scholarly journals Structure of an RNA Polymerase II–TFIIB Complex and the Transcription Initiation Mechanism

Science ◽  
2009 ◽  
Vol 327 (5962) ◽  
pp. 206-209 ◽  
Author(s):  
Xin Liu ◽  
David A. Bushnell ◽  
Dong Wang ◽  
Guillermo Calero ◽  
Roger D. Kornberg
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Active Center ◽  
Transcription Initiation ◽  
Terminal Region ◽  
Initiation Mechanism ◽  
Amino Terminal ◽  
Dna And Rna ◽  
General Transcription Factors ◽  
Carboxyl Terminal

Previous x-ray crystal structures have given insight into the mechanism of transcription and the role of general transcription factors in the initiation of the process. A structure of an RNA polymerase II–general transcription factor TFIIB complex at 4.5 angstrom resolution revealed the amino-terminal region of TFIIB, including a loop termed the “B finger,” reaching into the active center of the polymerase where it may interact with both DNA and RNA, but this structure showed little of the carboxyl-terminal region. A new crystal structure of the same complex at 3.8 angstrom resolution obtained under different solution conditions is complementary with the previous one, revealing the carboxyl-terminal region of TFIIB, located above the polymerase active center cleft, but showing none of the B finger. In the new structure, the linker between the amino- and carboxyl-terminal regions can also be seen, snaking down from above the cleft toward the active center. The two structures, taken together with others previously obtained, dispel long-standing mysteries of the transcription initiation process.

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.

scholarly journals Structure of TFIIK for phosphorylation of CTD of RNA polymerase II

2021 ◽  
Vol 7 (15) ◽  
pp. eabd4420
Author(s):  
Trevor van Eeuwen ◽  
Tao Li ◽  
Hee Jong Kim ◽  
Jose J. Gorbea Colón ◽  
Mitchell I. Parker ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Activation Loop ◽  
Active Conformation ◽  
General Transcription Factor ◽  
Cryo Electron Microscopy ◽  
Proposed Model ◽  
Carboxyl Terminal ◽  
Catalytic Cleft

During transcription initiation, the general transcription factor TFIIH marks RNA polymerase II by phosphorylating Ser5 of the carboxyl-terminal domain (CTD) of Rpb1, which is followed by extensive modifications coupled to transcription elongation, mRNA processing, and histone dynamics. We have determined a 3.5-Å resolution cryo–electron microscopy (cryo-EM) structure of the TFIIH kinase module (TFIIK in yeast), which is composed of Kin28, Ccl1, and Tfb3, yeast homologs of CDK7, cyclin H, and MAT1, respectively. The carboxyl-terminal region of Tfb3 was lying at the edge of catalytic cleft of Kin28, where a conserved Tfb3 helix served to stabilize the activation loop in its active conformation. By combining the structure of TFIIK with the previous cryo-EM structure of the preinitiation complex, we extend the previously proposed model of the CTD path to the active site of TFIIK.

The Long Road to Understanding RNAPII Transcription Initiation and Related Syndromes

2021 ◽  
Vol 90 (1) ◽  
pp. 193-219
Author(s):  
Emmanuel Compe ◽  
Jean-Marc Egly
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase Ii ◽  
Chromatin Remodeling ◽  
Transcription Initiation ◽  
Rna Synthesis ◽  
Regulatory Elements ◽  
Initiation Mechanism ◽  
Protein Coding ◽  
Core Promoters ◽  
General Transcription Factors

In eukaryotes, transcription of protein-coding genes requires the assembly at core promoters of a large preinitiation machinery containing RNA polymerase II (RNAPII) and general transcription factors (GTFs). Transcription is potentiated by regulatory elements called enhancers, which are recognized by specific DNA-binding transcription factors that recruit cofactors and convey, following chromatin remodeling, the activating cues to the preinitiation complex. This review summarizes nearly five decades of work on transcription initiation by describing the sequential recruitment of diverse molecular players including the GTFs, the Mediator complex, and DNA repair factors that support RNAPII to enable RNA synthesis. The elucidation of the transcription initiation mechanism has greatly benefited from the study of altered transcription components associated with human diseases that could be considered transcription syndromes.

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

1999 ◽  
Vol 19 (3) ◽  
pp. 2130-2141 ◽  
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.

scholarly journals Mutational analysis of the D1/E1 core helices and the conserved N-terminal region of yeast transcription factor IIB (TFIIB): identification of an N-terminal mutant that stabilizes TATA-binding protein-TFIIB-DNA complexes.

1997 ◽  
Vol 17 (12) ◽  
pp. 6784-6793 ◽  
Author(s):  
C S Bangur ◽  
T S Pardee ◽  
A S Ponticelli
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Terminal Region ◽  
Amino Terminal ◽  
Transcription Factor Iib

The general transcription factor IIB (TFIIB) plays an essential role in transcription of protein-coding genes by RNA polymerase II. We have used site-directed mutagenesis to assess the role of conserved amino acids in several important regions of yeast TFIIB. These include residues in the highly conserved amino-terminal region and basic residues in the D1 and E1 core domain alpha-helices. Acidic substitutions of residues K190 (D1) and K201 (E1) resulted in growth impairments in vivo, reduced basal transcriptional activity in vitro, and an inability to form stable TFIIB-TATA-binding protein-DNA (DB) complexes. Significantly, these mutants retained the ability to respond to acidic activators in vivo and to the Gal4-VP16 activator in vitro, supporting the view that these basic residues play a role in basal transcription. In addition, 14 single-amino-acid substitutions were introduced in the conserved amino-terminal region. Three of these mutants, the L50D, R64E, and R78L mutants, displayed altered growth properties in vivo and were compromised for supporting transcription in vitro. The L50D mutant was impaired for RNA polymerase II interaction, while the R64E mutant exhibited altered transcription start site selection both in vitro and in vivo and, surprisingly, was more active than the wild type in the formation of stable DB complexes. These results support the view that the amino-terminal domain is involved in the direct interaction between yeast TFIIB and RNA polymerase II and suggest that this domain may interact with DNA and/or modulate the formation of a DB complex.

scholarly journals Dynamic sumoylation of promoter-bound general transcription factors facilitates transcription by RNA polymerase II

PLoS Genetics ◽  
2021 ◽  
Vol 17 (9) ◽  
pp. e1009828
Author(s):  
Mohammad S. Baig ◽  
Yimo Dou ◽  
Benjamin G. Bergey ◽  
Russell Bahar ◽  
Justin M. Burgener ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Ribosomal Proteins ◽  
Protein Coding ◽  
General Transcription Factors ◽  
Genes Encoding ◽  
Associated Proteins ◽  
Related Proteins

Transcription-related proteins are frequently identified as targets of sumoylation, including multiple subunits of the RNA polymerase II (RNAPII) general transcription factors (GTFs). However, it is not known how sumoylation affects GTFs or whether they are sumoylated when they assemble at promoters to facilitate RNAPII recruitment and transcription initiation. To explore how sumoylation can regulate transcription genome-wide, we performed SUMO ChIP-seq in yeast and found, in agreement with others, that most chromatin-associated sumoylated proteins are detected at genes encoding tRNAs and ribosomal proteins (RPGs). However, we also detected 147 robust SUMO peaks at promoters of non-ribosomal protein-coding genes (non-RPGs), indicating that sumoylation also regulates this gene class. Importantly, SUMO peaks at non-RPGs align specifically with binding sites of GTFs, but not other promoter-associated proteins, indicating that it is GTFs specifically that are sumoylated there. Predominantly, non-RPGs with SUMO peaks are among the most highly transcribed, have high levels of TFIIF, and show reduced RNAPII levels when cellular sumoylation is impaired, linking sumoylation with elevated transcription. However, detection of promoter-associated SUMO by ChIP might be limited to sites with high levels of substrate GTFs, and promoter-associated sumoylation at non-RPGs may actually be far more widespread than we detected. Among GTFs, we found that TFIIF is a major target of sumoylation, specifically at lysines 60/61 of its Tfg1 subunit, and elevating Tfg1 sumoylation resulted in decreased interaction of TFIIF with RNAPII. Interestingly, both reducing promoter-associated sumoylation, in a sumoylation-deficient Tfg1-K60/61R mutant strain, and elevating promoter-associated SUMO levels, by constitutively tethering SUMO to Tfg1, resulted in reduced RNAPII occupancy at non-RPGs. This implies that dynamic GTF sumoylation at non-RPG promoters, not simply the presence or absence of SUMO, is important for maintaining elevated transcription. Together, our findings reveal a novel mechanism of regulating the basal transcription machinery through sumoylation of promoter-bound GTFs.

scholarly journals Construction and analysis of yeast RNA polymerase II CTD deletion and substitution mutations.

Genetics ◽  
1995 ◽  
Vol 140 (4) ◽  
pp. 1223-1233
Author(s):  
M L West ◽  
J L Corden
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Phosphorylation Sites ◽  
Terminal Domain ◽  
Carboxyl Terminal Domain ◽  
Carboxyl Terminal ◽  
Substitution Mutations ◽  
Rna Polymerase Ii Ctd

Abstract The carboxyl-terminal domain (CTD) of the RNA polymerase II largest subunit plays an essential but poorly understood role in transcription. The CTD is highly phosphorylated in vivo and this modification may be important in the transition from transcription initiation to elongation. We report here the development of a strategy for creating novel yeast CTDs. We have used this approach to show that the minimum viable CTD in yeast contains eight consensus (Tyr1Ser2Pro3Thr4Ser5Pro6Ser7) heptapeptide repeats. Substitution of alanine or glutamate for serines in positions two or five is lethal. In addition, changing tyrosine in position one to phenylalanine is lethal. The effects of mutations that alter potential phosphorylation sites are consistent with a requirement for CTD phosphorylation in vivo.

scholarly journals Structure of human Mediator-RNA polymerase II transcription pre-initiation complex

2021 ◽  
Author(s):  
Srinivasan Rengachari ◽  
Sandra Schilbach ◽  
Shintaro Aibara ◽  
Christian Dienemann ◽  
Patrick Cramer
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Complex Structure ◽  
Proximal Part ◽  
Initiation Complex ◽  
Protein Coding ◽  
Pol Ii ◽  
Promoter Escape ◽  
General Transcription Factors

Mediator is a conserved coactivator that enables regulated transcription initiation from eukaryotic protein-coding genes1-3. Mediator is recruited by transcriptional activators and binds the pre-initiation complex (PIC) to stimulate RNA polymerase II (Pol II) phosphorylation and promoter escape1-6. Here we prepare a 20-subunit recombinant human Mediator, reconstitute a 50-subunit Mediator-PIC complex, and resolve the complex structure by cryo-EM at an overall resolution of 4.5 Å. Mediator binds with its head module to the Pol II stalk and the general transcription factors TFIIB and TFIIE, resembling the Mediator-PIC interactions in the corresponding yeast complex7-9. One end of Mediator contains the metazoan-specific subunits MED27-MED30, which associate with exposed regions in MED14 and MED17 to form the proximal part of the tail module that binds activators. The opposite end of Mediator positions the flexibly linked CDK-activating kinase (CAK) of the general transcription factor TFIIH near the C-terminal repeat domain (CTD) of Pol II. The Mediator shoulder domain holds the CAK subunit CDK7, whereas the hook domain contacts a CDK7 element that flanks the kinase active site. The shoulder and hook reside in the Mediator head and middle modules, respectively, which can move relative to each other and may induce an active conformation of CDK7 to allosterically stimulate CTD phosphorylation and Pol II escape from the promoter.

scholarly journals DNA wrapping in transcription initiation by RNA polymerase II

1999 ◽  
Vol 77 (4) ◽  
pp. 257-264 ◽  
Author(s):  
Benoit Coulombe
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Dna Bending ◽  
Transcriptional Initiation ◽  
General Transcription Factors ◽  
Transcript Elongation ◽  
Dna Wrapping

The DNA wrapping model of transcription stipulates that DNA bending and wrapping around RNA polymerase causes an unwinding of the DNA helix at the enzyme catalytic center that stimulates strand separation prior to initiation and during transcript elongation. Recent experiments with mammalian RNA polymerase II indicate the significance of DNA bending and wrapping in transcriptional mechanisms. These findings have important implications in our understanding of the role of the general transcription factors in transcriptional initiation and the mechanisms underlying transcriptional regulation.Key words: mRNA synthesis, transcription initiation, RNA polymerase II, DNA wrapping, general transcription factors.

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