Genetic analysis of the large subunit of yeast transcription factor IIE reveals two regions with distinct functions.

Biochemical analysis of proteins necessary for transcription initiation by eukaryotic RNA polymerase II (pol II) has identified transcription factor IIE (TFIIE) as an essential component of the reaction. To better understand the role of TFIIE in transcription, we isolated conditional alleles of TFA1, the gene encoding the large subunit of TFIIE in the yeast Saccharomyces cerevisiae. The mutant Tfa1 proteins fall into two classes. The first class causes thermosensitive growth due to single amino acid substitutions of the cysteines comprising the Zn-binding motif. The second mutant class is made up of proteins that are C-terminally truncated and that cause a cold-sensitive growth phenotype. The behavior of these mutants suggests that Tfa1p possesses at least two domains with genetically distinct functions. The mutations in the Zn-binding motif do not affect the mutant protein's stability at the nonpermissive temperature or its ability to associate with the small subunit of TFIIE. Our studies further determined that wild-type TFIIE can bind to single-stranded DNA in vitro. However, this property is unaffected in the mutant TFIIE complexes. Finally, we have demonstrated the biological importance of TFIIE in pol II-mediated transcription by depleting the Tfa1 protein from the cells and observing a concomitant decrease in total poly(A)+ mRNA.

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Analysis of the yeast transcription factor TFIIA: distinct functional regions and a polymerase II-specific role in basal and activated transcription.

Molecular and Cellular Biology ◽

10.1128/mcb.15.3.1234 ◽

1995 ◽

Vol 15 (3) ◽

pp. 1234-1243 ◽

Cited By ~ 62

Author(s):

J J Kang ◽

D T Auble ◽

J A Ranish ◽

S Hahn

Keyword(s):

Transcription Factor ◽

Large Subunit ◽

Small Subunit ◽

Specific Factor ◽

Alanine Scanning Mutagenesis ◽

Alanine Scanning ◽

Pol Ii ◽

Conserved Regions

To probe the structure and function of the Saccharomyces cerevisiae general transcription factor TFIIA, we have systematically mutagenized the genes encoding both subunits and analyzed the effects of the mutations both in vivo and in vitro. We found that the central nonconserved region of the large subunit is not essential for function and likely acts as a spacer between the conserved N- and C-terminal regions. Deletion mutagenesis of the large subunit defined a region which is required for TATA binding protein (TBP) interaction. Alanine scanning mutagenesis defined a cluster of four basic residues which are likely required for interaction with DNA in the TBP-DNA complex. Much of the conserved regions of both subunits is required for subunit association, suggesting that these conserved regions fold into compact domains which extensively interact. In vitro transcription performed with extracts from yeast strains with mutations in either the large or the small TFIIA subunit demonstrated that TFIIA stimulates both basal and activated polymerase II (Pol II) transcription. The TFIIA-depleted extracts have normal Pol I and Pol III transcription activity, showing that TFIIA is a Pol II-specific factor. In vivo depletion of TFIIA activity reduced transcription from four different Pol II promoters. Finally, alanine scanning mutagenesis of TFIIA's small subunit has identified at least one mutation which is defective in transcription but which is not defective in subunit association or binding to TBP or TBP-DNA complexes.

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The general transcription factor RAP30 binds to RNA polymerase II and prevents it from binding nonspecifically to DNA

Molecular and Cellular Biology ◽

10.1128/mcb.12.1.30-37.1992 ◽

1992 ◽

Vol 12 (1) ◽

pp. 30-37

Author(s):

M T Killeen ◽

J F Greenblatt

Keyword(s):

Transcription Factor ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Small Subunit ◽

Glutathione S Transferase ◽

General Transcription Factor ◽

Indirect Consequence ◽

Transcription In Vitro ◽

Sigma 70

RAP30/74 is a human general transcription factor that binds to RNA polymerase II and is required for initiation of transcription in vitro regardless of whether the promoter has a recognizable TATA box (Z. F. Burton, M. Killeen, M. Sopta, L. G. Ortolan, and J. F. Greenblatt, Mol. Cell. Biol. 8:1602-1613, 1988). Part of the amino acid sequence of RAP30, the small subunit of RAP30/74, has limited homology with part of Escherichia coli sigma 70 (M. Sopta, Z. F. Burton, and J. Greenblatt, Nature (London) 341:410-414, 1989). To determine which sigmalike activities of RAP30/74 could be attributed to RAP30, we purified human RAP30 and a RAP30-glutathione-S-transferase fusion protein that had been produced in E. coli. Bacterially produced RAP30 bound to RNA polymerase II in the absence of RAP74. Both partially purified natural RAP30/74 and recombinant RAP30 prevented RNA polymerase II from binding nonspecifically to DNA. In addition, nonspecific transcription by RNA polymerase II was greatly inhibited by RAP30-glutathione-S-transferase. DNA-bound RNA polymerase II could be removed from DNA by partially purified RAP30/74 but not by bacterially expressed RAP30. Thus, the ability of RAP30/74 to recruit RNA polymerase II to a promoter-bound preinitiation complex may be an indirect consequence of its ability to suppress nonspecific binding of RNA polymerase II to DNA.

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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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Interaction of the Vaccinia Virus RNA Polymerase-Associated 94-Kilodalton Protein with the Early Transcription Factor

Journal of Virology ◽

10.1128/jvi.01653-09 ◽

2009 ◽

Vol 83 (23) ◽

pp. 12018-12026 ◽

Cited By ~ 23

Author(s):

Zhilong Yang ◽

Bernard Moss

Keyword(s):

Transcription Factor ◽

Rna Polymerase ◽

Vaccinia Virus ◽

Transcription Initiation ◽

Elongation Factor ◽

Small Subunit ◽

Functional Link ◽

Early Transcription

ABSTRACT A multisubunit RNA polymerase (RPO) encoded by vaccinia virus (VACV), in conjunction with specific factors, transcribes early, intermediate, and late viral genes. However, an additional virus-encoded polypeptide referred to as the RPO-associated protein of 94 kDa (RAP94) is tightly bound to the RPO for the transcription of early genes. Unlike the eight RPO core subunits, RAP94 is synthesized exclusively at late times after infection. Furthermore, RAP94 is necessary for the packaging of RPO and other components needed for early transcription in assembling virus particles. The direct association of RAP94 with NPH I, a DNA-dependent ATPase required for transcription termination, and the multifunctional poly(A) polymerase small subunit/2′-O-methyltransferase/elongation factor was previously demonstrated. That RAP94 provides a structural and functional link between the core RPO and the VACV early transcription factor (VETF) has been suspected but not previously demonstrated. Using VACV recombinants that constitutively or inducibly express VETF subunits and RAP94 with affinity tags, we showed that (i) VETF associates only with RPO containing RAP94 in vivo and in vitro, (ii) the association of RAP94 with VETF requires both subunits of the latter, (iii) neither viral DNA nor other virus-encoded late proteins are required for the interaction of RAP94 with VETF and core RPO subunits, (iv) different domains of RAP94 bind VETF and core subunits of RPO, and (v) NPH I and VETF bind independently and possibly simultaneously to the N-terminal region of RAP94. Thus, RAP94 provides the bridge between the RPO and proteins needed for transcription initiation, elongation, and termination.

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Archaeal DNA Replication: Identifying the Pieces to Solve a Puzzle

Genetics ◽

10.1093/genetics/152.4.1249 ◽

1999 ◽

Vol 152 (4) ◽

pp. 1249-1267 ◽

Cited By ~ 3

Author(s):

Isaac K O Cann ◽

Yoshizumi Ishino

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

Pyrococcus Furiosus ◽

Sequence Similarity ◽

Large Subunit ◽

Dna Polymerases ◽

Small Subunit ◽

Accessory Proteins ◽

Pol Ii

Abstract Archaeal organisms are currently recognized as very exciting and useful experimental materials. A major challenge to molecular biologists studying the biology of Archaea is their DNA replication mechanism. Undoubtedly, a full understanding of DNA replication in Archaea requires the identification of all the proteins involved. In each of four completely sequenced genomes, only one DNA polymerase (Pol BI proposed in this review from family B enzyme) was reported. This observation suggested that either a single DNA polymerase performs the task of replicating the genome and repairing the mutations or these genomes contain other DNA polymerases that cannot be identified by amino acid sequence. Recently, a heterodimeric DNA polymerase (Pol II, or Pol D as proposed in this review) was discovered in the hyperthermophilic archaeon, Pyrococcus furiosus. The genes coding for DP1 and DP2, the subunits of this DNA polymerase, are highly conserved in the Euryarchaeota. Euryarchaeotic DP1, the small subunit of Pol II (Pol D), has sequence similarity with the small subunit of eukaryotic DNA polymerase δ. DP2 protein, the large subunit of Pol II (Pol D), seems to be a catalytic subunit. Despite possessing an excellent primer extension ability in vitro, Pol II (Pol D) may yet require accessory proteins to perform all of its functions in euryarchaeotic cells. This review summarizes our present knowledge about archaeal DNA polymerases and their relationship with those accessory proteins, which were predicted from the genome sequences.

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Direct cleavage of human TATA-binding protein by poliovirus protease 3C in vivo and in vitro

Molecular and Cellular Biology ◽

10.1128/mcb.13.2.1232-1237.1993 ◽

1993 ◽

Vol 13 (2) ◽

pp. 1232-1237

Author(s):

M E Clark ◽

P M Lieberman ◽

A J Berk ◽

A Dasgupta

Keyword(s):

Transcription Factor ◽

Host Cell ◽

Rna Polymerase Ii ◽

Binding Protein ◽

Tata Binding Protein ◽

Significant Inhibition ◽

Pol Ii ◽

Poliovirus Infection

Host cell RNA polymerase II (Pol II)-mediated transcription is inhibited by poliovirus infection. This inhibition is correlated to a specific decrease in the activity of a chromatographic fraction which contains the transcription factor TFIID. To investigate the mechanism by which poliovirus infection results in a decrease of TFIID activity, we have analyzed a component of TFIID, the TATA-binding protein (TBP). Using Western immunoblot analysis, we show that TBP is cleaved in poliovirus-infected cells at the same time postinfection as when Pol II transcription is inhibited. Further, we show that one of the cleaved forms of TBP can be reproduced in vitro by incubating TBP with cloned, purified poliovirus encoded protease 3C. Protease 3C is a poliovirus-encoded protease that specifically cleaves glutamine-glycine bonds in the viral polyprotein. The cleavage of TBP by protease 3C occurs directly. Finally, incubation of an uninfected cell-derived TBP-containing fraction (TFIID) with protease 3C results in significant inhibition of Pol II-mediated transcription in vitro. These results demonstrate that a cellular transcription factor can be directly cleaved both in vitro and in vivo by a viral protease and suggest a role of the poliovirus proteinase 3C in host cell Pol II-mediated transcription shutoff.

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

Molecular and Cellular Biology ◽

10.1128/mcb.21.1.1-15.2001 ◽

2001 ◽

Vol 21 (1) ◽

pp. 1-15 ◽

Cited By ~ 25

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.

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Double-stranded DNA translocase activity of transcription factor TFIIH and the mechanism of RNA polymerase II open complex formation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1417709112 ◽

2015 ◽

Vol 112 (13) ◽

pp. 3961-3966 ◽

Cited By ~ 64

Author(s):

James Fishburn ◽

Eric Tomko ◽

Eric Galburt ◽

Steven Hahn

Keyword(s):

Transcription Factor ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription Initiation ◽

Atp Hydrolysis ◽

Dna Unwinding ◽

Pol Ii ◽

Torsional Strain ◽

Double Stranded Dna ◽

Dna Translocase

Formation of the RNA polymerase II (Pol II) open complex (OC) requires DNA unwinding mediated by the transcription factor TFIIH helicase-related subunit XPB/Ssl2. Because XPB/Ssl2 binds DNA downstream from the location of DNA unwinding, it cannot function using a conventional helicase mechanism. Here we show that yeast TFIIH contains an Ssl2-dependent double-stranded DNA translocase activity. Ssl2 tracks along one DNA strand in the 5′ → 3′ direction, implying it uses the nontemplate promoter strand to reel downstream DNA into the Pol II cleft, creating torsional strain and leading to DNA unwinding. Analysis of the Ssl2 and DNA-dependent ATPase activity of TFIIH suggests that Ssl2 has a processivity of approximately one DNA turn, consistent with the length of DNA unwound during transcription initiation. Our results can explain why maintaining the OC requires continuous ATP hydrolysis and the function of TFIIH in promoter escape. Our results also suggest that XPB/Ssl2 uses this translocase mechanism during DNA repair rather than physically wedging open damaged DNA.

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Prp5−Spt8/Spt3 interaction mediates a reciprocal coupling between splicing and transcription

Nucleic Acids Research ◽

10.1093/nar/gkaa311 ◽

2020 ◽

Vol 48 (11) ◽

pp. 5799-5813 ◽

Cited By ~ 4

Author(s):

Wei Shao ◽

Zhan Ding ◽

Zeng-Zhang Zheng ◽

Ji-Jia Shen ◽

Yu-Xian Shen ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Transcription Initiation ◽

In Vitro Assays ◽

Cold Sensitivity ◽

Saga Complex ◽

Spliceosome Assembly ◽

Pol Ii ◽

Genetic Suppressors ◽

Targeted Testing

Abstract Transcription and pre-mRNA splicing are coupled to promote gene expression and regulation. However, mechanisms by which transcription and splicing influence each other are still under investigation. The ATPase Prp5p is required for pre-spliceosome assembly and splicing proofreading at the branch-point region. From an open UV mutagenesis screen for genetic suppressors of prp5 defects and subsequent targeted testing, we identify components of the TBP-binding module of the Spt–Ada–Gcn5 Acetyltransferase (SAGA) complex, Spt8p and Spt3p. Spt8Δ and spt3Δ rescue the cold-sensitivity of prp5-GAR allele, and prp5 mutants restore growth of spt8Δ and spt3Δ strains on 6-azauracil. By chromatin immunoprecipitation (ChIP), we find that prp5 alleles decrease recruitment of RNA polymerase II (Pol II) to an intron-containing gene, which is rescued by spt8Δ. Further ChIP-seq reveals that global effects on Pol II-binding are mutually rescued by prp5-GAR and spt8Δ. Inhibited splicing caused by prp5-GAR is also restored by spt8Δ. In vitro assays indicate that Prp5p directly interacts with Spt8p, but not Spt3p. We demonstrate that Prp5p's splicing proofreading is modulated by Spt8p and Spt3p. Therefore, this study reveals that interactions between the TBP-binding module of SAGA and the spliceosomal ATPase Prp5p mediate a balance between transcription initiation/elongation and pre-spliceosome assembly.

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