scholarly journals Genetic Interactions of Spt4-Spt5 and TFIIS With the RNA Polymerase II CTD and CTD Modifying Enzymes in Saccharomyces cerevisiae

Genetics ◽  
2001 ◽  
Vol 159 (2) ◽  
pp. 487-497 ◽  
Author(s):  
Derek L Lindstrom ◽  
Grant A Hartzog
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Normal Function ◽  
Genetic Interactions ◽  
Elongation Factors ◽  
Repeat Domain ◽  
Ctd Phosphatase ◽  
Rna Polymerase Ii Ctd

Abstract Genetic and biochemical studies have identified many factors thought to be important for transcription elongation. We investigated relationships between three classes of these factors: (1) transcription elongation factors Spt4-Spt5, TFIIS, and Spt16; (2) the C-terminal heptapeptide repeat domain (CTD) of RNA polymerase II; and (3) protein kinases that phosphorylate the CTD and a phosphatase that dephosphorylates it. We observe that spt4 and spt5 mutations cause strong synthetic phenotypes in combination with mutations that shorten or alter the composition of the CTD; affect the Kin28, Bur1, or Ctk1 CTD kinases; and affect the CTD phosphatase Fcp1. We show that Spt5 co-immunoprecipitates with RNA polymerase II that has either a hyper- or a hypophosphorylated CTD. Furthermore, mutation of the CTD or of CTD modifying enzymes does not affect the ability of Spt5 to bind RNA polymerase II. We find a similar set of genetic interactions between the CTD, CTD modifying enzymes, and TFIIS. In contrast, an spt16 mutation did not show these interactions. These results suggest that the CTD plays a key role in modulating elongation in vivo and that at least a subset of elongation factors are dependent upon the CTD for their normal function.

Structural insight into nucleosome transcription by RNA polymerase II with elongation factors

Science ◽  
2019 ◽  
Vol 363 (6428) ◽  
pp. 744-747 ◽  
Author(s):  
Haruhiko Ehara ◽  
Tomoya Kujirai ◽  
Yuka Fujino ◽  
Mikako Shirouzu ◽  
Hitoshi Kurumizaka ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Chromosomal Dna ◽  
Elongation Factors ◽  
Cryo Electron Microscopy ◽  
Structural Insight ◽  
Insight Into

RNA polymerase II (RNAPII) transcribes chromosomal DNA that contains multiple nucleosomes. The nucleosome forms transcriptional barriers, and nucleosomal transcription requires several additional factors in vivo. We demonstrate that the transcription elongation factors Elf1 and Spt4/5 cooperatively lower the barriers and increase the RNAPII processivity in the nucleosome. The cryo–electron microscopy structures of the nucleosome-transcribing RNAPII elongation complexes (ECs) reveal that Elf1 and Spt4/5 reshape the EC downstream edge and intervene between RNAPII and the nucleosome. They facilitate RNAPII progression through superhelical location SHL(–1) by adjusting the nucleosome in favor of the forward progression. They suppress pausing at SHL(–5) by preventing the stable RNAPII-nucleosome interaction. Thus, the EC overcomes the nucleosomal barriers while providing a platform for various chromatin functions.

scholarly journals RNA Polymerase II CTD phosphatase Rtr1 fine-tunes transcription termination

PLoS Genetics ◽  
2020 ◽  
Vol 16 (3) ◽  
pp. e1008317 ◽  
Author(s):  
Jose F. Victorino ◽  
Melanie J. Fox ◽  
Whitney R. Smith-Kinnaman ◽  
Sarah A. Peck Justice ◽  
Katlyn H. Burriss ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Termination ◽  
Ctd Phosphatase ◽  
Rna Polymerase Ii Ctd

scholarly journals Genetic interaction mapping informs integrative structure determination of protein complexes

Science ◽  
2020 ◽  
Vol 370 (6522) ◽  
pp. eaaz4910 ◽  
Author(s):  
Hannes Braberg ◽  
Ignacia Echeverria ◽  
Stefan Bohn ◽  
Peter Cimermancic ◽  
Anthony Shiver ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Structure Determination ◽  
Genetic Interaction ◽  
Protein Complexes ◽  
Point Mutations ◽  
Genetic Interactions ◽  
Cellular Functions ◽  
Cross Links

Determining structures of protein complexes is crucial for understanding cellular functions. Here, we describe an integrative structure determination approach that relies on in vivo measurements of genetic interactions. We construct phenotypic profiles for point mutations crossed against gene deletions or exposed to environmental perturbations, followed by converting similarities between two profiles into an upper bound on the distance between the mutated residues. We determine the structure of the yeast histone H3-H4 complex based on ~500,000 genetic interactions of 350 mutants. We then apply the method to subunits Rpb1-Rpb2 of yeast RNA polymerase II and subunits RpoB-RpoC of bacterial RNA polymerase. The accuracy is comparable to that based on chemical cross-links; using restraints from both genetic interactions and cross-links further improves model accuracy and precision. The approach provides an efficient means to augment integrative structure determination with in vivo observations.

scholarly journals Requirement for Yeast RAD26, a Homolog of the HumanCSB Gene, in Elongation by RNA Polymerase II

2001 ◽  
Vol 21 (24) ◽  
pp. 8651-8656 ◽  
Author(s):  
Sung-Keun Lee ◽  
Sung-Lim Yu ◽  
Louise Prakash ◽  
Satya Prakash
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Rapid Synthesis ◽  
Developmental Defects ◽  
Developmental Abnormalities ◽  
Developmental Problems ◽  
Uv Lesions

ABSTRACT Mutations in the human CSB gene cause Cockayne syndrome (CS). In addition to increased photosensitivity, CS patients suffer from severe developmental abnormalities, including growth retardation and mental retardation. Whereas a deficiency in the preferential repair of UV lesions from the transcribed strand accounts for the increased photosensitivity of CS patients, the reason for developmental defects in these individuals has remained unclear. Here we provide in vivo evidence for a role of RAD26, the counterpart of the CSB gene in Saccharomyces cerevisiae, in transcription elongation by RNA polymerase II, and in addition we show that under conditions requiring rapid synthesis of new mRNAs, growth is considerably reduced in cells lackingRAD26. These findings implicate a role for CSB in transcription elongation, and they strongly suggest that impaired transcription elongation is the underlying cause of the developmental problems in CS patients.

scholarly journals FCP1, a Phosphatase Specific for the Heptapeptide Repeat of the Largest Subunit of RNA Polymerase II, Stimulates Transcription Elongation

2002 ◽  
Vol 22 (21) ◽  
pp. 7543-7552 ◽  
Author(s):  
Subhrangsu S. Mandal ◽  
Helen Cho ◽  
Sungjoon Kim ◽  
Kettly Cabane ◽  
Danny Reinberg
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Carboxy Terminal Domain ◽  
Transcription Factor Iif ◽  
Transcript Elongation ◽  
Rnap Ii ◽  
Carboxy Terminal

ABSTRACT FCP1, a phosphatase specific for the carboxy-terminal domain of RNA polymerase II (RNAP II), was found to stimulate transcript elongation by RNAP II in vitro and in vivo. This activity is independent of and distinct from the elongation-stimulatory activity associated with transcription factor IIF (TFIIF), and the elongation effects of TFIIF and FCP1 were found to be additive. Genetic experiments resulted in the isolation of several distinct fcp1 alleles. One of these alleles was found to suppress the slow-growth phenotype associated with either the reduction of intracellular nucleotide concentrations or the inhibition of other transcription elongation factors. Importantly, this allele of fcp1 was found to be lethal when combined individually with two mutations in the second-largest subunit of RNAP II, which had been shown previously to affect transcription elongation.

scholarly journals RNA Polymerase II Elongation Factors of Saccharomyces cerevisiae: a Targeted Proteomics Approach

2002 ◽  
Vol 22 (20) ◽  
pp. 6979-6992 ◽  
Author(s):  
Nevan J. Krogan ◽  
Minkyu Kim ◽  
Seong Hoon Ahn ◽  
Guoqing Zhong ◽  
Michael S. Kobor ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
The Novel ◽  
Coding Region ◽  
Elongation Factors ◽  
Uncharacterized Protein ◽  
Proteomics Approach

ABSTRACT To physically characterize the web of interactions connecting the Saccharomyces cerevisiae proteins suspected to be RNA polymerase II (RNAPII) elongation factors, subunits of Spt4/Spt5 and Spt16/Pob3 (corresponding to human DSIF and FACT), Spt6, TFIIF (Tfg1, -2, and -3), TFIIS, Rtf1, and Elongator (Elp1, -2, -3, -4, -5, and -6) were affinity purified under conditions designed to minimize loss of associated polypeptides and then identified by mass spectrometry. Spt16/Pob3 was discovered to associate with three distinct complexes: histones; Chd1/casein kinase II (CKII); and Rtf1, Paf1, Ctr9, Cdc73, and a previously uncharacterized protein, Leo1. Rtf1 and Chd1 have previously been implicated in the control of elongation, and the sensitivity to 6-azauracil of strains lacking Paf1, Cdc73, or Leo1 suggested that these proteins are involved in elongation by RNAPII as well. Confirmation came from chromatin immunoprecipitation (ChIP) assays demonstrating that all components of this complex, including Leo1, cross-linked to the promoter, coding region, and 3′ end of the ADH1 gene. In contrast, the three subunits of TFIIF cross-linked only to the promoter-containing fragment of ADH1. Spt6 interacted with the uncharacterized, essential protein Iws1 (interacts with Spt6), and Spt5 interacted either with Spt4 or with a truncated form of Spt6. ChIP on Spt6 and the novel protein Iws1 resulted in the cross-linking of both proteins to all three regions of the ADH1 gene, suggesting that Iws1 is likely an Spt6-interacting elongation factor. Spt5, Spt6, and Iws1 are phosphorylated on consensus CKII sites in vivo, conceivably by the Chd1/CKII associated with Spt16/Pob3. All the elongation factors but Elongator copurified with RNAPII.

scholarly journals The Structure of Fcp1, an Essential RNA Polymerase II CTD Phosphatase

2008 ◽  
Vol 32 (4) ◽  
pp. 478-490 ◽  
Author(s):  
Agnidipta Ghosh ◽  
Stewart Shuman ◽  
Christopher D. Lima
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Ctd Phosphatase ◽  
Rna Polymerase Ii Ctd

Inhibition of Tat transactivation by the RNA polymerase II CTD-phosphatase FCP1

AIDS ◽  
2001 ◽  
Vol 15 (3) ◽  
pp. 301-307 ◽  
Author(s):  
Paolo Licciardo ◽  
Giuliana Napolitano ◽  
Barbara Majello ◽  
Luigi Lania
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Ctd Phosphatase ◽  
Rna Polymerase Ii Ctd
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