scholarly journals Sub1 Globally Regulates RNA Polymerase II C-Terminal Domain Phosphorylation

2010 ◽  
Vol 30 (21) ◽  
pp. 5180-5193 ◽  
Author(s):  
Alicia García ◽  
Emanuel Rosonina ◽  
James L. Manley ◽  
Olga Calvo
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Large Subunit ◽  
Terminal Domain ◽  
Mrna Metabolism ◽  
Genes Encoding ◽  
The One ◽  
Ctd Phosphorylation ◽  
Transcription Cycle

ABSTRACT The transcriptional coactivator Sub1 has been implicated in several aspects of mRNA metabolism in yeast, such as activation of transcription, termination, and 3′-end formation. Here, we present evidence that Sub1 plays a significant role in controlling phosphorylation of the RNA polymerase II large subunit C-terminal domain (CTD). We show that SUB1 genetically interacts with the genes encoding all four known CTD kinases, SRB10, KIN28, BUR1, and CTK1, suggesting that Sub1 acts to influence CTD phosphorylation at more than one step of the transcription cycle. To address this directly, we first used in vitro kinase assays, and we show that, on the one hand, SUB1 deletion increased CTD phosphorylation by Kin28, Bur1, and Ctk1 but, on the other, it decreased CTD phosphorylation by Srb10. Second, chromatin immunoprecipitation assays revealed that SUB1 deletion decreased Srb10 chromatin association on the inducible GAL1 gene but increased Kin28 and Ctk1 chromatin association on actively transcribed genes. Taken together, our data point to multiple roles for Sub1 in the regulation of CTD phosphorylation throughout the transcription cycle.

scholarly journals Human Cyclin K, a Novel RNA Polymerase II-Associated Cyclin Possessing Both Carboxy-Terminal Domain Kinase and Cdk-Activating Kinase Activity

1998 ◽  
Vol 18 (7) ◽  
pp. 4291-4300 ◽  
Author(s):  
Michael C. Edwards ◽  
Calvin Wong ◽  
Stephen J. Elledge
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Cell Cycle Progression ◽  
Large Subunit ◽  
Terminal Domain ◽  
Gene Coding ◽  
Acid Protein ◽  
Rnap Ii ◽  
Carboxy Terminal

ABSTRACT The gene coding for human cyclin K was isolated as aCPR (cell-cycle progression restoration) gene by virtue of its ability to impart a Far− phenotype to the budding yeast Saccharomyces cerevisiae and to rescue the lethality of a deletion of the G1 cyclin genes CLN1,CLN2, and CLN3. The cyclin K gene encodes a 357-amino-acid protein most closely related to human cyclins C and H, which have been proposed to play a role in regulating basal transcription through their association with and activation of cyclin-dependent kinases (Cdks) that phosphorylate the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II (RNAP II). Murine and Drosophila melanogaster homologs of cyclin K have also been identified. Cyclin K mRNA is ubiquitously expressed in adult mouse and human tissues, but is most abundant in the developing germ cells of the adult testis and ovaries. Cyclin K is associated with potent CTD kinase and Cdk kinase (CAK) activity in vitro and coimmunoprecipitates with the large subunit of RNAP II. Thus, cyclin K represents a new member of the “transcription” cyclin family which may play a dual role in regulating Cdk and RNAP II activity.

scholarly journals Tyr1 phosphorylation promotes the phosphorylation of Ser2 on the C-terminal domain of RNA polymerase II by P-TEFb

2019 ◽  
Author(s):  
Joshua E. Mayfield ◽  
Seema Irani ◽  
Edwin E. Escobar ◽  
Zhao Zhang ◽  
Nathanial T. Burkholder ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Elongation Factor ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Terminal Domain ◽  
Ctd Phosphorylation

SummaryThe Positive Transcription Elongation Factor b (P-TEFb) phosphorylates Ser2 residues of RNA polymerase II’s C-terminal domain (CTD) and is essential for the transition from transcription initiation to elongation in vivo. Surprisingly, P-TEFb exhibits Ser5 phosphorylation activity in vitro. The mechanism garnering Ser2 specificity to P-TEFb remains elusive and hinders understanding of the transition from transcription initiation to elongation. Through in vitro reconstruction of CTD phosphorylation, mass spectrometry analysis, and chromatin immunoprecipitation sequencing (ChIP-seq) analysis we uncover a mechanism by which Tyr1 phosphorylation directs the kinase activity of P-TEFb and alters its specificity from Ser5 to Ser2. The loss of Tyr1 phosphorylation causes a reduction of phosphorylated Ser2 and accumulation of RNA polymerase II in the promoter region as detected by ChIP-seq. We demonstrate the ability of Tyr1 phosphorylation to generate a heterogeneous CTD modification landscape that expands the CTD’s coding potential. These findings provide direct experimental evidence for a combinatorial CTD phosphorylation code wherein previously installed modifications direct the identity and abundance of subsequent coding events by influencing the behavior of downstream enzymes.

scholarly journals Role for the Ssu72 C-Terminal Domain Phosphatase in RNA Polymerase II Transcription Elongation

2006 ◽  
Vol 27 (3) ◽  
pp. 926-936 ◽  
Author(s):  
Mariela Reyes-Reyes ◽  
Michael Hampsey
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
Elongation Complex ◽  
Terminal Domain ◽  
Rnap Ii ◽  
Transcription Cycle

ABSTRACT The RNA polymerase II (RNAP II) transcription cycle is accompanied by changes in the phosphorylation status of the C-terminal domain (CTD), a reiterated heptapeptide sequence (Y1S2P3T4S5P6S7) present at the C terminus of the largest RNAP II subunit. One of the enzymes involved in this process is Ssu72, a CTD phosphatase with specificity for serine-5-P. Here we report that the ssu72-2-encoded Ssu72-R129A protein is catalytically impaired in vitro and that the ssu72-2 mutant accumulates the serine-5-P form of RNAP II in vivo. An in vitro transcription system derived from the ssu72-2 mutant exhibits impaired elongation efficiency. Mutations in RPB1 and RPB2, the genes encoding the two largest subunits of RNAP II, were identified as suppressors of ssu72-2. The rpb1-1001 suppressor encodes an R1281A replacement, whereas rpb2-1001 encodes an R983G replacement. This information led us to identify the previously defined rpb2-4 and rpb2-10 alleles, which encode catalytically slow forms of RNAP II, as additional suppressors of ssu72-2. Furthermore, deletion of SPT4, which encodes a subunit of the Spt4-Spt5 early elongation complex, also suppresses ssu72-2, whereas the spt5-242 allele is suppressed by rpb2-1001. These results define Ssu72 as a transcription elongation factor. We propose a model in which Ssu72 catalyzes serine-5-P dephosphorylation subsequent to addition of the 7-methylguanosine cap on pre-mRNA in a manner that facilitates the RNAP II transition into the elongation stage of the transcription cycle.

scholarly journals Wwp2-Mediated Ubiquitination of the RNA Polymerase II Large Subunit in Mouse Embryonic Pluripotent Stem Cells

2007 ◽  
Vol 27 (15) ◽  
pp. 5296-5305 ◽  
Author(s):  
Hui Li ◽  
Zhihong Zhang ◽  
Beibei Wang ◽  
Junmei Zhang ◽  
Yingming Zhao ◽  
...  
Keyword(s):  
Dna Damage ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Mammalian Cells ◽  
Large Subunit ◽  
Terminal Domain ◽  
Ubiquitin E3 Ligase ◽  
Lysine Residues

ABSTRACT Ubiquitination and the degradation of the large subunit of RNA polymerase II, Rpb1, is not only involved in DNA damage-induced arrest but also in other transcription-obstructing events. However, the ubiquitin ligases responsible for DNA damage-independent processes in mammalian cells remain to be identified. Here, we identified Wwp2, a mouse HECT domain ubiquitin E3 ligase, as a novel ubiquitin ligase of Rpb1. We found that Wwp2 specifically interacted with mouse Rpb1 and targeted it for ubiquitination both in vitro and in vivo. Interestingly, the interaction with and ubiquitination of Rpb1 was dependent neither on its phosphorylation state nor on DNA damage. However, the enzymatic activity of Wwp2 was absolutely required for its ubiquitin modification of Rpb1. Furthermore, our study indicates that the interaction between Wwp2 and Rpb1 was mediated through WW domain of Wwp2 and C-terminal domain of Rpb1, respectively. Strikingly, downregulation of Wwp2 expression compromised Rpb1 ubiquitination and elevated its intracellular steady-state protein level significantly. Importantly, we identified six lysine residues in the C-terminal domain of Rpb1 as ubiquitin acceptor sites mediated by Wwp2. These results indicate that Wwp2 plays an important role in regulating expression of Rpb1 in normal physiological conditions.

scholarly journals BRCA1 Can Modulate RNA Polymerase II Carboxy-Terminal Domain Phosphorylation Levels

2004 ◽  
Vol 24 (16) ◽  
pp. 6947-6956 ◽  
Author(s):  
Annie Moisan ◽  
Chantal Larochelle ◽  
Benoît Guillemette ◽  
Luc Gaudreau
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Terminal Region ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Hcc1937 Cell ◽  
Carboxy Terminal ◽  
Cycle Regulation ◽  
Ctd Phosphorylation

ABSTRACT A high incidence of breast and ovarian cancers has been linked to mutations in the BRCA1 gene. BRCA1 has been shown to be involved in both positive and negative regulation of gene activity as well as in numerous other processes such as DNA repair and cell cycle regulation. Since modulation of the RNA polymerase II carboxy-terminal domain (CTD) phosphorylation levels could constitute an interface to all these functions, we wanted to directly test the possibility that BRCA1 might regulate the phosphorylation state of the CTD. We have shown that the BRCA1 C-terminal region can negatively modulate phosphorylation levels of the RNA polymerase II CTD by the Cdk-activating kinase (CAK) in vitro. Interestingly, the BRCA1 C-terminal region can directly interact with CAK and inhibit CAK activity by competing with ATP. Finally, we demonstrated that full-length BRCA1 can inhibit CTD phosphorylation when introduced in the BRCA1−/− HCC1937 cell line. Our results suggest that BRCA1 could play its ascribed roles, at least in part, by modulating CTD kinase components.

scholarly journals Tyr1 phosphorylation promotes phosphorylation of Ser2 on the C-terminal domain of eukaryotic RNA polymerase II by P-TEFb

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Joshua E Mayfield ◽  
Seema Irani ◽  
Edwin E Escobar ◽  
Zhao Zhang ◽  
Nathaniel T Burkholder ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Elongation Factor ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Terminal Domain ◽  
Ctd Phosphorylation

The Positive Transcription Elongation Factor b (P-TEFb) phosphorylates Ser2 residues of the C-terminal domain (CTD) of the largest subunit (RPB1) of RNA polymerase II and is essential for the transition from transcription initiation to elongation in vivo. Surprisingly, P-TEFb exhibits Ser5 phosphorylation activity in vitro. The mechanism garnering Ser2 specificity to P-TEFb remains elusive and hinders understanding of the transition from transcription initiation to elongation. Through in vitro reconstruction of CTD phosphorylation, mass spectrometry analysis, and chromatin immunoprecipitation sequencing (ChIP-seq) analysis, we uncover a mechanism by which Tyr1 phosphorylation directs the kinase activity of P-TEFb and alters its specificity from Ser5 to Ser2. The loss of Tyr1 phosphorylation causes an accumulation of RNA polymerase II in the promoter region as detected by ChIP-seq. We demonstrate the ability of Tyr1 phosphorylation to generate a heterogeneous CTD modification landscape that expands the CTD’s coding potential. These findings provide direct experimental evidence for a combinatorial CTD phosphorylation code wherein previously installed modifications direct the identity and abundance of subsequent coding events by influencing the behavior of downstream enzymes.

Mapping mutations in genes encoding the two large subunits of Drosophila RNA polymerase II defines domains essential for basic transcription functions and for proper expression of developmental genes

1993 ◽  
Vol 13 (7) ◽  
pp. 4214-4222
Author(s):  
Y Chen ◽  
J Weeks ◽  
M A Mortin ◽  
A L Greenleaf
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dna Sequence ◽  
Pcr Amplification ◽  
Single Strand Conformation Polymorphism ◽  
Chain Elongation ◽  
Developmental Defect ◽  
Genes Encoding ◽  
Transcript Elongation

We have mapped a number of mutations at the DNA sequence level in genes encoding the largest (RpII215) and second-largest (RpII140) subunits of Drosophila melanogaster RNA polymerase II. Using polymerase chain reaction (PCR) amplification and single-strand conformation polymorphism (SSCP) analysis, we detected 12 mutations from 14 mutant alleles (86%) as mobility shifts in nondenaturing gel electrophoresis, thus localizing the mutations to the corresponding PCR fragments of about 350 bp. We then determined the mutations at the DNA sequence level by directly subcloning the PCR fragments and sequencing them. The five mapped RpII140 mutations clustered in a C-terminal portion of the second-largest subunit, indicating the functional importance of this region of the subunit. The RpII215 mutations were distributed more broadly, although six of eight clustered in a central region of the subunit. One notable mutation that we localized to this region was the alpha-amanitin-resistant mutation RpII215C4, which also affects RNA chain elongation in vitro. RpII215C4 mapped to a position near the sites of corresponding mutations in mouse and in Caenorhabditis elegans genes, reinforcing the idea that this region is involved in amatoxin binding and transcript elongation. We also mapped mutations in both RpII215 and RpII140 that cause a developmental defect known as the Ubx effect. The clustering of these mutations in each gene suggests that they define functional domains in each subunit whose alteration induces the mutant phenotype.

Transcription initiation complexes and upstream activation with RNA polymerase II lacking the C-terminal domain of the largest subunit

1990 ◽  
Vol 10 (10) ◽  
pp. 5562-5564
Author(s):  
S Buratowski ◽  
P A Sharp
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Adenovirus Type ◽  
Late Promoter ◽  
Terminal Domain ◽  
Late Transcription ◽  
Major Late Promoter

RNA polymerase II assembles with other factors on the adenovirus type 2 major late promoter to generate pairs of transcription initiation complexes resolvable by nondenaturing gel electrophoresis. The pairing of the complexes is caused by the presence or absence of the C-terminal domain of the largest subunit. This domain is not required for transcription stimulation by the major late transcription factor in vitro.

scholarly journals Functions of the N- and C-Terminal Domains of Human RAP74 in Transcriptional Initiation, Elongation, and Recycling of RNA Polymerase II

1998 ◽  
Vol 18 (4) ◽  
pp. 2130-2142 ◽  
Author(s):  
Lei Lei ◽  
Delin Ren ◽  
Ann Finkelstein ◽  
Zachary F. Burton
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Initiation ◽  
Pol Ii ◽  
Terminal Domain ◽  
Multiple Round ◽  
Major Late Promoter ◽  
Central Loop ◽  
Transcription Cycle ◽  
Terminal Domains

ABSTRACT Transcription factor IIF (TFIIF) cooperates with RNA polymerase II (pol II) during multiple stages of the transcription cycle including preinitiation complex assembly, initiation, elongation, and possibly termination and recycling. Human TFIIF appears to be an α2β2 heterotetramer of RNA polymerase II-associating protein 74- and 30-kDa subunits (RAP74 and RAP30). From inspection of its 517-amino-acid (aa) sequence, the RAP74 subunit appears to comprise separate N- and C-terminal domains connected by a flexible loop. In this study, we present functional data that strongly support this model for RAP74 architecture and further show that the N- and C-terminal domains and the central loop of RAP74 have distinct roles during separate phases of the transcription cycle. The N-terminal domain of RAP74 (minimally aa 1 to 172) is sufficient to deliver pol II into a complex formed on the adenovirus major late promoter with the TATA-binding protein, TFIIB, and RAP30. A more complete N-terminal domain fragment (aa 1 to 217) strongly stimulates both accurate initiation and elongation by pol II. The region of RAP74 between aa 172 and 205 and a subregion between aa 170 and 178 are critical for both accurate initiation and elongation, and mutations in these regions have similar effects on initiation and elongation. Based on these observations, RAP74 appears to have similar functions in initiation and elongation. The central region and the C-terminal domain of RAP74 do not contribute strongly to single-round accurate initiation or elongation stimulation but do stimulate multiple-round transcription in an extract system.

scholarly journals Mapping mutations in genes encoding the two large subunits of Drosophila RNA polymerase II defines domains essential for basic transcription functions and for proper expression of developmental genes.

1993 ◽  
Vol 13 (7) ◽  
pp. 4214-4222 ◽  
Author(s):  
Y Chen ◽  
J Weeks ◽  
M A Mortin ◽  
A L Greenleaf
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dna Sequence ◽  
Pcr Amplification ◽  
Single Strand Conformation Polymorphism ◽  
Chain Elongation ◽  
Developmental Defect ◽  
Genes Encoding ◽  
Transcript Elongation

We have mapped a number of mutations at the DNA sequence level in genes encoding the largest (RpII215) and second-largest (RpII140) subunits of Drosophila melanogaster RNA polymerase II. Using polymerase chain reaction (PCR) amplification and single-strand conformation polymorphism (SSCP) analysis, we detected 12 mutations from 14 mutant alleles (86%) as mobility shifts in nondenaturing gel electrophoresis, thus localizing the mutations to the corresponding PCR fragments of about 350 bp. We then determined the mutations at the DNA sequence level by directly subcloning the PCR fragments and sequencing them. The five mapped RpII140 mutations clustered in a C-terminal portion of the second-largest subunit, indicating the functional importance of this region of the subunit. The RpII215 mutations were distributed more broadly, although six of eight clustered in a central region of the subunit. One notable mutation that we localized to this region was the alpha-amanitin-resistant mutation RpII215C4, which also affects RNA chain elongation in vitro. RpII215C4 mapped to a position near the sites of corresponding mutations in mouse and in Caenorhabditis elegans genes, reinforcing the idea that this region is involved in amatoxin binding and transcript elongation. We also mapped mutations in both RpII215 and RpII140 that cause a developmental defect known as the Ubx effect. The clustering of these mutations in each gene suggests that they define functional domains in each subunit whose alteration induces the mutant phenotype.

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