scholarly journals A three-step pathway of transcription initiation leading to promoter clearance at an activation RNA polymerase II promoter.

1996 ◽  
Vol 16 (4) ◽  
pp. 1614-1621 ◽  
Author(s):  
Y Jiang ◽  
M Yan ◽  
J D Gralla
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Potential Effect ◽  
Multiple Roles ◽  
Elongation Complex ◽  
Terminal Domain ◽  
Step Model ◽  
Dna Strands

The progress of transcription bubbles during inhibition in vitro was followed in order to learn how RNA polymerase II begins transcription at the activated adenovirus E4 promoter. The issues addressed include the multiple roles of ATP, the potential effect of polymerase C-terminal domain phosphorylation, and the ability of polymerase to clear the promoter for reinitiation. The results lead to a three-step model for the transition from closed complex to elongation complex, two steps of which use ATP independently. In the first step, studied previously, ATP is hydrolyzed to open the DNA strands over the start site. In a second step, apparently independent of ATP, transcription bubbles move into the initial transcribed region where RNA synthesis can stall. In the third step, transcripts can be made as polymerase is released from these stalled positions with the assistance of an ATP-dependent process, likely phosphorylation of the polymerase C-terminal domain. After this third step, the promoter becomes cleared, allowing for the reinitiation of transcription.

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 RNAP II CTD tyrosine 1 performs diverse functions in vertebrate cells

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Jing-Ping Hsin ◽  
Wencheng Li ◽  
Mainul Hoque ◽  
Bin Tian ◽  
James L Manley
Keyword(s):  
Rna Polymerase ◽  
Cell Viability ◽  
Rna Polymerase Ii ◽  
Multiple Roles ◽  
Terminal Domain ◽  
Report Analysis ◽  
Rnap Ii ◽  
Y Cells ◽  
Dt40 Cells

The RNA polymerase II largest subunit (Rpb1) contains a unique C-terminal domain (CTD) that plays multiple roles during transcription. The CTD is composed of consensus Y1S2P3T4S5P6S7 repeats, in which Ser, Thr and Tyr residues can all be phosphorylated. Here we report analysis of CTD Tyr1 using genetically tractable chicken DT40 cells. Cells expressing an Rpb1 derivative with all Tyr residues mutated to Phe (Rpb1-Y1F) were inviable. Remarkably, Rpb1-Y1F was unstable, degraded to a CTD-less form; however stability, but not cell viability, was fully rescued by restoration of a single C-terminal Tyr (Rpb1-25F+Y). Cytoplasmic and nucleoplasmic Rpb1 was phosphorylated exclusively on Tyr1, and phosphorylation specifically of Tyr1 prevented CTD degradation by the proteasome in vitro. Tyr1 phosphorylation was also detected on chromatin-associated, hyperphosphorylated Rpb1, consistent with a role in transcription. Indeed, we detected accumulation of upstream antisense (ua) RNAs in Rpb1-25F+Y cells, indicating a role for Tyr1 in uaRNA expression.

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 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 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 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.

Crystal structure of Ssu72, an essential eukaryotic phosphatase specific for the C-terminal domain of RNA polymerase II, in complex with a transition state analogue

2011 ◽  
Vol 434 (3) ◽  
pp. 435-444 ◽  
Author(s):  
Yong Zhang ◽  
Mengmeng Zhang ◽  
Yan Zhang
Keyword(s):  
Rna Polymerase ◽  
Transition State ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Complex Structure ◽  
Protein Tyrosine Phosphatases ◽  
Cysteine Residue ◽  
Phosphoryl Transfer ◽  
Terminal Domain ◽  
Deep Groove

Reversible phosphorylation of the CTD (C-terminal domain) of the eukaryotic RNA polymerase II largest subunit represents a critical regulatory mechanism during the transcription cycle and mRNA processing. Ssu72 is an essential phosphatase conserved in eukaryotes that dephosphorylates phosphorylated Ser5 of the CTD heptapeptide. Its function is implicated in transcription initiation, elongation and termination, as well as RNA processing. In the present paper we report the high resolution X-ray crystal structures of Drosophila melanogaster Ssu72 phosphatase in the apo form and in complex with an inhibitor mimicking the transition state of phosphoryl transfer. Ssu72 facilitates dephosphorylation of the substrate through a phosphoryl-enzyme intermediate, as visualized in the complex structure of Ssu72 with the oxo-anion compound inhibitor vanadate at a 2.35 Å (1 Å=0.1 nm) resolution. The structure resembles the transition state of the phosphoryl transfer with vanadate exhibiting a trigonal bi-pyramidal geometry covalently bonded to the nucleophilic cysteine residue. Interestingly, the incorporation of oxo-anion compounds greatly stabilizes a flexible loop containing the general acid, as detected by an increase of melting temperature of Ssu72 detected by differential scanning fluorimetry. The Ssu72 structure exhibits a core fold with a similar topology to that of LMWPTPs [low-molecular-mass PTPs (protein tyrosine phosphatases)], but with an insertion of a unique ‘cap’ domain to shelter the active site from the solvent with a deep groove in between where the CTD substrates bind. Mutagenesis studies in this groove established the functional roles of five residues (Met17, Pro46, Asp51, Tyr77 and Met85) that are essential specifically for substrate recognition.

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.

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