Contractions of the C-Terminal Domain of Saccharomyces cerevisiae Rpb1p Are Mediated by Rad5p

The C-terminal domain (CTD) is an essential domain of the largest subunit of RNA polymerase II, Rpb1p, and is composed of 26 tandem repeats of a seven-amino acid sequence, YSPTSPS. Despite being an essential domain within an essential gene, we have previously demonstrated that the CTD coding region is genetically unstable. Furthermore, yeast with a truncated or mutated CTD sequence are capable of promoting spontaneous genetic expansion or contraction of this coding region to improve fitness. We investigated the mechanism by which the CTD contracts using a tet-off reporter system for RPB1 to monitor genetic instability within the CTD coding region. We report that contractions require the post-replication repair factor Rad5p but, unlike expansions, not the homologous recombination factors Rad51p and Rad52p. Sequence analysis of contraction events reveals that deleted regions are flanked by microhomologies. We also find that G-quadruplex forming sequences predicted by the QGRS Mapper are enriched on the noncoding strand of the CTD compared to the body of RPB1. Formation of G-quadruplexes in the CTD coding region could block the replication fork, necessitating post-replication repair. We propose that contractions of the CTD result when microhomologies misalign during Rad5p-dependent template switching via fork reversal.

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Template Switching Mediates Contractions of a Repetitive Coding Region in S. cerevisiae

10.1101/712588 ◽

2019 ◽

Author(s):

Taylor Stewart ◽

Alexandra E. Exner ◽

Paras Patnaik ◽

Stephen M. Fuchs

Keyword(s):

Rna Polymerase Ii ◽

Tandem Repeats ◽

Essential Gene ◽

The Body ◽

Template Switching ◽

Coding Region ◽

Reporter System ◽

G Quadruplex ◽

Post Replication Repair ◽

Essential Domain

ABSTRACTThe C-terminal domain (CTD) is an essential domain of the largest subunit of RNA polymerase II, Rpb1p, and is composed of 26 tandem repeats of a seven-amino acid sequence, YSPTSPS. Despite being an essential domain within an essential gene, we have previously demonstrated that the CTD coding region is genetically unstable. Furthermore, yeast with a truncated or mutated CTD sequence are capable of promoting spontaneous genetic expansion or contraction of this coding region to improve fitness. We investigated the mechanism by which the CTD contracts using a tet-off reporter system for RPB1 to monitor genetic instability within the CTD coding region. We report that contractions require the post-replication repair factor Rad5p but, unlike expansions, not the homologous recombination factors Rad51p and Rad52p. Sequence analysis of contraction events reveals that deleted regions are flanked by microhomologies. We also find that G-quadruplex forming sequences predicted by the QGRS Mapper are enriched on the noncoding strand of the CTD compared to the body of RPB1. Formation of G-quadruplexes in the CTD coding region could block the replication fork, necessitating post-replication repair by template switching. We propose that contractions of the CTD result when microhomologies misalign during Rad5p-dependent template switching via fork reversal.

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Tyrosine phosphorylation of mammalian RNA polymerase II carboxyl-terminal domain

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.90.23.11167 ◽

1993 ◽

Vol 90 (23) ◽

pp. 11167-11171 ◽

Cited By ~ 142

Author(s):

R Baskaran ◽

M E Dahmus ◽

J Y Wang

Keyword(s):

Tyrosine Kinase ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Tandem Repeats ◽

Consensus Sequence ◽

Src Tyrosine Kinase ◽

Terminal Domain ◽

Carboxyl Terminal Domain ◽

Carboxyl Terminal

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II is composed of tandem repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. Phosphorylation of the CTD occurs during formation of the initiation complex and is correlated with the transition from complex assembly to elongation. Previously, serine and threonine residues within the CTD have been shown to be modified by the addition of phosphate and by the addition of O-linked GlcNAc. Our results establish that the CTD is also modified in vivo by phosphorylation on tyrosine. Furthermore, a nuclear tyrosine kinase encoded by the c-abl protooncogene phosphorylates the CTD to a high stoichiometry in vitro. Under conditions of maximum phosphorylation, approximately 30 mol of phosphate are incorporated per mol of CTD. The observation that the CTD is not phosphorylated by c-Src tyrosine kinase under identical conditions indicates that the CTD is not a substrate of all tyrosine kinases. Phosphorylation of tyrosine residues within the CTD may modulate the interaction of RNA polymerase II with the preinitiation complex and, hence, may be important in regulating gene expression.

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Rct1, a Nuclear RNA Recognition Motif-Containing Cyclophilin, Regulates Phosphorylation of the RNA Polymerase II C-Terminal Domain

Molecular and Cellular Biology ◽

10.1128/mcb.02187-06 ◽

2007 ◽

Vol 27 (10) ◽

pp. 3601-3611 ◽

Cited By ~ 13

Author(s):

Monika Gullerova ◽

Andrea Barta ◽

Zdravko J. Lorkovic

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Essential Gene ◽

Close Association ◽

Rna Recognition Motif ◽

Rna Recognition ◽

Terminal Domain ◽

Important Player ◽

Rnap Ii

ABSTRACT Phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (RNAP II) is a dynamic process that regulates transcription and coordinates it with pre-mRNA processing. We show here that Rct1, a nuclear multidomain cyclophilin from Schizosaccharomyces pombe, is encoded by an essential gene that interacts with the CTD and regulates its phosphorylation in vivo. Downregulation of Rct1 levels results in increased phosphorylation of the CTD at both Ser2 and Ser5 and in a commensurate decrease in RNAP II transcription. In contrast, overexpression of Rct1 decreases phosphorylation on both sites. The close association of Rct1 with transcriptionally active chromatin suggests a role in regulation of RNAP II transcriptional activity. These data, together with the pleiotropic phenotype upon Rct1 deregulation, suggest that this multidomain cyclophilin is an important player in maintaining the correct phosphorylation code of the CTD and thereby regulating CTD function.

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Interactions of Sen1, Nrd1, and Nab3 with Multiple Phosphorylated Forms of the Rpb1 C-Terminal Domain in Saccharomyces cerevisiae

Eukaryotic Cell ◽

10.1128/ec.05320-11 ◽

2012 ◽

Vol 11 (4) ◽

pp. 417-429 ◽

Cited By ~ 39

Author(s):

Karen Chinchilla ◽

Juan B. Rodriguez-Molina ◽

Doris Ursic ◽

Jonathan S. Finkel ◽

Aseem Z. Ansari ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Rna Polymerase Ii ◽

Protein Interactions ◽

Noncoding Rna ◽

Protein Protein Interactions ◽

Coding Region ◽

Protein Coding ◽

Content Type ◽

Protein Coding Genes ◽

Terminal Domain

ABSTRACT The Saccharomyces cerevisiae SEN1 gene codes for a nuclear, ATP-dependent helicase which is embedded in a complex network of protein-protein interactions. Pleiotropic phenotypes of mutations in SEN1 suggest that Sen1 functions in many nuclear processes, including transcription termination, DNA repair, and RNA processing. Sen1, along with termination factors Nrd1 and Nab3, is required for the termination of noncoding RNA transcripts, but Sen1 is associated during transcription with coding and noncoding genes. Sen1 and Nrd1 both interact directly with Nab3, as well as with the C-terminal domain (CTD) of Rpb1, the largest subunit of RNA polymerase II. It has been proposed that Sen1, Nab3, and Nrd1 form a complex that associates with Rpb1 through an interaction between Nrd1 and the Ser 5 -phosphorylated (Ser 5 -P) CTD. To further study the relationship between the termination factors and Rpb1, we used two-hybrid analysis and immunoprecipitation to characterize sen1-R302W , a mutation that impairs an interaction between Sen1 and the Ser 2 -phosphorylated CTD. Chromatin immunoprecipitation indicates that the impairment of the interaction between Sen1 and Ser 2 -P causes the reduced occupancy of mutant Sen1 across the entire length of noncoding genes. For protein-coding genes, mutant Sen1 occupancy is reduced early and late in transcription but is similar to that of the wild type across most of the coding region. The combined data suggest a handoff model in which proteins differentially transfer from the Ser 5 - to the Ser 2 -phosphorylated CTD to promote the termination of noncoding transcripts or other cotranscriptional events for protein-coding genes.

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A rule-based kinetic model of RNA polymerase II C-terminal domain phosphorylation

Journal of The Royal Society Interface ◽

10.1098/rsif.2013.0438 ◽

2013 ◽

Vol 10 (86) ◽

pp. 20130438 ◽

Cited By ~ 5

Author(s):

Stuart Aitken ◽

Ross D. Alexander ◽

Jean D. Beggs

Keyword(s):

Kinetic Model ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Messenger Rna ◽

Transcriptional Elongation ◽

Coding Region ◽

Detailed Model ◽

Rule Based ◽

Terminal Domain ◽

Processing Pathways

The complexity of many RNA processing pathways is such that a conventional systems modelling approach is inadequate to represent all the molecular species involved. We demonstrate that rule-based modelling permits a detailed model of a complex RNA signalling pathway to be defined. Phosphorylation of the RNA polymerase II (RNAPII) C-terminal domain (CTD; a flexible tail-like extension of the largest subunit) couples pre-messenger RNA capping, splicing and 3′ end maturation to transcriptional elongation and termination, and plays a central role in integrating these processes. The phosphorylation states of the serine residues of many heptapeptide repeats of the CTD alter along the coding region of genes as a function of distance from the promoter. From a mechanistic perspective, both the changes in phosphorylation and the location at which they take place on the genes are a function of the time spent by RNAPII in elongation as this interval provides the opportunity for the kinases and phosphatases to interact with the CTD. On this basis, we synthesize the available data to create a kinetic model of the action of the known kinases and phosphatases to resolve the phosphorylation pathways and their kinetics.

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Suppression Analysis Reveals a Functional Difference Between the Serines in Positions Two and Five in the Consensus Sequence of the C-Terminal Domain of Yeast RNA Polymerase II

Genetics ◽

10.1093/genetics/143.2.661 ◽

1996 ◽

Vol 143 (2) ◽

pp. 661-671 ◽

Cited By ~ 6

Author(s):

Anton Yuryev ◽

Jeffry L Corden

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Tandem Repeats ◽

Consensus Sequence ◽

Functional Difference ◽

Terminal Domain ◽

Truncation Mutation ◽

Cold Sensitive ◽

Substitution Mutations ◽

Multiple Substitution

Abstract The largest subunit of RNA polymerase II contains a repetitive C-terminal domain (CTD) consisting of tandem repeats of the consensus sequence TyrlSer2Pro3Thr4Ser5Pro6Ser7. Substitution of nonphosphe rylatable amino acids at positions two or five of the Saccharomyces cerevisiae CTD is lethal. We developed a selection ssytem for isolating suppressors of this lethal phenotype and cloned a gene, SCA1 (suppressor of CTD alanine), which complements recessive suppressors of lethal multiple-substitution mutations. A partial deletion of SCA1 (sca1Δ::hisG) suppresses alanine or glutamate substitutions at position two of the consensus CTD sequence, and a lethal CTD truncation mutation, but SCA1 deletion does not suppress alanine or glutamate substitutions at position five. SCA1 is identical to SRB9, a suppressor of a cold-sensitive CTD truncation mutation. Strains carrying dominant SRB mutations have the same suppression properties as a sca1Δ::hisG strain. These results reveal a functional difference between positions two and five of the consensus CTD heptapeptide repeat. The ability of SCA1 and SRB mutant alleles to suppress CTD truncation mutations suggest that substitutions at position two, but not at position five, cause a defect in RNA polymerase II function similar to that introduced by CTD truncation.

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Tandem repeats structure of gel-forming mucin domains could be revealed by SMRT sequencing data

10.21203/rs.3.rs-112828/v2 ◽

2021 ◽

Author(s):

Tiange Lang

Keyword(s):

Single Molecule ◽

Tandem Repeats ◽

The Body ◽

Nucleotide Polymorphisms ◽

Sequencing Data ◽

Coding Region ◽

Smrt Sequencing ◽

Epithelial Surface ◽

Sequencing Technologies ◽

Long Reads

Abstract Mucins are large glycoproteins that cover and protect epithelial surface of the body. Gel-forming mucin domains of mucin genes are rich in proline, threonine, and serine that are heavily glycosylate. These domains show great complexity with tandem repeats (TRs), thus make it difficult to study the sequences. With the coming of single molecule real-time (SMRT) sequencing technologies, we manage to present sequence structure of mucin domains via SMRT long reads for gel-forming mucins MUC2, MUC5AC, MUC5B and MUC6. Our study shows that for different individuals, single nucleotide polymorphisms (SNPs) could be found in mucin domains of MUC2, MUC5AC, MUC5B and MUC6, while different number of tandem repeats could be found in mucin domains of MUC2 and MUC6. Furthermore, we get the sequence of MUC2, MUC5AC, and MUC5B mucin domain in a Chinese individual at accuracy of possibly maximum 99.98%, 99.93%, and 99.76%, respectively. We report a new method to obtain DNA sequence of gel-forming mucin domains. This method will provided new insights on getting the sequence for Tandem Repeat parts which locate in coding region. With the sequences we obtained with this method, we can give more information for people to study the sequences of gel-forming mucin domains.

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