Phosphorylation of the RNA polymerase II C-terminal domain by TFIIH kinase is not essential for transcription of
            <i>Saccharomyces cerevisiae</i>
            genome

Hong, Sun Woo; Hong, Seong Min; Yoo, Jae Wook; Lee, Young Chul; Kim, Soyoun; Lis, John T.; Lee, Dong-Ki

doi:10.1073/pnas.0903642106

Cited by 42 publications

(51 citation statements)

References 30 publications

(29 reference statements)

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“…The Pol IIO was indistinguishable from that generated with the yeast TFIIK kinase, a subcomplex of TFIIH (supplemental Fig. S1B), which is responsible for in vivo phosphorylation of the CTD and the recruitment of CE (29,54). As such, the Pol IIO generated in vitro was suitable for binding studies with CE.…”

Section: Methodsmentioning

confidence: 88%

A Dual Interface Determines the Recognition of RNA Polymerase II by RNA Capping Enzyme*

Suh

Meyer

et al. 2010

Journal of Biological Chemistry

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RNA capping enzyme (CE) is recruited specifically to RNA polymerase II (Pol II) transcription sites to facilitate cotranscriptional 5-capping of pre-mRNA and other Pol II transcripts. The current model to explain this specific recruitment of CE to Pol II as opposed to Pol I and Pol III rests on the interaction between CE and the phosphorylated C-terminal domain (CTD) of Pol II largest subunit Rpb1 and more specifically between the CE nucleotidyltransferase domain and the phosphorylated CTD. Through biochemical and diffraction analyses, we demonstrate the existence of a distinctive stoichiometric complex between CE and the phosphorylated Pol II (Pol IIO). Analysis of the complex revealed an additional and unexpected polymerase-CE interface (PCI) located on the multihelical Foot domain of Rpb1. We name this interface PCI1 and the previously known nucleotidyltransferase/phosphorylated CTD interface PCI2. Although PCI1 and PCI2 individually contribute to only weak interactions with CE, a dramatically stabilized and stoichiometric complex is formed when PCI1 and PCI2 are combined in cis as they occur in an intact phosphorylated Pol II molecule. Disrupting either PCI1 or PCI2 by alanine substitution or deletion diminishes CE association with Pol II and causes severe growth defects in vivo. Evidence from manipulating PCI1 indicates that the Foot domain contributes to the specificity in CE interaction with Pol II as opposed to Pol I and Pol III. Our results indicate that the dual interface based on combining PCI1 and PCI2 is required for directing CE to Pol II elongation complexes.In eukaryotic cells, RNA polymerase II (RNA Pol II) 5 and its associated factors carry out transcription of pre-mRNAs, snRNAs, telomerase RNA, and other noncoding RNAs. Pol II also couples transcription to nuclear processes including pre-mRNA modifications (1-4), mRNA export, and chromatin reconfiguration (5-7). Coupling RNA processing with synthesis is presumed to be critical in restricting the temporal window during which unmodified transcripts are vulnerable to degradation by endogenous ribonuclease activities (8 -10) and directing RNA processing factors to sites of Pol II transcription at specific steps during Pol II progression through a gene. The Pol II elongation complex coordinates these transactions to help orchestrate control over gene expression (for review, see Ref. 11). Such coordination is mediated by specific and reversible interactions among Pol II and the factors involved in elongation.The formation of RNA 5Ј cap structure, m 7 GpppN, is the first transcription-coordinated RNA modification event, and it occurs as soon as the transcript attains ϳ25 nucleotides (12-15). The RNA cap is formed in three enzymatic steps (16, 17); (i) removal of the 5Ј ␥-phosphate catalyzed by the RNA triphosphatase; (ii) attachment of a GMP to the 5Ј diphosphate by the guanylyltransferase; (iii) methylation of the 5Ј guanine by the cap methyltransferase. The first two of these steps are closely linked and coupled to Pol II transcription in most org...

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Section: Methodsmentioning

confidence: 88%

A Dual Interface Determines the Recognition of RNA Polymerase II by RNA Capping Enzyme*

Suh

Meyer

et al. 2010

Journal of Biological Chemistry

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“…These interactions and others noted above underlie the proposal of a transcription elongation checkpoint that ensures a temporal window for capping of nascent mRNAs (27,35,36,45). The checkpoint model is attractive insofar as there is evidence that positive and negative regulation of transcription can occur at the step of capping enzyme recruitment (6,12) and that either diminished cap guanylylation activity or defective installation of the guanylyltransferase-recruiting Ser5-PO 4 Pol II CTD mark can result in the production of uncapped transcripts that suffer premature 5Ј exonucleolytic decay (20,41). However, recent studies highlight that the putative checkpoint is either not enforced or not required on a large fraction of cellular transcription units.…”

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confidence: 99%

“…The inherently plastic CTD structure is sculpted by cyclin-dependent kinases (Cdks) that have various positional specificities and act at different stages of the transcription cycle. The Cdk7 kinase (a component of transcription factor TFIIH) acts at or shortly after initiation to install Ser5-PO 4 and Ser7-PO 4 marks on the CTD (1), of which Ser5-PO 4 is a critical determinant of capping enzyme recruitment and mRNA capping in vivo (20,22,49).Capping enzymes may also access the transcription complex by binding to the Pol II elongation factor Spt5 (32, 52). Spt5 is a large polypeptide (ϳ1,000 to 1,200 amino acids [aa]) composed of multiple domain modules, including a distinctive Cterminal repeat domain (the "Spt5 CTD") that directly binds RNA capping enzymes and is targeted for threonine phosphorylation by the Cdk9 protein kinase (33, 56).…”

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confidence: 99%

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confidence: 99%

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Separable Functions of the Fission Yeast Spt5 Carboxyl-Terminal Domain (CTD) in Capping Enzyme Binding and Transcription Elongation Overlap with Those of the RNA Polymerase II CTD

Schneider

Pei

Shuman

et al. 2010

Molecular and Cellular Biology

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An interaction network connecting mRNA capping enzymes, the RNA polymerase II (Pol II) carboxylterminal domain (CTD), elongation factor Spt5, and the Cdk7 and Cdk9 protein kinases is thought to comprise a transcription elongation checkpoint. A crux of this network is Spt5, which regulates early transcription elongation and has an imputed role in pre-mRNA processing via its physical association with capping enzymes. Schizosaccharomyces pombe Spt5 has a distinctive CTD composed of tandem nonapeptide repeats of the consensus sequence 1 TPAWNSGSK 9 . The Spt5 CTD binds the capping enzymes and is a substrate for threonine phosphorylation by the Cdk9 kinase. Here we report that deletion of the S. pombe Spt5 CTD results in slow growth and aberrant cell morphology. The severity of the spt5-⌬CTD phenotype is exacerbated by truncation of the Pol II CTD and ameliorated by overexpression of the capping enzymes RNA triphosphatase and RNA guanylyltransferase. These results suggest that the Spt5 and Pol II CTDs play functionally overlapping roles in capping enzyme recruitment. We probed structure-activity relations of the Spt5 CTD by alanine scanning of the consensus nonapeptide. The T1A change abolished CTD phosphorylation by Cdk9 but did not affect CTD binding to the capping enzymes. The T1A and P2A mutations elicited cold-sensitive (cs) and temperature-sensitive (ts) growth defects and conferred sensitivity to growth inhibition by 6-azauracil that was exacerbated by partial truncations of the Pol II CTD. The T1A phenotypes were rescued by a phosphomimetic T1E change but not by capping enzyme overexpression. These results imply a positive role for Spt5 CTD phosphorylation in Pol Il transcription elongation in fission yeast, distinct from its capping enzyme interactions. Viability of yeast cells bearing both Spt5 CTD T1A and Pol II CTD S2A mutations heralds that the Cdk9 kinase has an essential target other than Spt5 and Pol II CTD-Ser2.Eukaryal mRNA processing is linked physically and temporally to transcription elongation. The earliest processing step is mRNA capping, which can occur as soon as the 5Ј triphosphate terminus of the nascent RNA extrudes from elongating RNA polymerase (6, 15). The cellular RNA capping enzymes are directed to nascent mRNAs by binding to the phosphorylated carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) (7,8,18,34,29,57). The Pol II CTD, consisting of tandem heptapeptide repeats of the consensus sequence Y 1 S 2 P 3 T 4 S 5 P 6 S 7 , functions as a landing pad for diverse cellular proteins that regulate the initiation, elongation, and termination steps of Pol II transcription, modify chromatin structure, and catalyze or regulate RNA capping, splicing, and polyadenylation (30, 37). The inherently plastic CTD structure is sculpted by cyclin-dependent kinases (Cdks) that have various positional specificities and act at different stages of the transcription cycle. The Cdk7 kinase (a component of transcription factor TFIIH) acts at or shortly after initiation to ins...

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“…Expression data was normalized through quantile normalization and Robust Multichip Average (RMA) algorithm. 26 To visualize overall changes in gene expression MA plots were generated, where X axis represents the average of log2 expression of control and test samples and Y axis corresponds to ratio of log2 expression value of test to control. Fold changes in expression for 24,000 genes were calculated for each nanoparticle relative to untreated control.…”

Section: Methodsmentioning

confidence: 99%

Evaluation of Toxicity and Gene Expression Changes Triggered by Quantum Dots

Dua¹,

Jeong²,

Lee³

et al. 2010

Bulletin of the Korean Chemical Society

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Quantum dots (QDs) are extensively employed for biomedical research as a fluorescence reporter and their use for various labeling applications will continue to increase as they are preferred over conventional labeling methods for various reasons. However, concerns have been raised over the toxicity of these particles in the biological system. Till date no thorough investigation has been carried out to identify the molecular signatures of QD mediated toxicity. In this study we evaluated the toxicity of CdSe, Cd1-xZnxS/ZnS and CdSe/ZnS quantum dots having different spectral properties (red, blue, green) using human embryonic kidney fibroblast cells (HEK293). Cell viability assay for both short and long duration exposure show concentration material dependent toxicity, in the order of CdSe > Cd1-xZnxS/ ZnS > CdSe/ZnS. Genome wide changes in the expression of genes upon QD exposure was also analyzed by wholegenome microarray. All the three QDs show increase in the expression of genes related to apoptosis, inflammation and response towards stress and wounding. Further comparison of coated versus uncoated CdSe QD-mediated cell death and molecular changes suggests that ZnS coating could reduce QD mediated cytotoxicity to some extent only.

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Phosphorylation of the RNA polymerase II C-terminal domain by TFIIH kinase is not essential for transcription of Saccharomyces cerevisiae genome

Cited by 42 publications

References 30 publications

A Dual Interface Determines the Recognition of RNA Polymerase II by RNA Capping Enzyme*

A Dual Interface Determines the Recognition of RNA Polymerase II by RNA Capping Enzyme*

Separable Functions of the Fission Yeast Spt5 Carboxyl-Terminal Domain (CTD) in Capping Enzyme Binding and Transcription Elongation Overlap with Those of the RNA Polymerase II CTD

Evaluation of Toxicity and Gene Expression Changes Triggered by Quantum Dots

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