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 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 Direct Interaction between the Subunit RAP30 of Transcription Factor IIF (TFIIF) and RNA Polymerase Subunit 5, Which Contributes to the Association between TFIIF and RNA Polymerase II

2001 ◽  
Vol 276 (15) ◽  
pp. 12266-12273 ◽  
Author(s):  
Wenxiang Wei ◽  
Dorjbal Dorjsuren ◽  
Yong Lin ◽  
Weiping Qin ◽  
Takahiro Nomura ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Middle Part ◽  
Wild Type ◽  
Binding Region ◽  
Pol Ii ◽  
Transcription Factor Iif ◽  
B Virus

The general transcription factor IIF (TFIIF) assembled in the initiation complex, and RAP30 of TFIIF, have been shown to associate with RNA polymerase II (pol II), although it remains unclear which pol II subunit is responsible for the interaction. We examined whether TFIIF interacts with RNA polymerase II subunit 5 (RPB5), the exposed domain of which binds transcriptional regulatory factors such as hepatitis B virus X protein and a novel regulatory protein, RPB5-mediating protein. The results demonstrated that RPB5 directly binds RAP30in vitrousing purified recombinant proteins andin vivoin COS1 cells transiently expressing recombinant RAP30 and RPB5. The RAP30-binding region was mapped to the central region (amino acids (aa) 47–120) of RPB5, which partly overlaps the hepatitis B virus X protein-binding region. Although the middle part (aa 101–170) and the N-terminus (aa 1–100) of RAP30 independently bound RPB5, the latter was not involved in the RPB5 binding when RAP30 was present in TFIIF complex. Scanning of the middle part of RAP30 by clustered alanine substitutions and then point alanine substitutions pinpointed two residues critical for the RPB5 binding inin vitroandin vivoassays. Wild type but not mutants Y124A and Q131A of RAP30 coexpressed with FLAG-RAP74 efficiently recovered endogenous RPB5 to the FLAG-RAP74-bound anti-FLAG M2 resin. The recovered endogenous RPB5 is assembled in pol II as demonstrated immunologically. Interestingly, coexpression of the central region of RPB5 and wild type RAP30 inhibited recovery of endogenous pol II to the FLAG-RAP74-bound M2 resin, strongly suggesting that the RAP30-binding region of RPB5 inhibited the association of TFIIF and pol II. The exposed domain of RPB5 interacts with RAP30 of TFIIF and is important for the association between pol II and TFIIF.

scholarly journals CTCF Interacts with and Recruits the Largest Subunit of RNA Polymerase II to CTCF Target Sites Genome-Wide

2007 ◽  
Vol 27 (5) ◽  
pp. 1631-1648 ◽  
Author(s):  
Igor Chernukhin ◽  
Shaharum Shamsuddin ◽  
Sung Yun Kang ◽  
Rosita Bergström ◽  
Yoo-Wook Kwon ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Gene Activation ◽  
Ctcf Binding ◽  
Pol Ii ◽  
Genome Wide ◽  
Target Sites ◽  
On Chip

ABSTRACT CTCF is a transcription factor with highly versatile functions ranging from gene activation and repression to the regulation of insulator function and imprinting. Although many of these functions rely on CTCF-DNA interactions, it is an emerging realization that CTCF-dependent molecular processes involve CTCF interactions with other proteins. In this study, we report the association of a subpopulation of CTCF with the RNA polymerase II (Pol II) protein complex. We identified the largest subunit of Pol II (LS Pol II) as a protein significantly colocalizing with CTCF in the nucleus and specifically interacting with CTCF in vivo and in vitro. The role of CTCF as a link between DNA and LS Pol II has been reinforced by the observation that the association of LS Pol II with CTCF target sites in vivo depends on intact CTCF binding sequences. “Serial” chromatin immunoprecipitation (ChIP) analysis revealed that both CTCF and LS Pol II were present at the β-globin insulator in proliferating HD3 cells but not in differentiated globin synthesizing HD3 cells. Further, a single wild-type CTCF target site (N-Myc-CTCF), but not the mutant site deficient for CTCF binding, was sufficient to activate the transcription from the promoterless reporter gene in stably transfected cells. Finally, a ChIP-on-ChIP hybridization assay using microarrays of a library of CTCF target sites revealed that many intergenic CTCF target sequences interacted with both CTCF and LS Pol II. We discuss the possible implications of our observations with respect to plausible mechanisms of transcriptional regulation via a CTCF-mediated direct link of LS Pol II to the DNA.

scholarly journals Yeast NC2 Associates with the RNA Polymerase II Preinitiation Complex and Selectively Affects Transcription In Vivo

2001 ◽  
Vol 21 (8) ◽  
pp. 2736-2742 ◽  
Author(s):  
Joseph V. Geisberg ◽  
Frank C. Holstege ◽  
Richard A. Young ◽  
Kevin Struhl
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Negative Regulator ◽  
Preinitiation Complex ◽  
Positive Role ◽  
Pol Ii ◽  
Basal Transcription Factors

ABSTRACT NC2 (Dr1-Drap1 or Bur6-Ydr1) has been characterized in vitro as a general negative regulator of RNA polymerase II (Pol II) transcription that interacts with TATA-binding protein (TBP) and inhibits its function. Here, we show that NC2 associates with promoters in vivo in a manner that correlates with transcriptional activity and with occupancy by basal transcription factors. NC2 rapidly associates with promoters in response to transcriptional activation, and it remains associated under conditions in which transcription is blocked after assembly of the Pol II preinitiation complex. NC2 positively and negatively affects approximately 17% of Saccharomyces cerevisiaegenes in a pattern that resembles the response to general environmental stress. Relative to TBP, NC2 occupancy is high at promoters where NC2 is positively required for normal levels of transcription. Thus, NC2 is associated with the Pol II preinitiation complex, and it can play a direct and positive role at certain promoters in vivo.

scholarly journals TFIIH-Associated Cdk7 Kinase Functions in Phosphorylation of C-Terminal Domain Ser7 Residues, Promoter-Proximal Pausing, and Termination by RNA Polymerase II

2009 ◽  
Vol 29 (20) ◽  
pp. 5455-5464 ◽  
Author(s):  
Kira Glover-Cutter ◽  
Stéphane Larochelle ◽  
Benjamin Erickson ◽  
Chao Zhang ◽  
Kevan Shokat ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Histone H4 ◽  
Pol Ii ◽  
Terminal Domain ◽  
Histone H4 Acetylation ◽  
Flanking Regions

ABSTRACT The function of human TFIIH-associated Cdk7 in RNA polymerase II (Pol II) transcription and C-terminal domain (CTD) phosphorylation was investigated in analogue-sensitive Cdk7 as/as mutant cells where the kinase can be inhibited without disrupting TFIIH. We show that both Cdk7 and Cdk9/PTEFb contribute to phosphorylation of Pol II CTD Ser5 residues on transcribed genes. Cdk7 is also a major kinase of CTD Ser7 on Pol II at the c-fos and U snRNA genes. Furthermore, TFIIH and recombinant Cdk7-CycH-Mat1 as well as recombinant Cdk9-CycT1 phosphorylated CTD Ser7 and Ser5 residues in vitro. Inhibition of Cdk7 in vivo suppressed the amount of Pol II accumulated at 5′ ends on several genes including c-myc, p21, and glyceraldehyde-3-phosphate dehydrogenase genes, indicating reduced promoter-proximal pausing or polymerase “leaking” into the gene. Consistent with a 5′ pausing defect, Cdk7 inhibition reduced recruitment of the negative elongation factor NELF at start sites. A role of Cdk7 in regulating elongation is further suggested by enhanced histone H4 acetylation and diminished histone H4 trimethylation on lysine 36—two marks of elongation—within genes when the kinase was inhibited. Consistent with a new role for TFIIH at 3′ ends, it was detected within genes and 3′-flanking regions, and Cdk7 inhibition delayed pausing and transcription termination.

scholarly journals Studies of Nematode TFIIE Function Reveal a Link between Ser-5 Phosphorylation of RNA Polymerase II and the Transition from Transcription Initiation to Elongation

2001 ◽  
Vol 21 (1) ◽  
pp. 1-15 ◽  
Author(s):  
Seiji Yamamoto ◽  
Yoshinori Watanabe ◽  
Peter J. van der Spek ◽  
Tomomichi Watanabe ◽  
Hiroyuki Fujimoto ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
In Vitro Transcription ◽  
Pol Ii ◽  
Transcription System ◽  
Carboxy Terminal ◽  
Transition To Elongation ◽  
Ctd Phosphorylation

ABSTRACT The general transcription factor TFIIE plays important roles in transcription initiation and in the transition to elongation. However, little is known about its function during these steps. Here we demonstrate for the first time that TFIIH-mediated phosphorylation of RNA polymerase II (Pol II) is essential for the transition to elongation. This phosphorylation occurs at serine position 5 (Ser-5) of the carboxy-terminal domain (CTD) heptapeptide sequence of the largest subunit of Pol II. In a human in vitro transcription system with a supercoiled template, this process was studied using a human TFIIE (hTFIIE) homolog from Caenorhabditis elegans (ceTFIIEα and ceTFIIEβ). ceTFIIEβ could partially replace hTFIIEβ, whereas ceTFIIEα could not replace hTFIIEα. We present the studies of TFIIE binding to general transcription factors and the effects of subunit substitution on CTD phosphorylation. As a result, ceTFIIEα did not bind tightly to hTFIIEβ, and ceTFIIEβ showed a similar profile for binding to its human counterpart and supported an intermediate level of CTD phosphorylation. Using antibodies against phosphorylated serine at either Ser-2 or Ser-5 of the CTD, we found that ceTFIIEβ induced Ser-5 phosphorylation very little but induced Ser-2 phosphorylation normally, in contrast to wild-type hTFIIE, which induced phosphorylation at both Ser-2 and Ser-5. In transcription transition assays using a linear template, ceTFIIEβ was markedly defective in its ability to support the transition to elongation. These observations provide evidence of TFIIE involvement in the transition and suggest that Ser-5 phosphorylation is essential for Pol II to be in the processive elongation form.

scholarly journals Subnuclear Localization of Ku Protein: Functional Association with RNA Polymerase II Elongation Sites

2002 ◽  
Vol 22 (22) ◽  
pp. 8088-8099 ◽  
Author(s):  
Xianming Mo ◽  
William S. Dynan
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Genome Stability ◽  
Strand Break ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Rnap Ii

ABSTRACT Ku is an abundant nuclear protein with an essential function in the repair of DNA double-strand breaks. Various observations suggest that Ku also interacts with the cellular transcription machinery, although the mechanism and significance of this interaction are not well understood. In the present study, we investigated the subnuclear distribution of Ku in normally growing human cells by using confocal microscopy, chromatin immunoprecipitation, and protein immunoprecipitation. All three approaches indicated association of Ku with RNA polymerase II (RNAP II) elongation sites. This association occurred independently of the DNA-dependent protein kinase catalytic subunit and was highly selective. There was no detectable association with the initiating isoform of RNAP II or with the general transcription initiation factors. In vitro protein-protein interaction assays demonstrated that the association of Ku with elongation proteins is mediated, in part, by a discrete C-terminal domain in the Ku80 subunit. Functional disruption of this interaction with a dominant-negative mutant inhibited transcription in vitro and in vivo and suppressed cell growth. These results suggest that association of Ku with transcription sites is important for maintenance of global transcription levels. Tethering of double-strand break repair proteins to defined subnuclear structures may also be advantageous in maintenance of genome stability.

scholarly journals Requirements for RNA polymerase II preinitiation complex formation in vivo

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Natalia Petrenko ◽  
Yi Jin ◽  
Liguo Dong ◽  
Koon Ho Wong ◽  
Kevin Struhl
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Associated Factors ◽  
Yeast Cells ◽  
Preinitiation Complex ◽  
Pol Ii ◽  
General Transcription Factors ◽  
Nucleotide Triphosphates

Transcription by RNA polymerase II requires assembly of a preinitiation complex (PIC) composed of general transcription factors (GTFs) bound at the promoter. In vitro, some GTFs are essential for transcription, whereas others are not required under certain conditions. PICs are stable in the absence of nucleotide triphosphates, and subsets of GTFs can form partial PICs. By depleting individual GTFs in yeast cells, we show that all GTFs are essential for TBP binding and transcription, suggesting that partial PICs do not exist at appreciable levels in vivo. Depletion of FACT, a histone chaperone that travels with elongating Pol II, strongly reduces PIC formation and transcription. In contrast, TBP-associated factors (TAFs) contribute to transcription of most genes, but TAF-independent transcription occurs at substantial levels, preferentially at promoters containing TATA elements. PICs are absent in cells deprived of uracil, and presumably UTP, suggesting that transcriptionally inactive PICs are removed from promoters in vivo.

scholarly journals RPAP2 regulates a transcription initiation checkpoint by prohibiting assembly of preinitiation complex

2021 ◽  
Author(s):  
Xizi Chen ◽  
Yilun Qi ◽  
Xinxin Wang ◽  
Zhenning Wang ◽  
Li Wang ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Phosphatase Activity ◽  
Transcription Initiation ◽  
Critical Role ◽  
In Vitro Transcription ◽  
Pol Ii ◽  
Activity Structure

RNA polymerase II (Pol II)-mediated transcription in metazoan requires precise regulation. RNA polymerase II-associated protein 2 (RPAP2) was previously identified to transport Pol II from cytoplasm to nucleus and dephosphorylates Pol II C-terminal domain (CTD). We found that RPAP2 binds hypo/hyper-phosphorylated Pol II with undetectable phosphatase activity. Structure of RPAP2-Pol II shows mutually exclusive assembly of RPAP2-Pol II and pre-initiation complex (PIC) due to three steric clashes. RPAP2 prevents/disrupts Pol II-TFIIF interaction and impairs in vitro transcription initiation, suggesting a function in prohibiting PIC assembly. Loss of RPAP2 in cells leads to global accumulation of TFIIF and Pol II at promoters, indicating critical role of RPAP2 in inhibiting PIC assembly independent of its putative phosphatase activity. Our study indicates that RPAP2 functions as a gatekeeper to prohibit PIC assembly and transcription initiation and suggests a novel transcription checkpoint.

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