A Novel Subunit of Yeast RNA Polymerase III Interacts with the TFIIB-Related Domain of TFIIIB70

ABSTRACT There is limited information on how eukaryotic RNA polymerases (Pol) recognize their cognate preinitiation complex. We have characterized a polypeptide copurifying with yeast Pol III. This protein, C17, was found to be homologous to a mammalian protein described as a hormone receptor. Deletion of the corresponding gene,RPC17, was lethal and its regulated extinction caused a selective defect in transcription of class III genes in vivo. Two-hybrid and coimmunoprecipitation experiments indicated that C17 interacts with two Pol III subunits, one of which, C31, is important for the initiation reaction. C17 also interacted with TFIIIB70, the TFIIB-related component of TFIIIB. The interaction domain was found to be in the N-terminal, TFIIB-like half of TFIIIB70, downstream of the zinc ribbon and first imperfect repeat. Although Pol II similarly interacts with TFIIB, it is notable that C17 has no similarity to any Pol II subunit. The data indicate that C17 is a novel specific subunit of Pol III which participates together with C34 in the recruitment of Pol III by the preinitiation complex.

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Retinoblastoma Protein Disrupts Interactions Required for RNA Polymerase III Transcription

Molecular and Cellular Biology ◽

10.1128/mcb.20.24.9192-9202.2000 ◽

2000 ◽

Vol 20 (24) ◽

pp. 9192-9202 ◽

Cited By ~ 47

Author(s):

Josephine E. Sutcliffe ◽

Timothy R. P. Brown ◽

Simon J. Allison ◽

Pamela H. Scott ◽

Robert J. White

Keyword(s):

Rna Polymerase ◽

Transcriptional Repression ◽

Histone Deacetylases ◽

Trichostatin A ◽

Rna Polymerase Iii ◽

Retinoblastoma Protein ◽

Class Iii ◽

Negative Effect ◽

Pol Iii

ABSTRACT The retinoblastoma protein (RB) has been shown to suppress RNA polymerase (Pol) III transcription in vivo (R. J. White, D. Trouche, K. Martin, S. P. Jackson, and T. Kouzarides, Nature 382:88–90, 1996). This regulation involves interaction with TFIIIB, a multisubunit factor that is required for the expression of all Pol III templates (C. G. C. Larminie, C. A. Cairns, R. Mital, K. Martin, T. Kouzarides, S. P. Jackson, and R. J. White, EMBO J. 16:2061–2071, 1997; W.-M. Chu, Z. Wang, R. G. Roeder, and C. W. Schmid, J. Biol. Chem. 272:14755–14761, 1997). However, it has not been established why RB binding to TFIIIB results in transcriptional repression. For several Pol II-transcribed genes, RB has been shown to inhibit expression by recruiting histone deacetylases, which are thought to decrease promoter accessibility. We present evidence that histone deacetylases exert a negative effect on Pol III activity in vivo. However, RB remains able to regulate Pol III transcription in the presence of the histone deacetylase inhibitor trichostatin A. Instead, RB represses by disrupting interactions between TFIIIB and other components of the basal Pol III transcription apparatus. Recruitment of TFIIIB to most class III genes requires its binding to TFIIIC2, but this can be blocked by RB. In addition, RB disrupts the interaction between TFIIIB and Pol III that is essential for transcription. The ability of RB to inhibit these key interactions can explain its action as a potent repressor of class III gene expression.

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Functional mRNA can be generated by RNA polymerase III.

Molecular and Cellular Biology ◽

10.1128/mcb.15.7.3597 ◽

1995 ◽

Vol 15 (7) ◽

pp. 3597-3607 ◽

Cited By ~ 41

Author(s):

S Gunnery ◽

M B Mathews

Keyword(s):

Human Immunodeficiency Virus ◽

Rna Polymerase ◽

Rna Polymerase Iii ◽

Chloramphenicol Acetyltransferase ◽

Pol Ii ◽

Pol I ◽

Immunodeficiency Virus ◽

Pol Iii

Eukaryotic cellular mRNA is believed to be synthesized exclusively by RNA polymerase II (pol II), whereas pol I produces long rRNAs and pol III produces 5S rRNA, tRNA, and other small RNAs. To determine whether this functional differentiation is obligatory, we examined the translational potential of an artificial pol III transcript. The coding region of the human immunodeficiency virus type 1 tat gene was placed under the control of a strong pol III promoter from the adenovirus type 2 VA RNAI gene. The resultant chimera, pVA-Tat, was transcribed accurately in vivo and in vitro and gave rise to Tat protein, which transactivated a human immunodeficiency virus-driven chloramphenicol acetyltransferase reporter construct in transfected HeLa cells. pol III-specific mutations down-regulated VA-Tat RNA production in vivo and in vitro and dramatically reduced chloramphenicol acetyltransferase transactivation. As expected for a pol III transcript, VA-Tat RNA was not detectably capped at its 5' end or polyadenylated at its 3' end, but, like mRNA, it was associated with polysomes in a salt-stable manner. Mutational analysis of a short open reading frame upstream of the Tat-coding sequence implicates scanning in the initiation of VA-Tat RNA translation despite the absence of a cap. In comparison with tat mRNA generated by pol II, VA-Tat RNA was present on smaller polysomes and was apparently translated less efficiently, which is consistent with a relatively low initiation rate. Evidently, human cells are capable of utilizing pol III transcripts as functional mRNAs, and neither a cap nor a poly(A) tail is essential for translation, although they may be stimulatory. These findings raise the possibility that some cellular mRNAs are made by pol I or pol III.

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Maf1p, a Negative Effector of RNA Polymerase III inSaccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.21.15.5031-5040.2001 ◽

2001 ◽

Vol 21 (15) ◽

pp. 5031-5040 ◽

Cited By ~ 109

Author(s):

Krzysztof Pluta ◽

Olivier Lefebvre ◽

Nancy C. Martin ◽

Wieslaw J. Smagowicz ◽

David R. Stanford ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Iii ◽

Growth Defect ◽

Class Iii ◽

Gene Encoding ◽

Cell Extracts ◽

Polymerase Iii ◽

Pol Iii

ABSTRACT Although yeast RNA polymerase III (Pol III) and the auxiliary factors TFIIIC and TFIIIB are well characterized, the mechanisms of class III gene regulation are poorly understood. Previous studies identified MAF1, a gene that affects tRNA suppressor efficiency and interacts genetically with Pol III. We show here that tRNA levels are elevated in maf1 mutant cells. In keeping with the higher levels of tRNA observed in vivo, the in vitro rate of Pol III RNA synthesis is significantly increased in maf1cell extracts. Mutations in the RPC160 gene encoding the largest subunit of Pol III which reduce tRNA levels were identified as suppressors of the maf1 growth defect. Interestingly, Maf1p is located in the nucleus and coimmunopurifies with epitope-tagged RNA Pol III. These results indicate that Maf1p acts as a negative effector of Pol III synthesis. This potential regulator of Pol III transcription is likely conserved since orthologs of Maf1p are present in other eukaryotes, including humans.

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tRNA genes as transcriptional repressor elements

Molecular and Cellular Biology ◽

10.1128/mcb.14.2.1266-1277.1994 ◽

1994 ◽

Vol 14 (2) ◽

pp. 1266-1277

Author(s):

M W Hull ◽

J Erickson ◽

M Johnston ◽

D R Engelke

Keyword(s):

Trna Gene ◽

Rna Polymerase Iii ◽

Trna Genes ◽

Temperature Sensitive ◽

Regulatory Relationship ◽

Yeast Saccharomyces Cerevisiae ◽

Pol Ii ◽

Active Transcription ◽

Pol Iii

Eukaryotic genomes frequently contain large numbers of repetitive RNA polymerase III (pol III) promoter elements interspersed between and within RNA pol II transcription units, and in several instances a regulatory relationship between the two types of promoter has been postulated. In the budding yeast Saccharomyces cerevisiae, tRNA genes are the only known interspersed pol III promoter-containing repetitive elements, and we find that they strongly inhibit transcription from adjacent pol II promoters in vivo. This inhibition requires active transcription of the upstream tRNA gene but is independent of its orientation and appears not to involve simple steric blockage of the pol II upstream activator sites. Evidence is presented that different pol II promoters can be repressed by different tRNA genes placed upstream at varied distances in both orientations. To test whether this phenomenon functions in naturally occurring instances in which tRNA genes and pol II promoters are juxtaposed, we examined the sigma and Ty3 elements. This class of retrotransposons is always found integrated immediately upstream of different tRNA genes. Weakening tRNA gene transcription by means of a temperature-sensitive mutation in RNA pol III increases the pheromone-inducible expression of sigma and Ty3 elements up to 60-fold.

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tRNA genes as transcriptional repressor elements.

Molecular and Cellular Biology ◽

10.1128/mcb.14.2.1266 ◽

1994 ◽

Vol 14 (2) ◽

pp. 1266-1277 ◽

Cited By ~ 84

Author(s):

M W Hull ◽

J Erickson ◽

M Johnston ◽

D R Engelke

Keyword(s):

Trna Gene ◽

Rna Polymerase Iii ◽

Trna Genes ◽

Temperature Sensitive ◽

Regulatory Relationship ◽

Yeast Saccharomyces Cerevisiae ◽

Pol Ii ◽

Active Transcription ◽

Pol Iii

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In vitro analysis of elongation and termination by mutant RNA polymerases with altered termination behavior.

Molecular and Cellular Biology ◽

10.1128/mcb.16.11.6468 ◽

1996 ◽

Vol 16 (11) ◽

pp. 6468-6476 ◽

Cited By ~ 21

Author(s):

S A Shaaban ◽

E V Bobkova ◽

D M Chudzik ◽

B D Hall

Keyword(s):

Rna Polymerase Iii ◽

Multiple Roles ◽

Wild Type ◽

Protein Motifs ◽

Study Support ◽

Vitro Analysis ◽

Pol Iii ◽

In Vitro Analysis

We have studied the in vitro elongation and termination properties of several yeast RNA polymerase III (pol III) mutant enzymes that have altered in vivo termination behavior (S. A. Shaaban, B. M. Krupp, and B. D. Hall, Mol. Cell. Biol. 15:1467-1478, 1995). The pattern of completed-transcript release was also characterized for three of the mutant enzymes. The mutations studied occupy amino acid regions 300 to 325, 455 to 521, and 1061 to 1082 of the RET1 protein (P. James, S. Whelen, and B. D. Hall, J. Biol. Chem. 266:5616-5624, 1991), the second largest subunit of yeast RNA pol III. In general, mutant enzymes which have increased termination require a longer time to traverse a template gene than does wild-type pol III; the converse holds true for most decreased-termination mutants. One increased-termination mutant (K310T I324K) was faster and two reduced termination mutants (K512N and T455I E478K) were slower than the wild-type enzyme. In most cases, these changes in overall elongation kinetics can be accounted for by a correspondingly longer or shorter dwell time at pause sites within the SUP4 tRNA(Tyr) gene. Of the three mutants analyzed for RNA release, one (T455I) was similar to the wild type while the two others (T455I E478K and E478K) bound the completed SUP4 pre-tRNA more avidly. The results of this study support the view that termination is a multistep pathway in which several different regions of the RET1 protein are actively involved. Region 300 to 325 likely affects a step involved in RNA release, while the Rif homology region, amino acids 455 to 521, interacts with the nascent RNA 3' end. The dual effects of several mutations on both elongation kinetics and RNA release suggest that the protein motifs affected by them have multiple roles in the steps leading to transcription termination.

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Eukaryotic transcription termination factor La mediates transcript release and facilitates reinitiation by RNA polymerase III

Molecular and Cellular Biology ◽

10.1128/mcb.14.3.2147-2158.1994 ◽

1994 ◽

Vol 14 (3) ◽

pp. 2147-2158

Author(s):

R J Maraia ◽

D J Kenan ◽

J D Keene

Keyword(s):

Rna Polymerase ◽

Rna Binding ◽

Transcription Termination ◽

Rna Polymerase Iii ◽

Class Iii ◽

Ample Evidence ◽

Polymerase Iii ◽

Transcription Complexes ◽

Pol Iii

Ample evidence indicates that Alu family interspersed elements retrotranspose via primary transcripts synthesized by RNA polymerase III (pol III) and that this transposition sometimes results in genetic disorders in humans. However, Alu primary transcripts can be processed posttranscriptionally, diverting them away from the transposition pathway. The pol III termination signal of a well-characterized murine B1 (Alu-equivalent) element inhibits RNA 3' processing, thereby stabilizing the putative transposition intermediary. We used an immobilized template-based assay to examine transcription termination by VA1, 7SL, and Alu class III templates and the role of transcript release in the pol III terminator-dependent inhibition of processing of B1-Alu transcripts. We found that the RNA-binding protein La confers this terminator-dependent 3' processing inhibition on transcripts released from the B1-Alu template. Using pure recombinant La protein and affinity-purified transcription complexes, we also demonstrate that La facilitates multiple rounds of transcription reinitiation by pol III. These results illustrate an important role for La in RNA production by demonstrating its ability to clear the termination sites of class III templates, thereby promoting efficient use of transcription complexes by pol III. The role of La as a potential regulatory factor in transcript maturation and how this might apply to Alu interspersed elements is discussed.

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Proximal sequence element-binding transcription factor (PTF) is a multisubunit complex required for transcription of both RNA polymerase II- and RNA polymerase III-dependent small nuclear RNA genes.

Molecular and Cellular Biology ◽

10.1128/mcb.15.4.2019 ◽

1995 ◽

Vol 15 (4) ◽

pp. 2019-2027 ◽

Cited By ~ 73

Author(s):

J B Yoon ◽

S Murphy ◽

L Bai ◽

Z Wang ◽

R G Roeder

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Small Nuclear Rna ◽

Class Iii ◽

Sequence Element ◽

Pol Ii ◽

Proximal Sequence Element ◽

Nuclear Rna ◽

Class Iii Genes ◽

Pol Iii

The proximal sequence element (PSE), found in both RNA polymerase II (Pol II)- and RNA Pol III-transcribed small nuclear RNA (snRNA) genes, is specifically bound by the PSE-binding transcription factor (PTF). We have purified PTF to near homogeneity from HeLa cell extracts by using a combination of conventional and affinity chromatographic methods. Purified PTF is composed of four polypeptides with apparent molecular masses of 180, 55, 45, and 44 kDa. A combination of preparative electrophoretic mobility shift and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses has conclusively identified these four polypeptides as subunits of human PTF, while UV cross-linking experiments demonstrate that the largest subunit of PTF is in close contact with the PSE. The purified PTF activates transcription from promoters of both Pol II- and Pol III-transcribed snRNA genes in a PSE-dependent manner. In addition, we have investigated factor requirements in transcription of Pol III-dependent snRNA genes. We show that in extracts that have been depleted of TATA-binding protein (TBP) and associated factors, recombinant TBP restores transcription from U6 and 7SK promoters but not from the VAI promoter, whereas the highly purified TBP-TBP-associated factor complex TFIIIB restores transcription from the VAI but not the U6 or 7SK promoter. Furthermore, by complementation of heat-treated extracts lacking TFIIIC activity, we show that TFIIIC1 is required for transcription of both the 7SK and VAI genes, whereas TFIIIC2 is required only for transcription of the VAI gene. From these observations, we conclude (i) that PTF and TFIIIC2 function as gene-specific as gene-specific factors for PSE-and B-box-containing Pol III genes, respectively, (ii) that the form of TBP used by class III genes with upstream promoter elements differs from the from used by class III genes with internal promoters, and (iii) that TFIIIC1 is required for both internal and external Pol III promoters.

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

Molecular and Cellular Biology ◽

10.1128/mcb.21.8.2736-2742.2001 ◽

2001 ◽

Vol 21 (8) ◽

pp. 2736-2742 ◽

Cited By ~ 51

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.

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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.

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