
Topoisomerase Ii ◽  
Chromatin Condensation ◽  
Rna Polymerase Iii ◽  
Protein Kinase Activity ◽  
Cdc2 Kinase ◽  
Polymerase Iii ◽  
Transcription In Vitro

A normal consequence of mitosis in eukaryotes is the repression of transcription. Using Xenopus egg extracts shifted to a mitotic state by the addition of purified cyclin, we have for the first time been able to reproduce a mitotic repression of transcription in vitro. Active RNA polymerase III transcription is observed in interphase extracts, but strongly repressed in extracts converted to mitosis. With the topoisomerase II inhibitor VM-26, we demonstrate that this mitotic repression of RNA polymerase III transcription does not require normal chromatin condensation. Similarly; in vitro mitotic repression of transcription does not require the presence of nucleosome structure or involve a general repressive chromatin-binding protein, as inhibition of chromatin formation with saturating amounts of non-specific DNA has no effect on repression. Instead, the mitotic repression of transcription appears to be due to phosphorylation of a component of the transcription machinery by a mitotic protein kinase, either cdc2 kinase and/or a kinase activated by it. Mitotic repression of RNA polymerase III transcription is observed both in complete mitotic cytosol and when a kinase-enriched mitotic fraction is added to a highly simplified 5S RNA transcription reaction. We present evidence that, upon depletion of cdc2 kinase, a secondary protein kinase activity remains and can mediate this in vitro mitotic repression of transcription.

DNA methylation inhibits transcription by RNA polymerase III of a tRNA gene, but not of a 5S rRNA gene

FEBS Letters ◽  
1990 ◽  
Vol 269 (2) ◽  
pp. 358-362 ◽  
Author(s):  
Daniel Besser ◽  
Frank Götz ◽  
Kai Schulze-Forster ◽  
Herbert Wagner ◽  
Hans Kröger ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Rna Polymerase ◽  
Trna Gene ◽  
Rna Polymerase Iii ◽  
5S Rrna ◽  
Rrna Gene ◽  
Polymerase Iii ◽  
5S Rrna Gene

Molecular mechanisms of chromatin transcription by RNA polymerase III. Part 1

2012 ◽  
Vol 67 (3-4) ◽  
pp. 96-100 ◽  
Author(s):  
V. M. Studitskii ◽  
I. V. Orlovskii ◽  
O. V. Chertkov ◽  
N. S. Efimova ◽  
M. A. Loginova ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Molecular Mechanisms ◽  
Rna Polymerase Iii ◽  
Polymerase Iii

Transcription termination by RNA polymerase III: uncoupling of polymerase release from termination signal recognition

1992 ◽  
Vol 12 (5) ◽  
pp. 2260-2272
Author(s):  
F E Campbell ◽  
D R Setzer
Keyword(s):  
Rna Polymerase ◽  
Transcription Termination ◽  
Transcription Elongation ◽  
Rna Polymerase Iii ◽  
5S Rrna ◽  
Rrna Gene ◽  
Signal Recognition ◽  
Polymerase Iii ◽  
5S Rrna Gene ◽  
Nascent Rna

Xenopus RNA polymerase III specifically initiates transcription on poly(dC)-tailed DNA templates in the absence of other class III transcription factors normally required for transcription initiation. In experimental analyses of transcription termination using DNA fragments with a 5S rRNA gene positioned downstream of the tailed end, only 40% of the transcribing polymerase molecules terminate at the normally efficient Xenopus borealis somatic-type 5S rRNA terminators; the remaining 60% read through these signals and give rise to runoff transcripts. We find that the nascent RNA strand is inefficiently displaced from the DNA template during transcription elongation. Interestingly, only polymerases synthesizing a displaced RNA terminate at the 5S rRNA gene terminators; when the nascent RNA is not displaced from the template, read-through transcripts are synthesized. RNAs with 3' ends at the 5S rRNA gene terminators are judged to result from authentic termination events on the basis of multiple criteria, including kinetic properties, the precise 3' ends generated, release of transcripts from the template, and recycling of the polymerase. Even though only 40% of the polymerase molecules ultimately terminate at either of the tandem 5S rRNA gene terminators, virtually all polymerases pause there, demonstrating that termination signal recognition can be experimentally uncoupled from polymerase release. Thus, termination is dependent on RNA strand displacement during transcription elongation, whereas termination signal recognition is not. We interpret our results in terms of a two-step model for transcription termination in which polymerase release is dependent on the fate of the nascent RNA strand during transcription elongation.

scholarly journals Displacement of Xenopus transcription factor IIIA from a 5S rRNA gene by a transcribing RNA polymerase.

1991 ◽  
Vol 11 (8) ◽  
pp. 3978-3986 ◽  
Author(s):  
F E Campbell ◽  
D R Setzer
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Rna Polymerase ◽  
Internal Control ◽  
Rna Polymerase Iii ◽  
5S Rrna ◽  
Rrna Gene ◽  
Transcription Factor Iiia ◽  
Polymerase Iii ◽  
5S Rrna Gene

In the absence of other components of the RNA polymerase III transcription machinery, transcription factor IIIA (TFIIIA) can be displaced from both strands of its DNA-binding site (the internal control region) on the somatic-type 5S rRNA gene of Xenopus borealis during transcription elongation by bacteriophage T7 RNA polymerase, regardless of which DNA strand is transcribed. Furthermore, substantial displacement is observed after the template has been transcribed only once. Since the complete 5S rRNA transcription complex has previously been shown to remain stably bound to the gene during repeated rounds of transcription by either RNA polymerase III or bacteriophage SP6 RNA polymerase, these results indicate that a factor(s) in addition to TFIIIA is required to create a complex that will remain stably associated with the template during transcription. Thus, transcription complex stability during passage of RNA polymerase cannot be explained solely on the basis of the DNA-binding properties of TFIIIA.

scholarly journals Molecular mechanisms of Bdp1 in TFIIIB assembly and RNA polymerase III transcription initiation

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Jerome Gouge ◽  
Nicolas Guthertz ◽  
Kevin Kramm ◽  
Oleksandr Dergai ◽  
Guillermo Abascal-Palacios ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Rna Polymerase Iii ◽  
Polymerase Iii

Transcription factor requirements for in vitro formation of transcriptionally competent 5S rRNA gene chromatin

1990 ◽  
Vol 10 (5) ◽  
pp. 2390-2401
Author(s):  
S J Felts ◽  
P A Weil ◽  
R Chalkley
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
5S Rrna ◽  
Rrna Gene ◽  
Active Chromatin ◽  
Polymerase Iii ◽  
Protein Dna Interactions ◽  
5S Rrna Gene

The Saccharomyces cerevisiae 5S rRNA gene was used as a model system to study the requirements for assembling transcriptionally active chromatin in vitro with purified components. When a plasmid containing yeast 5S rDNA was assembled into chromatin with purified core histones, the gene was inaccessible to the yeast class III gene transcription machinery. Preformation of a 5S rRNA gene-TFIIIA complex was not sufficient for the formation of active chromatin in this in vitro system. Instead, a complete transcription factor complex consisting of TFIIIA, TFIIIB, and TFIIIC needed to be formed before the addition of histones in order for the 5S chromatin to subsequently be transcribed by RNA polymerase III. Various 5S rRNA maxigenes were constructed and used for chromatin assembly studies. In vitro transcription from these assembled 5S maxigenes revealed that RNA polymerase III was readily able to transcribe through one, two, or four nucleosomes. However, we found that RNA polymerase III was not able to efficiently transcribe a chromatin template containing a more extended array of nucleosomes. In vivo expression experiments indicated that all in vitro-constructed maxigenes were transcriptionally competent. Analyses of protein-DNA interactions formed on these maxigenes in vivo by indirect end labeling indicated that there are extensive interactions throughout the length of these maxigenes. The patterns of protein-DNA interactions formed on these genes are consistent with these DNAs being assembled into extensive nucleosomal arrays.

scholarly journals Transcription factor requirements for in vitro formation of transcriptionally competent 5S rRNA gene chromatin.

1990 ◽  
Vol 10 (5) ◽  
pp. 2390-2401 ◽  
Author(s):  
S J Felts ◽  
P A Weil ◽  
R Chalkley
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
5S Rrna ◽  
Rrna Gene ◽  
Active Chromatin ◽  
Polymerase Iii ◽  
Protein Dna Interactions ◽  
5S Rrna Gene

The Saccharomyces cerevisiae 5S rRNA gene was used as a model system to study the requirements for assembling transcriptionally active chromatin in vitro with purified components. When a plasmid containing yeast 5S rDNA was assembled into chromatin with purified core histones, the gene was inaccessible to the yeast class III gene transcription machinery. Preformation of a 5S rRNA gene-TFIIIA complex was not sufficient for the formation of active chromatin in this in vitro system. Instead, a complete transcription factor complex consisting of TFIIIA, TFIIIB, and TFIIIC needed to be formed before the addition of histones in order for the 5S chromatin to subsequently be transcribed by RNA polymerase III. Various 5S rRNA maxigenes were constructed and used for chromatin assembly studies. In vitro transcription from these assembled 5S maxigenes revealed that RNA polymerase III was readily able to transcribe through one, two, or four nucleosomes. However, we found that RNA polymerase III was not able to efficiently transcribe a chromatin template containing a more extended array of nucleosomes. In vivo expression experiments indicated that all in vitro-constructed maxigenes were transcriptionally competent. Analyses of protein-DNA interactions formed on these maxigenes in vivo by indirect end labeling indicated that there are extensive interactions throughout the length of these maxigenes. The patterns of protein-DNA interactions formed on these genes are consistent with these DNAs being assembled into extensive nucleosomal arrays.

Molecular mechanisms of transcription through chromatin by RNA polymerase III: Part 2

2012 ◽  
Vol 67 (3-4) ◽  
pp. 101-106 ◽  
Author(s):  
V. M. Studitskii ◽  
I. V. Orlovskii ◽  
O. V. Chertkov ◽  
N. S. Efimova ◽  
M. A. Loginova ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Molecular Mechanisms ◽  
Rna Polymerase Iii ◽  
Polymerase Iii

scholarly journals Ex vivo visualization of RNA polymerase III-specific gene activity with electron microscopy

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Sina Manger ◽  
Utz H. Ermel ◽  
Achilleas S. Frangakis
Keyword(s):  
Electron Microscopy ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Gene Clusters ◽  
Gene Activity ◽  
Foreign Gene ◽  
Specific Gene ◽  
Rrna Gene ◽  
Direct Visualization ◽  
Polymerase Iii

AbstractThe direct study of transcription or DNA–protein-binding events, requires imaging of individual genes at molecular resolution. Electron microscopy (EM) can show local detail of the genome. However, direct visualization and analysis of specific individual genes is currently not feasible as they cannot be unambiguously localized in the crowded, landmark-free environment of the nucleus. Here, we present a method for the genomic insertion of gene clusters that can be localized and imaged together with their associated protein complexes in the EM. The method uses CRISPR/Cas9 technology to incorporate several genes of interest near the 35S rRNA gene, which is a frequently occurring, easy-to-identify genomic locus within the nucleolus that can be used as a landmark in micrographs. As a proof of principle, we demonstrate the incorporation of the locus-native gene RDN5 and the locus-foreign gene HSX1. This led to a greater than 7-fold enrichment of RNA polymerase III (Pol III) complexes associated with the genes within the field of view, allowing for a significant increase in the analysis yield. This method thereby allows for the insertion and direct visualization of gene clusters for a range of analyses, such as changes in gene activity upon alteration of cellular or external factors.

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