Stimulation of RNA polymerase II transcription initiation by recruitment of TBP in vivo

ABSTRACT Transcription factor IIF (TFIIF) is required for transcription of protein-encoding genes by eukaryotic RNA polymerase II. In contrast to numerous studies establishing a role for higher eukaryotic TFIIF in multiple steps of the transcription cycle, relatively little has been reported regarding the functions of TFIIF in the yeast Saccharomyces cerevisiae. In this study, site-directed mutagenesis, plasmid shuffle complementation assays, and primer extension analyses were employed to probe the functional domains of the S. cerevisiae TFIIF subunits Tfg1 and Tfg2. Analyses of 35 Tfg1 alanine substitution mutants and 19 Tfg2 substitution mutants identified 5 mutants exhibiting altered properties in vivo. Primer extension analyses revealed that the conditional growth properties exhibited by the tfg1-E346A, tfg1-W350A, and tfg2-L59K mutants were associated with pronounced upstream shifts in transcription initiation in vivo. Analyses of double mutant strains demonstrated functional interactions between the Tfg1 mutations and mutations in Tfg2, TFIIB, and RNA polymerase II. Importantly, biochemical results demonstrated an altered interaction between mutant TFIIF protein and RNA polymerase II. These results provide direct evidence for the involvement of S. cerevisiae TFIIF in the mechanism of transcription start site utilization and support the view that a TFIIF-RNA polymerase II interaction is a determinant in this process.

Download Full-text

Structural Biology of RNA Polymerase II Transcription: 20 Years On

Annual Review of Cell and Developmental Biology ◽

10.1146/annurev-cellbio-042020-021954 ◽

2020 ◽

Vol 36 (1) ◽

pp. 1-34 ◽

Cited By ~ 1

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.

Download Full-text

Positionally exact initiation is required for the formation of a stable RNA polymerase II transcription complex in vivo.

The EMBO Journal ◽

10.1002/j.1460-2075.1988.tb03263.x ◽

1988 ◽

Vol 7 (12) ◽

pp. 3785-3792 ◽

Cited By ~ 13

Author(s):

G. Tebb ◽

I. W. Mattaj

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Rna Polymerase Ii Transcription

Download Full-text

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

Molecular and Cellular Biology ◽

10.1128/mcb.24.7.2863-2874.2004 ◽

2004 ◽

Vol 24 (7) ◽

pp. 2863-2874 ◽

Cited By ~ 17

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.

Download Full-text

Promoter-Specific Shifts in Transcription Initiation Conferred by Yeast TFIIB Mutations Are Determined by the Sequence in the Immediate Vicinity of the Start Sites

Molecular and Cellular Biology ◽

10.1128/mcb.21.14.4427-4440.2001 ◽

2001 ◽

Vol 21 (14) ◽

pp. 4427-4440 ◽

Cited By ~ 40

Author(s):

Silviu L. Faitar ◽

Seth A. Brodie ◽

Alfred S. Ponticelli

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription Initiation ◽

Genetic Selection ◽

Functional Domain ◽

Start Site ◽

Recessive Mutations ◽

Transcription Factor Iib ◽

General Transcription Factor

ABSTRACT The general transcription factor IIB (TFIIB) is required for transcription of class II genes by RNA polymerase II. Previous studies demonstrated that mutations in the Saccharomyces cerevisiae SUA7 gene, which encodes TFIIB, can alter transcription initiation patterns in vivo. To further delineate the functional domain and residues of TFIIB involved in transcription start site utilization, a genetic selection was used to isolate S. cerevisiae TFIIB mutants exhibiting downstream shifts in transcription initiation in vivo. Both dominant and recessive mutations conferring downstream shifts were identified at multiple positions within a highly conserved homology block in the N-terminal region of the protein. The TFIIB mutations conferred downstream shifts in transcription initiation at the ADH1 and CYC1 promoters, whereas no significant shifts were observed at the HIS3 promoter. Analysis of a series of ADH1-HIS3 hybrid promoters and variant ADH1 and HIS3 promoters containing insertions, deletions, or site-directed base substitutions revealed that the feature that renders a promoter sensitive to TFIIB mutations is the sequence in the immediate vicinity of the normal start sites. We discuss these results in light of possible models for the mechanism of start site utilization by S. cerevisiae RNA polymerase II and the role played by TFIIB.

Download Full-text

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

Molecular and Cellular Biology ◽

10.1128/mcb.19.3.2130 ◽

1999 ◽

Vol 19 (3) ◽

pp. 2130-2141 ◽

Cited By ~ 39

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.

Download Full-text