Mutations in a conserved region of RNA polymerase II influence the accuracy of mRNA start site selection

A sensitive phenotypic assay has been used to identify mutations affecting transcription initiation in the genes encoding the two large subunits of Saccharomyces cerevisiae RNA polymerase II (RPB1 and RPB2). The rpb1 and rpb2 mutations alter the ratio of transcripts initiated at two adjacent start sites of a delta-insertion promoter. Of a large number of rpb1 and rpb2 mutations screened, only a few affect transcription initiation patterns at delta-insertion promoters, and these mutations are in close proximity to each other within both RPB1 and RPB2. The two rpb1 mutations alter amino acid residues within homology block G, a region conserved in the large subunits of all RNA polymerases. The three strong rpb2 mutations alter adjacent amino acids. At a wild-type promoter, the rpb1 mutations affect the accuracy of mRNA start site selection by producing a small but detectable increase in the 5'-end heterogeneity of transcripts. These RNA polymerase II mutations implicate specific portions of the enzyme in aspects of transcription initiation.

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Functional Interaction between TFIIB and the Rpb2 Subunit of RNA Polymerase II: Implications for the Mechanism of Transcription Initiation

Molecular and Cellular Biology ◽

10.1128/mcb.24.9.3983-3991.2004 ◽

2004 ◽

Vol 24 (9) ◽

pp. 3983-3991 ◽

Cited By ~ 33

Author(s):

Bo-Shiun Chen ◽

Michael Hampsey

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Site Selection ◽

Transcription Initiation ◽

Growth Defect ◽

Nucleoside Triphosphate ◽

Start Site ◽

Functional Interaction ◽

Rnap Ii

ABSTRACT The general transcription factor TFIIB is required for accurate initiation, although the mechanism by which RNA polymerase II (RNAP II) identifies initiation sites is not well understood. Here we describe results from genetic and biochemical analyses of an altered form of yeast TFIIB containing an arginine-78 → cysteine (R78C) replacement in the “B-finger” domain. TFIIB R78C shifts start site selection downstream of normal and confers a cold-sensitive growth defect (Csm−). Suppression of the R78C Csm− phenotype identified a functional interaction between TFIIB and the Rpb2 subunit of RNAP II and defined a novel role for Rpb2 in start site selection. The rpb2 suppressor encodes a glycine-369 → serine (G369S) replacement, located in the “lobe” domain of Rpb2 and near the Rpb9 subunit, which was identified previously as an effector of start site selection. The Rpb2-Rpb9 “lobe-jaw” region of RNAP II is downstream of the catalytic center and distal to the site of RNAP II-TFIIB interaction. A TFIIB R78C mutant extract was defective for promoter-specific run-on transcription but yielded an altered pattern of abortive initiation products, indicating that the R78C defect does not preclude initiation. The sua7-3 rpb2-101 double mutant was sensitive to 6-azauracil in vivo and to nucleoside triphosphate substrate depletion in vitro. In the context of the recent X-ray structure of the yeast RNAP II-TFIIB complex, these results define a functional interaction between the B-finger domain of TFIIB and the distal lobe-jaw region of RNAP II and provide insight into the mechanism of start site selection.

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Mutational Analysis of Yeast TFIIB: A Functional Relationship Between Ssu72 and Sub1/Tsp1 Defined by Allele-Specific Interactions With TFIIB

Genetics ◽

10.1093/genetics/153.2.643 ◽

1999 ◽

Vol 153 (2) ◽

pp. 643-652

Author(s):

Wei-Hua Wu ◽

Inés Pinto ◽

Bo-Shiun Chen ◽

Michael Hampsey

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Site Selection ◽

Transcription Initiation ◽

Mutational Analysis ◽

Single Amino Acid ◽

Structural Defects ◽

Start Site ◽

Allele Specific

Abstract TFIIB is an essential component of the RNA polymerase II core transcriptional machinery. Previous studies have defined TFIIB domains required for interaction with other transcription factors and for basal transcription in vitro. In the study reported here we investigated the TFIIB structural requirements for transcription initiation in vivo. A library of sua7 mutations encoding altered forms of yeast TFIIB was generated by error-prone polymerase chain reaction and screened for conditional growth defects. Twenty-two single amino acid replacements in TFIIB were defined and characterized. These replacements are distributed throughout the protein and occur primarily at phylogenetically conserved positions. Most replacements have little or no effect on the steady-state protein levels, implying that each affects TFIIB function rather than synthesis or stability. In contrast to the initial sua7 mutants, all replacements, with one exception, have no effect on start site selection, indicating that specific TFIIB structural defects affect transcriptional accuracy. This collection of sua7 alleles, including the initial sua7 alleles, was used to investigate the allele specificity of interactions between ssu72 and sub1, both of which were initially identified as either suppressors (SUB1 2μ) or enhancers (sub1Δ, ssu72-1) of sua7 mutations. We show that the interactions of ssu72-1 and sub1Δ with sua7 are allele specific; that the allele specificities of ssu72 and sub1 overlap; and that each of the sua7 alleles that interacts with ssu72 and sub1 affects the accuracy of transcription start site selection. These results demonstrate functional interactions among TFIIB, Ssu72, and Sub1 and suggest that these interactions play a role in the mechanism of start site selection by RNA polymerase II.

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Faculty Opinions recommendation of Mechanism of start site selection by RNA polymerase II: interplay between TFIIB and Ssl2/XPB helicase subunit of TFIIH.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.13968976.15426085 ◽

2012 ◽

Author(s):

Daniel Reines

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Site Selection ◽

Start Site

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Mechanism of Start Site Selection by RNA Polymerase II

Journal of Biological Chemistry ◽

10.1074/jbc.m111.281576 ◽

2011 ◽

Vol 287 (1) ◽

pp. 557-567 ◽

Cited By ~ 18

Author(s):

Shivani Goel ◽

Shankarling Krishnamurthy ◽

Michael Hampsey

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Site Selection ◽

Start Site

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Core Promoter-Dependent TFIIB Conformation and a Role for TFIIB Conformation in Transcription Start Site Selection

Molecular and Cellular Biology ◽

10.1128/mcb.22.19.6697-6705.2002 ◽

2002 ◽

Vol 22 (19) ◽

pp. 6697-6705 ◽

Cited By ~ 24

Author(s):

Jennifer A. Fairley ◽

Rachel Evans ◽

Nicola A. Hawkes ◽

Stefan G. E. Roberts

Keyword(s):

Transcription Start Site ◽

Site Selection ◽

Transcription Initiation ◽

Core Promoter ◽

Initiation Site ◽

Start Site ◽

Wild Type ◽

Transcription Start ◽

General Transcription Factor ◽

Selection Of

ABSTRACT The general transcription factor TFIIB plays a central role in the selection of the transcription initiation site. The mechanisms involved are not clear, however. In this study, we analyze core promoter features that are responsible for the susceptibility to mutations in TFIIB and cause a shift in the transcription start site. We show that TFIIB can modulate both the 5′ and 3′ parameters of transcription start site selection in a manner dependent upon the sequence of the initiator. Mutations in TFIIB that cause aberrant transcription start site selection concentrate in a region that plays a pivotal role in modulating TFIIB conformation. Using epitope-specific antibody probes, we show that a TFIIB mutant that causes aberrant transcription start site selection assembles at the promoter in a conformation different from that for wild-type TFIIB. In addition, we uncover a core promoter-dependent effect on TFIIB conformation and provide evidence for novel sequence-specific TFIIB promoter contacts.

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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.

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Saccharomyces cerevisiae HMO1 interacts with TFIID and participates in start site selection by RNA polymerase II

Nucleic Acids Research ◽

10.1093/nar/gkm1068 ◽

2008 ◽

Vol 36 (4) ◽

pp. 1343-1357 ◽

Cited By ~ 20

Author(s):

Koji Kasahara ◽

Sewon Ki ◽

Kayo Aoyama ◽

Hiroyuki Takahashi ◽

Tetsuro Kokubo

Keyword(s):

Saccharomyces Cerevisiae ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Site Selection ◽

Start Site

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In vitro transcription of a Drosophila U1 small nuclear RNA gene requires TATA box-binding protein and two proximal cis-acting elements with stringent spacing requirements

Molecular and Cellular Biology ◽

10.1128/mcb.13.9.5918-5927.1993 ◽

1993 ◽

Vol 13 (9) ◽

pp. 5918-5927

Author(s):

Z Zamrod ◽

C M Tyree ◽

Y Song ◽

W E Stumph

Keyword(s):

Rna Polymerase Ii ◽

Transcription Initiation ◽

Tata Box ◽

Start Site ◽

Small Nuclear Rna ◽

Wild Type ◽

Nuclear Rna ◽

Tata Sequence ◽

Tata Box Binding Protein

Transcription of a Drosophila U1 small nuclear RNA gene was functionally analyzed in cell extracts derived from 0- to 12-h embryos. Two promoter elements essential for efficient initiation of transcription in vitro by RNA polymerase II were identified. The first, termed PSEA, is located between positions -41 and -61 relative to the transcription start site, is crucial for promoter activity, and is the dominant element for specifying the transcription initiation site. PSEA thus appears to be functionally homologous to the proximal sequence element of vertebrate small nuclear RNA genes. The second element, termed PSEB, is located at positions -25 to -32 and is required for an efficient level of transcription initiation because mutation of PSEB, or alteration of the spacing between PSEA and PSEB, severely reduced transcriptional activity relative to that of the wild-type promoter. Although the PSEB sequence does not have any obvious sequence similarity to a TATA box, conversion of PSEB to the canonical TATA sequence dramatically increased the efficiency of the U1 promoter and simultaneously relieved the requirement for the upstream PSEA. Despite these effects, introduction of the TATA sequence into the U1 promoter had no effect on the choice of start site or on the RNA polymerase II specificity of the promoter. Finally, evidence is presented that the TATA box-binding protein is required for transcription from the wild-type U1 promoter as well as from the TATA-containing U1 promoter.

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