scholarly journals TbISWI Regulates Multiple Polymerase I (Pol I)-Transcribed Loci and Is Present at Pol II Transcription Boundaries in Trypanosoma brucei

2011 ◽  
Vol 10 (7) ◽  
pp. 964-976 ◽  
Author(s):  
Tara M. Stanne ◽  
Manish Kushwaha ◽  
Matthew Wand ◽  
Jesse E. Taylor ◽  
Gloria Rudenko
Keyword(s):  
Rna Polymerase Ii ◽  
Trypanosoma Brucei ◽  
Transcription Initiation ◽  
Transcriptional Control ◽  
Histone Variants ◽  
Unicellular Eukaryote ◽  
Pol Ii ◽  
Content Type ◽  
Pol I ◽  
Switch Regions

ABSTRACTThe unicellular eukaryoteTrypanosoma bruceiis unusual in having very little transcriptional control. The bulk of theT. bruceigenome is constitutively transcribed by RNA polymerase II (Pol II) as extensive polycistronic transcription units. Exceptions to this rule include several RNA Pol I transcription units such as theVSGexpression sites (ESs), which are mono-allelically expressed. TbISWI, a member of the SWI2/SNF2 related chromatin remodeling ATPases, plays a role in repression of Pol I-transcribed ESs in both bloodstream- and procyclic-formT. brucei. We show that TbISWI binds both active and silent ESs but is depleted from the ES promoters themselves. TbISWI knockdown results in an increase inVSGtranscripts from the silentVSGESs. In addition to its role in the repression of the silent ESs, TbISWI also contributes to the downregulation of the Pol I-transcribed procyclin loci, as well as nontranscribedVSGbasic copy arrays and minichromosomes. We also show that TbISWI is enriched at a number of strand switch regions which form the boundaries between Pol II transcription units. These strand switch regions are the presumed sites of Pol II transcription initiation and termination and are enriched in modified histones and histone variants. Our results indicate that TbISWI is a versatile chromatin remodeler that regulates transcription at multiple Pol I loci and is particularly abundant at many Pol II transcription boundaries inT. brucei.

scholarly journals Antagonistic cotranscriptional regulation through ARGONAUTE1 and the THO/TREX complex orchestrates FLC transcriptional output

2021 ◽  
Vol 118 (47) ◽  
pp. e2113757118
Author(s):  
Congyao Xu ◽  
Xiaofeng Fang ◽  
Tiancong Lu ◽  
Caroline Dean
Keyword(s):  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Transcriptional Control ◽  
Antisense Transcript ◽  
Elongation Rate ◽  
Pol Ii ◽  
Alternative Processing ◽  
Floral Repressor ◽  
Related Proteins ◽  
Transcriptional Output

Quantitative transcriptional control is essential for physiological and developmental processes in many organisms. Transcriptional output is influenced by cotranscriptional processes interconnected to chromatin regulation, but how the functions of different cotranscriptional regulators are integrated is poorly understood. The Arabidopsis floral repressor locus FLOWERING LOCUS C (FLC) is cotranscriptionally repressed by alternative processing of the antisense transcript COOLAIR. Proximal 3′-end processing of COOLAIR resolves a cotranscriptionally formed R-loop, and this process physically links to a histone-modifying complex FLD/SDG26/LD. This induces a chromatin environment locally that determines low transcription initiation and a slow elongation rate to both sense and antisense strands. Here, we show that ARGONAUTE1 (AGO1) genetically functions in this cotranscriptional repression mechanism. AGO1 associates with COOLAIR and influences COOLAIR splicing dynamics to promote proximal COOLAIR, R-loop resolution, and chromatin silencing. Proteomic analyses revealed physical associations between AGO1, subunits of RNA Polymerase II (Pol II), the splicing-related proteins—the spliceosome NineTeen Complex (NTC) and related proteins (NTR)—and the THO/TREX complex. We connect these activities by demonstrating that the THO/TREX complex activates FLC expression acting antagonistically to AGO1 in COOLAIR processing. Together these data reveal that antagonistic cotranscriptional regulation through AGO1 or THO/TREX influences COOLAIR processing to deliver a local chromatin environment that determines FLC transcriptional output. The involvement of these conserved cotranscriptional regulators suggests similar mechanisms may underpin quantitative transcriptional regulation generally.

scholarly journals Disruption of largest subunit RNA polymerase II genes in Trypanosoma brucei.

1993 ◽  
Vol 13 (6) ◽  
pp. 3734-3743 ◽  
Author(s):  
H M Chung ◽  
M G Lee ◽  
P Dietrich ◽  
J Huang ◽  
L H Van der Ploeg
Keyword(s):  
Amino Acid ◽  
Rna Polymerase Ii ◽  
Trypanosoma Brucei ◽  
Promoter Region ◽  
Gene Promoter ◽  
Amino Acid Substitutions ◽  
Pol Ii ◽  
Pol I ◽  
Alpha Amanitin ◽  
Gene Promoter Region

Two types of largest subunit RNA polymerase II (pol II) genes (pol IIA and pol IIB), differing in 3 amino acid substitutions, are encoded in the Trypanosoma brucei (stock 427-60) genome. As a result, the alpha-amanitin-resistant transcription of the procyclic acidic repetitive protein (PARP) and variant surface glycoprotein (VSG) genes was proposed to involve a modified, alpha-amanitin-resistant form of the largest subunit of pol II. Alternatively, pol I could transcribe the PARP and VSG genes. To discriminate between these two models, we deleted the N-terminal domain (about one-third of the polypeptide), which encodes the amino acid substitutions which discriminated the pol IIA and pol IIB genes, at both pol IIB alleles. The pol IIB- trypanosomes still transcribe the PARP genes and the VSG gene promoter region in insect-form trypanosomes by alpha-amanitin-resistant RNA polymerases, while control housekeeping genes are transcribed in an alpha-amanitin-sensitive manner, presumably by pol IIA. We conclude that the alpha-amanitin-resistant transcription of protein coding genes in T. brucei is not mediated by a diverged form of the largest subunit of pol II and that the presence of both the pol IIA and pol IIB genes is not essential for trypanosome viability. This conclusion was further supported by the finding that individual trypanosome variants exhibited allelic heterogeneity for the previously identified amino acid substitutions and that various permutations of the polymorphic amino acids generate at least four different types of largest subunit pol II genes. The expression of the PARP genes and the VSG gene promoter region by alpha-amanitin-resistant RNA polymerases in the pol IIB- trypanosomes provides evidence for transcription of these genes by pol I.

scholarly journals Trypanosoma brucei ribonuclease H2A is an essential R-loop processing enzyme whose loss causes DNA damage during transcription initiation and antigenic variation

2019 ◽  
Vol 47 (17) ◽  
pp. 9180-9197 ◽  
Author(s):  
Emma Briggs ◽  
Kathryn Crouch ◽  
Leandro Lemgruber ◽  
Graham Hamilton ◽  
Craig Lapsley ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Damage ◽  
Trypanosoma Brucei ◽  
Transcription Initiation ◽  
Critical Role ◽  
Rnase H ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Pol I ◽  
Rnase H2

Abstract Ribonucleotides represent a threat to DNA genome stability and transmission. Two types of Ribonuclease H (RNase H) excise ribonucleotides when they form part of the DNA strand, or hydrolyse RNA when it base-pairs with DNA in structures termed R-loops. Loss of either RNase H is lethal in mammals, whereas yeast survives the absence of both enzymes. RNase H1 loss is tolerated by the parasite Trypanosoma brucei but no work has examined the function of RNase H2. Here we show that loss of T. brucei RNase H2 (TbRH2A) leads to growth and cell cycle arrest that is concomitant with accumulation of nuclear damage at sites of RNA polymerase (Pol) II transcription initiation, revealing a novel and critical role for RNase H2. Differential gene expression analysis reveals limited overall changes in RNA levels for RNA Pol II genes after TbRH2A loss, but increased perturbation of nucleotide metabolic genes. Finally, we show that TbRH2A loss causes R-loop and DNA damage accumulation in telomeric RNA Pol I transcription sites, also leading to altered gene expression. Thus, we demonstrate separation of function between two nuclear T. brucei RNase H enzymes during RNA Pol II transcription, but overlap in function during RNA Pol I-mediated gene expression during host immune evasion.

scholarly journals Trypanosoma brucei ribonuclease H2A is an essential enzyme that resolves R-loops associated with transcription initiation and antigenic variation

2019 ◽  
Author(s):  
Emma Briggs ◽  
Kathryn Crouch ◽  
Leandro Lemgruber ◽  
Graham Hamilton ◽  
Craig Lapsley ◽  
...  
Keyword(s):  
Gene Expression ◽  
Trypanosoma Brucei ◽  
Transcription Initiation ◽  
Antigenic Variation ◽  
Critical Role ◽  
Rnase H ◽  
Surface Glycoprotein ◽  
Pol Ii ◽  
Pol I ◽  
Rnase H2

In every cell ribonucleotides represent a threat to the stability and transmission of the DNA genome. Two types of Ribonuclease H (RNase H) tackle such ribonucleotides, either by excision when they form part of the DNA strand, or by hydrolysing RNA when it base-pairs with DNA, in structures termed R-loops. Loss of either RNase H is lethal in mammals, whereas yeast can prosper in the absence of both enzymes. Removal of RNase H1 is tolerated by the parasite Trypanosoma brucei but no work has examined the function of RNase H2. Here we show that loss of the catalytic subunit of T. brucei RNase H2 (TbRH2A) leads to growth and cell cycle arrest that is concomitant with accumulation of nuclear damage at sites of RNA polymerase (Pol) II transcription initiation, revealing a novel and critical role for RNase H2. In addition, differential gene expression of both RNA Pol I and II transcribed genes occurs after TbRH2A loss, including patterns that may relate to cytosolic DNA accumulation in humans with autoimmune disease. Finally, we show that TbRH2A loss causes R-loop and DNA damage accumulation in telomeric RNA Pol I transcription sites, leading to altered variant surface glycoprotein expression. Thus, we demonstrate a separation of function between the two nuclear T. brucei RNase H enzymes during RNA Pol II transcription, but overlap in function during RNA Pol I-mediated gene expression during host immune evasion.

Disruption of largest subunit RNA polymerase II genes in Trypanosoma brucei

1993 ◽  
Vol 13 (6) ◽  
pp. 3734-3743
Author(s):  
H M Chung ◽  
M G Lee ◽  
P Dietrich ◽  
J Huang ◽  
L H Van der Ploeg
Keyword(s):  
Amino Acid ◽  
Rna Polymerase Ii ◽  
Trypanosoma Brucei ◽  
Promoter Region ◽  
Gene Promoter ◽  
Amino Acid Substitutions ◽  
Pol Ii ◽  
Pol I ◽  
Alpha Amanitin ◽  
Gene Promoter Region

Two types of largest subunit RNA polymerase II (pol II) genes (pol IIA and pol IIB), differing in 3 amino acid substitutions, are encoded in the Trypanosoma brucei (stock 427-60) genome. As a result, the alpha-amanitin-resistant transcription of the procyclic acidic repetitive protein (PARP) and variant surface glycoprotein (VSG) genes was proposed to involve a modified, alpha-amanitin-resistant form of the largest subunit of pol II. Alternatively, pol I could transcribe the PARP and VSG genes. To discriminate between these two models, we deleted the N-terminal domain (about one-third of the polypeptide), which encodes the amino acid substitutions which discriminated the pol IIA and pol IIB genes, at both pol IIB alleles. The pol IIB- trypanosomes still transcribe the PARP genes and the VSG gene promoter region in insect-form trypanosomes by alpha-amanitin-resistant RNA polymerases, while control housekeeping genes are transcribed in an alpha-amanitin-sensitive manner, presumably by pol IIA. We conclude that the alpha-amanitin-resistant transcription of protein coding genes in T. brucei is not mediated by a diverged form of the largest subunit of pol II and that the presence of both the pol IIA and pol IIB genes is not essential for trypanosome viability. This conclusion was further supported by the finding that individual trypanosome variants exhibited allelic heterogeneity for the previously identified amino acid substitutions and that various permutations of the polymorphic amino acids generate at least four different types of largest subunit pol II genes. The expression of the PARP genes and the VSG gene promoter region by alpha-amanitin-resistant RNA polymerases in the pol IIB- trypanosomes provides evidence for transcription of these genes by pol I.

scholarly journals The cryoelectron microscopy structure of the human CDK-activating kinase

2020 ◽  
Vol 117 (37) ◽  
pp. 22849-22857 ◽  
Author(s):  
Basil J. Greber ◽  
Juan M. Perez-Bertoldi ◽  
Kif Lim ◽  
Anthony T. Iavarone ◽  
Daniel B. Toso ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Rational Design ◽  
Control Cell ◽  
3D Structure ◽  
Structural Basis ◽  
Pol Ii ◽  
Insight Into ◽  
Cdk Activating Kinase

The human CDK-activating kinase (CAK), a complex composed of cyclin-dependent kinase (CDK) 7, cyclin H, and MAT1, is a critical regulator of transcription initiation and the cell cycle. It acts by phosphorylating the C-terminal heptapeptide repeat domain of the RNA polymerase II (Pol II) subunit RPB1, which is an important regulatory event in transcription initiation by Pol II, and it phosphorylates the regulatory T-loop of CDKs that control cell cycle progression. Here, we have determined the three-dimensional (3D) structure of the catalytic module of human CAK, revealing the structural basis of its assembly and providing insight into CDK7 activation in this context. The unique third component of the complex, MAT1, substantially extends the interaction interface between CDK7 and cyclin H, explaining its role as a CAK assembly factor, and it forms interactions with the CDK7 T-loop, which may contribute to enhancing CAK activity. We have also determined the structure of the CAK in complex with the covalently bound inhibitor THZ1 in order to provide insight into the binding of inhibitors at the CDK7 active site and to aid in the rational design of therapeutic compounds.

Structural Biology of RNA Polymerase II Transcription: 20 Years On

2020 ◽  
Vol 36 (1) ◽  
pp. 1-34 ◽  
Author(s):  
Sara Osman ◽  
Patrick Cramer
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Focal Point ◽  
Dna Lesions ◽  
Cellular Regulation ◽  
Pol Ii ◽  
Associated Proteins ◽  
Rna Polymerase Ii Transcription

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.

scholarly journals Sir2 Silences Gene Transcription by Targeting the Transition between RNA Polymerase II Initiation and Elongation

2008 ◽  
Vol 28 (12) ◽  
pp. 3979-3994 ◽  
Author(s):  
Lu Gao ◽  
David S. Gross
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Transcriptional Silencing ◽  
Pol Ii ◽  
Initiation Factors ◽  
Lysine Deacetylase ◽  
Evolutionarily Conserved ◽  
Mrna Capping ◽  
Capping Enzyme

ABSTRACT It is well accepted that for transcriptional silencing in budding yeast, the evolutionarily conserved lysine deacetylase Sir2, in concert with its partner proteins Sir3 and Sir4, establishes a chromatin structure that prevents RNA polymerase II (Pol II) transcription. However, the mechanism of repression remains controversial. Here, we show that the recruitment of Pol II, as well as that of the general initiation factors TBP and TFIIH, occurs unimpeded to the silent HMR a 1 and HMLα1/HMLα2 mating promoters. This, together with the fact that Pol II is Ser5 phosphorylated, implies that SIR-mediated silencing is permissive to both preinitiation complex (PIC) assembly and transcription initiation. In contrast, the occupancy of factors critical to both mRNA capping and Pol II elongation, including Cet1, Abd1, Spt5, Paf1C, and TFIIS, is virtually abolished. In agreement with this, efficiency of silencing correlates not with a restriction in Pol II promoter occupancy but with a restriction in capping enzyme recruitment. These observations pinpoint the transition between polymerase initiation and elongation as the step targeted by Sir2 and indicate that transcriptional silencing is achieved through the differential accessibility of initiation and capping/elongation factors to chromatin. We compare Sir2-mediated transcriptional silencing to a second repression mechanism, mediated by Tup1. In contrast to Sir2, Tup1 prevents TBP, Pol II, and TFIIH recruitment to the HMLα1 promoter, thereby abrogating PIC formation.

scholarly journals A Nonconserved Surface of the TFIIB Zinc Ribbon Domain Plays a Direct Role in RNA Polymerase II Recruitment

2004 ◽  
Vol 24 (7) ◽  
pp. 2863-2874 ◽  
Author(s):  
Thomas C. Tubon ◽  
William P. Tansey ◽  
Winship Herr
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Terminal Region ◽  
General Role ◽  
Pol Ii ◽  
Amino Terminal ◽  
Zinc Ribbon

ABSTRACT The general transcription factor TFIIB is a highly conserved and essential component of the eukaryotic RNA polymerase II (pol II) transcription initiation machinery. It consists of a single polypeptide with two conserved structural domains: an amino-terminal zinc ribbon structure (TFIIBZR) and a carboxy-terminal core (TFIIBCORE). We have analyzed the role of the amino-terminal region of human TFIIB in transcription in vivo and in vitro. We identified a small nonconserved surface of the TFIIBZR that is required for pol II transcription in vivo and for different types of basal pol II transcription in vitro. Consistent with a general role in transcription, this TFIIBZR surface is directly involved in the recruitment of pol II to a TATA box-containing promoter. Curiously, although the amino-terminal human TFIIBZR domain can recruit both human pol II and yeast (Saccharomyces cerevisiae) pol II, the yeast TFIIB amino-terminal region recruits yeast pol II but not human pol II. Thus, a critical process in transcription from many different promoters—pol II recruitment—has changed in sequence specificity during eukaryotic evolution.

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