scholarly journals 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 ◽  
1996 ◽  
Vol 143 (2) ◽  
pp. 661-671 ◽  
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.

scholarly journals Tyrosine phosphorylation of mammalian RNA polymerase II carboxyl-terminal domain

1993 ◽  
Vol 90 (23) ◽  
pp. 11167-11171 ◽  
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.

Genetic analysis of the repetitive carboxyl-terminal domain of the largest subunit of mouse RNA polymerase II

1988 ◽  
Vol 8 (1) ◽  
pp. 330-339 ◽  
Author(s):  
M S Bartolomei ◽  
N F Halden ◽  
C R Cullen ◽  
J L Corden
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Consensus Sequence ◽  
Loss Of Function ◽  
Terminal Domain ◽  
Carboxyl Terminal Domain ◽  
Carboxyl Terminal ◽  
Substitution Mutations ◽  
Mutant Genes ◽  
Alpha Amanitin

The carboxyl-terminal domain (CTD) of the mouse RNA polymerase II largest subunit consists of 52 repeats of a seven-amino-acid block with the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. A genetic approach was used to determine whether the CTD plays an essential role in RNA polymerase function. Deletion, insertion, and substitution mutations were created in the repetitive region of an alpha-amanitin-resistant largest-subunit gene. The effects of these mutations on RNA polymerase II activity were assayed by measuring the ability of mutant genes to confer alpha-amanitin resistance after transfection of susceptible rodent cells. Mutations that resulted in CTDs containing between 36 and 78 repeats had no effect on the transfer of alpha-amanitin resistance, whereas mutations with 25 or fewer repeats were inactive in this assay. Mutations that contained 29, 31, or 32 repeats had an intermediate effect; the number of alpha-amanitin-resistant colonies was lower and the colonies obtained were smaller, indicating that the mutant RNA polymerase II was defective. In addition, not all of the heptameric repeats were functionally equivalent in that repeats that diverged in up to three amino acids from the consensus sequence could not substitute for the conserved heptamer repeats. We concluded that the CTD is essential for RNA polymerase II activity, since substantial mutations in this region result in loss of function.

scholarly journals Genetic analysis of the repetitive carboxyl-terminal domain of the largest subunit of mouse RNA polymerase II.

1988 ◽  
Vol 8 (1) ◽  
pp. 330-339 ◽  
Author(s):  
M S Bartolomei ◽  
N F Halden ◽  
C R Cullen ◽  
J L Corden
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Consensus Sequence ◽  
Loss Of Function ◽  
Terminal Domain ◽  
Carboxyl Terminal Domain ◽  
Carboxyl Terminal ◽  
Substitution Mutations ◽  
Mutant Genes ◽  
Alpha Amanitin

The carboxyl-terminal domain (CTD) of the mouse RNA polymerase II largest subunit consists of 52 repeats of a seven-amino-acid block with the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. A genetic approach was used to determine whether the CTD plays an essential role in RNA polymerase function. Deletion, insertion, and substitution mutations were created in the repetitive region of an alpha-amanitin-resistant largest-subunit gene. The effects of these mutations on RNA polymerase II activity were assayed by measuring the ability of mutant genes to confer alpha-amanitin resistance after transfection of susceptible rodent cells. Mutations that resulted in CTDs containing between 36 and 78 repeats had no effect on the transfer of alpha-amanitin resistance, whereas mutations with 25 or fewer repeats were inactive in this assay. Mutations that contained 29, 31, or 32 repeats had an intermediate effect; the number of alpha-amanitin-resistant colonies was lower and the colonies obtained were smaller, indicating that the mutant RNA polymerase II was defective. In addition, not all of the heptameric repeats were functionally equivalent in that repeats that diverged in up to three amino acids from the consensus sequence could not substitute for the conserved heptamer repeats. We concluded that the CTD is essential for RNA polymerase II activity, since substantial mutations in this region result in loss of function.

scholarly journals Role of the Mammalian RNA Polymerase II C-Terminal Domain (CTD) Nonconsensus Repeats in CTD Stability and Cell Proliferation

2005 ◽  
Vol 25 (17) ◽  
pp. 7665-7674 ◽  
Author(s):  
Rob D. Chapman ◽  
Marcus Conrad ◽  
Dirk Eick
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Consensus Sequence ◽  
Normal Growth ◽  
Mrna Processing ◽  
Functional Importance ◽  
Pol Ii ◽  
Terminal Domain

ABSTRACT The C-terminal domain (CTD) of mammalian RNA polymerase II (Pol II) consists of 52 repeats of the consensus heptapeptide YSPTSPS and links transcription to the processing of pre-mRNA. The length of the CTD and the number of repeats diverging from the consensus sequence have increased through evolution, but their functional importance remains unknown. Here, we show that the deletion of repeats 1 to 3 or 52 leads to cleavage and degradation of the CTD from Pol II in vivo. Including these repeats, however, allowed the construction of stable, synthetic CTDs. To our surprise, polymerases consisting of just consensus repeats could support normal growth and viability of cells. We conclude that all other nonconsensus CTD repeats are dispensable for the transcription and pre-mRNA processing of genes essential for proliferation.

scholarly journals Functional Unit of the RNA Polymerase II C-Terminal Domain Lies within Heptapeptide Pairs

2004 ◽  
Vol 3 (3) ◽  
pp. 735-740 ◽  
Author(s):  
John W. Stiller ◽  
Matthew S. Cook
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Functional Unit ◽  
Consensus Sequence ◽  
Stabilizing Selection ◽  
Structural Constraints ◽  
Phenotypic Effect ◽  
Terminal Domain ◽  
Tandem Structure ◽  
Rnap Ii

ABSTRACT Unlike all other RNA polymerases, the largest subunit (RPB1) of eukaryotic DNA-dependent RNA polymerase II (RNAP II) has a C-terminal domain (CTD) comprising tandemly repeated heptapeptides with the consensus sequence Y-S-P-T-S-P-S. The tandem structure, heptad consensus, and most key functions of the CTD are conserved between yeast and mammals. In fact, all metazoans, fungi, and green plants examined to date, as well as the nearest protistan relatives of these multicellular groups, contain a tandemly repeated CTD. In contrast, the RNAP II largest subunits from many other eukaryotic organisms have a highly degenerate C terminus or show no semblance of the CTD whatsoever. The reasons for intense stabilizing selection on CTD structure in certain eukaryotes, and its apparent absence in others, are unknown. Here we demonstrate, through in vivo genetic complementation, that the essential functional unit of the yeast CTD is contained within pairs of heptapeptides. Insertion of a single alanine residue between diheptads has little phenotypic effect, while increasing the distance between diheptads produces a mostly quantitative effect on yeast cell growth. We further explore structural constraints on the CTD within an evolutionary context and propose selective mechanisms that could maintain a global tandem structure across hundreds of millions of years of eukaryotic evolution.

scholarly journals Construction and analysis of yeast RNA polymerase II CTD deletion and substitution mutations.

Genetics ◽  
1995 ◽  
Vol 140 (4) ◽  
pp. 1223-1233
Author(s):  
M L West ◽  
J L Corden
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Phosphorylation Sites ◽  
Terminal Domain ◽  
Carboxyl Terminal Domain ◽  
Carboxyl Terminal ◽  
Substitution Mutations ◽  
Rna Polymerase Ii Ctd

Abstract The carboxyl-terminal domain (CTD) of the RNA polymerase II largest subunit plays an essential but poorly understood role in transcription. The CTD is highly phosphorylated in vivo and this modification may be important in the transition from transcription initiation to elongation. We report here the development of a strategy for creating novel yeast CTDs. We have used this approach to show that the minimum viable CTD in yeast contains eight consensus (Tyr1Ser2Pro3Thr4Ser5Pro6Ser7) heptapeptide repeats. Substitution of alanine or glutamate for serines in positions two or five is lethal. In addition, changing tyrosine in position one to phenylalanine is lethal. The effects of mutations that alter potential phosphorylation sites are consistent with a requirement for CTD phosphorylation in vivo.

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