Control of transcriptional elongation and cotranscriptional histone modification by the yeast BUR kinase substrate Spt5

Elongation by RNA polymerase II (RNAPII) is a finely regulated process in which many elongation factors contribute to gene regulation. Among these factors are the polymerase-associated factor (PAF) complex, which associates with RNAPII, and several cyclin-dependent kinases, including positive transcription elongation factor b (P-TEFb) in humans and BUR kinase (Bur1–Bur2) and C-terminal domain (CTD) kinase 1 (CTDK1) in Saccharomyces cerevisiae. An important target of P-TEFb and CTDK1, but not BUR kinase, is the CTD of the Rpb1 subunit of RNAPII. Although the essential BUR kinase phosphorylates Rad6, which is required for histone H2B ubiquitination on K123, Rad6 is not essential, leaving a critical substrate(s) of BUR kinase unidentified. Here we show that BUR kinase is important for the phosphorylation in vivo of Spt5, a subunit of the essential yeast RNAPII elongation factor Spt4/Spt5, whose human orthologue is DRB sensitivity-inducing factor. BUR kinase can also phosphorylate the C-terminal region (CTR) of Spt5 in vitro. Like BUR kinase, the Spt5 CTR is important for promoting elongation by RNAPII and recruiting the PAF complex to transcribed regions. Also like BUR kinase and the PAF complex, the Spt5 CTR is important for histone H2B K123 monoubiquitination and histone H3 K4 and K36 trimethylation during transcription elongation. Our results suggest that the Spt5 CTR, which contains 15 repeats of a hexapeptide whose consensus sequence is S[T/A]WGG[A/Q], is a substrate of BUR kinase and a platform for the association of proteins that promote both transcription elongation and histone modification in transcribed regions.

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Regulation of P-TEFb Elongation Complex Activity by CDK9 Acetylation

Molecular and Cellular Biology ◽

10.1128/mcb.00857-06 ◽

2007 ◽

Vol 27 (13) ◽

pp. 4641-4651 ◽

Cited By ~ 40

Author(s):

Junjiang Fu ◽

Ho-Geun Yoon ◽

Jun Qin ◽

Jiemin Wong

Keyword(s):

Rna Polymerase Ii ◽

Elongation Factor ◽

Transcriptional Elongation ◽

Elongation Complex ◽

Complex Activity ◽

Interacting Protein ◽

Pol Ii ◽

Carboxy Terminal

ABSTRACT P-TEFb, comprised of CDK9 and a cyclin T subunit, is a global transcriptional elongation factor important for most RNA polymerase II (pol II) transcription. P-TEFb facilitates transcription elongation in part by phosphorylating Ser2 of the heptapeptide repeat of the carboxy-terminal domain (CTD) of the largest subunit of pol II. Previous studies have shown that P-TEFb is subjected to negative regulation by forming an inactive complex with 7SK small RNA and HEXIM1. In an effort to investigate the molecular mechanism by which corepressor N-CoR mediates transcription repression, we identified HEXIM1 as an N-CoR-interacting protein. This finding led us to test whether the P-TEFb complex is regulated by acetylation. We demonstrate that CDK9 is an acetylated protein in cells and can be acetylated by p300 in vitro. Through both in vitro and in vivo assays, we identified lysine 44 of CDK9 as a major acetylation site. We present evidence that CDK9 is regulated by N-CoR and its associated HDAC3 and that acetylation of CDK9 affects its ability to phosphorylate the CTD of pol II. These results suggest that acetylation of CDK9 is an important posttranslational modification that is involved in regulating P-TEFb transcriptional elongation function.

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Identification of rpo30, a vaccinia virus RNA polymerase gene with structural similarity to a eucaryotic transcription elongation factor

Molecular and Cellular Biology ◽

10.1128/mcb.10.10.5433-5441.1990 ◽

1990 ◽

Vol 10 (10) ◽

pp. 5433-5441

Author(s):

B Y Ahn ◽

P D Gershon ◽

E V Jones ◽

B Moss

Keyword(s):

Rna Polymerase ◽

Vaccinia Virus ◽

Rna Polymerase Ii ◽

Elongation Factor ◽

Viral Rna ◽

In Vitro Translation ◽

Virus Protein ◽

Transcriptional Elongation

Eucaryotic transcription factors that stimulate RNA polymerase II by increasing the efficiency of elongation of specifically or randomly initiated RNA chains have been isolated and characterized. We have identified a 30-kilodalton (kDa) vaccinia virus-encoded protein with apparent homology to SII, a 34-kDa mammalian transcriptional elongation factor. In addition to amino acid sequence similarities, both proteins contain C-terminal putative zinc finger domains. Identification of the gene, rpo30, encoding the vaccinia virus protein was achieved by using antibody to the purified viral RNA polymerase for immunoprecipitation of the in vitro translation products of in vivo-synthesized early mRNA selected by hybridization to cloned DNA fragments of the viral genome. Western immunoblot analysis using antiserum made to the vaccinia rpo30 protein expressed in bacteria indicated that the 30-kDa protein remains associated with highly purified viral RNA polymerase. Thus, the vaccinia virus protein, unlike its eucaryotic homolog, is an integral RNA polymerase subunit rather than a readily separable transcription factor. Further studies showed that the expression of rpo30 is regulated by dual early and later promoters.

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Identification of rpo30, a vaccinia virus RNA polymerase gene with structural similarity to a eucaryotic transcription elongation factor.

Molecular and Cellular Biology ◽

10.1128/mcb.10.10.5433 ◽

1990 ◽

Vol 10 (10) ◽

pp. 5433-5441 ◽

Cited By ~ 62

Author(s):

B Y Ahn ◽

P D Gershon ◽

E V Jones ◽

B Moss

Keyword(s):

Rna Polymerase ◽

Vaccinia Virus ◽

Rna Polymerase Ii ◽

Elongation Factor ◽

Viral Rna ◽

In Vitro Translation ◽

Virus Protein ◽

Transcriptional Elongation

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JMJD6 cleaves MePCE to release positive transcription elongation factor b (P-TEFb) in higher eukaryotes

eLife ◽

10.7554/elife.53930 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 3

Author(s):

Schuyler Lee ◽

Haolin Liu ◽

Ryan Hill ◽

Chunjing Chen ◽

Xia Hong ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Transcription Elongation ◽

Elongation Factor ◽

Factor B ◽

Pol Ii ◽

Positive Transcription Elongation Factor ◽

Transcription Elongation Factor ◽

Higher Eukaryotes

More than 30% of genes in higher eukaryotes are regulated by promoter-proximal pausing of RNA polymerase II (Pol II). Phosphorylation of Pol II CTD by positive transcription elongation factor b (P-TEFb) is a necessary precursor event that enables productive transcription elongation. The exact mechanism on how the sequestered P-TEFb is released from the 7SK snRNP complex and recruited to Pol II CTD remains unknown. In this report, we utilize mouse and human models to reveal methylphosphate capping enzyme (MePCE), a core component of the 7SK snRNP complex, as the cognate substrate for Jumonji domain-containing 6 (JMJD6)’s novel proteolytic function. Our evidences consist of a crystal structure of JMJD6 bound to methyl-arginine, enzymatic assays of JMJD6 cleaving MePCE in vivo and in vitro, binding assays, and downstream effects of Jmjd6 knockout and overexpression on Pol II CTD phosphorylation. We propose that JMJD6 assists bromodomain containing 4 (BRD4) to recruit P-TEFb to Pol II CTD by disrupting the 7SK snRNP complex.

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JMJD6 Cleaves MePCE to Release P-TEFb

10.1101/725853 ◽

2019 ◽

Author(s):

Schuyler Lee ◽

Haolin Liu ◽

Ryan Hill ◽

Xia Hong ◽

Xinjian Liu ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Transcription Elongation ◽

Elongation Factor ◽

Binding Assays ◽

Enzymatic Assays ◽

Pol Ii ◽

Downstream Effects ◽

Capping Enzyme

AbstractMore than 30% of genes in higher eukaryotes are regulated by promoter-proximal pausing of RNA polymerase II (Pol II). Phosphorylation of Pol II-CTD by positive transcription elongation factor (P-TEFb) is a necessary precursor event that enables productive transcription elongation. The exact mechanism on how the sequestered P-TEFb is released from the 7SK snRNP complex and recruited to Pol II-CTD remains unknown. In this report, we reveal methylphosphate capping enzyme (MePCE), a core component of the 7SK snRNP complex, as the cognate substrate for Jumonji domain-containing 6 (JMJD6)’s novel proteolytic function. Our evidences consist of a crystal structure of JMJD6 bound to methyl-arginine, enzymatic assays of JMJD6 cleaving MePCE in vivo and in vitro, binding assays, and downstream effects of Jmjd6 knockout and overexpression on Pol II-CTD phosphorylation. We propose that JMJD6 assists bromodomain containing 4 (BRD4) to recruit P-TEFb to Pol II-CTD by disrupting the 7SK snRNP complex.

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Cyclin-Dependent Kinase 9 (Cdk9) of Fission Yeast Is Activated by the CDK-Activating Kinase Csk1, Overlaps Functionally with the TFIIH-Associated Kinase Mcs6, and Associates with the mRNA Cap Methyltransferase Pcm1 In Vivo

Molecular and Cellular Biology ◽

10.1128/mcb.26.3.777-788.2006 ◽

2006 ◽

Vol 26 (3) ◽

pp. 777-788 ◽

Cited By ~ 39

Author(s):

Yi Pei ◽

Hongyan Du ◽

Juliet Singer ◽

Courtney St. Amour ◽

Selena Granitto ◽

...

Keyword(s):

Fission Yeast ◽

Rna Polymerase Ii ◽

Elongation Factor ◽

Growth Defect ◽

Transcriptional Elongation ◽

Cyclin Dependent Kinase ◽

Elongation Complex ◽

Pol Ii

ABSTRACT Cyclin-dependent kinase 9 (Cdk9) of fission yeast is an essential ortholog of metazoan positive transcription elongation factor b (P-TEFb), which is proposed to coordinate capping and elongation of RNA polymerase II (Pol II) transcripts. Here we show that Cdk9 is activated to phosphorylate Pol II and the elongation factor Spt5 by Csk1, one of two fission yeast CDK-activating kinases (CAKs). Activation depends on Cdk9 T-loop residue Thr-212. The other CAK—Mcs6, the kinase component of transcription factor IIH (TFIIH)—cannot activate Cdk9. Consistent with the specificities of the two CAKs in vitro, the kinase activity of Cdk9 is reduced ∼10-fold by csk1 deletion, and Cdk9 complexes from csk1Δ but not csk1 + cells can be activated by Csk1 in vitro. A cdk9 T212A mutant is viable but phenocopies conditional growth defects of csk1Δ strains, indicating a role for Csk1-dependent activation of Cdk9 in vivo. A cdk9 T212A mcs6 S165A strain, in which neither Cdk9 nor Mcs6 can be activated by CAK, has a synthetic growth defect, implying functional overlap between the two CDKs, which have distinct but overlapping substrate specificities. Cdk9 forms complexes in vivo with the essential cyclin Pch1 and with Pcm1, the mRNA cap methyltransferase. The carboxyl-terminal region of Cdk9, through which it interacts with another capping enzyme, the RNA triphosphatase Pct1, is essential. Together, the data support a proposed model whereby Cdk9/Pch1—the third essential CDK-cyclin complex described in fission yeast—helps to target the capping apparatus to the transcriptional elongation complex.

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Structures and Functions of the Multiple KOW Domains of Transcription Elongation Factor Spt5

Molecular and Cellular Biology ◽

10.1128/mcb.00520-15 ◽

2015 ◽

Vol 35 (19) ◽

pp. 3354-3369 ◽

Cited By ~ 17

Author(s):

Peter A. Meyer ◽

Sheng Li ◽

Mincheng Zhang ◽

Kentaro Yamada ◽

Yuichiro Takagi ◽

...

Keyword(s):

High Resolution ◽

Nucleic Acids ◽

Rna Polymerase Ii ◽

Transcription Elongation ◽

Chromatin Modification ◽

Elongation Factor ◽

Content Type ◽

Processing Pathways

The eukaryotic Spt4-Spt5 heterodimer forms a higher-order complex with RNA polymerase II (and I) to regulate transcription elongation. Extensive genetic and functional data have revealed diverse roles of Spt4-Spt5 in coupling elongation with chromatin modification and RNA-processing pathways. A mechanistic understanding of the diverse functions of Spt4-Spt5 is hampered by challenges in resolving the distribution of functions among its structural domains, including the five KOW domains in Spt5, and a lack of their high-resolution structures. We present high-resolution crystallographic results demonstrating that distinct structures are formed by the first through third KOW domains (KOW1-Linker1 [K1L1] and KOW2-KOW3) ofSaccharomyces cerevisiaeSpt5. The structure reveals that K1L1 displays a positively charged patch (PCP) on its surface, which binds nucleic acidsin vitro, as shown in biochemical assays, and is important forin vivofunction, as shown in growth assays. Furthermore, assays in yeast have shown that the PCP has a function that partially overlaps that of Spt4. Synthesis of our results with previous evidence suggests a model in which Spt4 and the K1L1 domain of Spt5 form functionally overlapping interactions with nucleic acids upstream of the transcription bubble, and this mechanism may confer robustness on processes associated with transcription elongation.

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

Molecular and Cellular Biology ◽

10.1128/mcb.01361-06 ◽

2006 ◽

Vol 27 (3) ◽

pp. 926-936 ◽

Cited By ~ 39

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.

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Human Transcription Elongation Factor CA150 Localizes to Splicing Factor-Rich Nuclear Speckles and Assembles Transcription and Splicing Components into Complexes through Its Amino and Carboxyl Regions

Molecular and Cellular Biology ◽

10.1128/mcb.01991-05 ◽

2006 ◽

Vol 26 (13) ◽

pp. 4998-5014 ◽

Cited By ~ 40

Author(s):

Miguel Sánchez-Álvarez ◽

Aaron C. Goldstrohm ◽

Mariano A. Garcia-Blanco ◽

Carlos Suñé

Keyword(s):

Rna Polymerase Ii ◽

Transcription Elongation ◽

Elongation Factor ◽

Splicing Factor ◽

Transcriptional Elongation ◽

Nuclear Speckles ◽

Ww Domains ◽

Amino Terminal ◽

Transcription Elongation Factor

ABSTRACT The human transcription elongation factor CA150 contains three N-terminal WW domains and six consecutive FF domains. WW and FF domains, versatile modules that mediate protein-protein interactions, are found in nuclear proteins involved in transcription and splicing. CA150 interacts with the splicing factor SF1 and with the phosphorylated C-terminal repeat domain (CTD) of RNA polymerase II (RNAPII) through its WW and FF domains, respectively. WW and FF domains may, therefore, serve to link transcription and splicing components and play a role in coupling transcription and splicing in vivo. In the study presented here, we investigated the subcellular localization and association of CA150 with factors involved in pre-mRNA transcriptional elongation and splicing. Endogenous CA150 colocalized with nuclear speckles, and this was not affected either by inhibition of cellular transcription or by RNAPII CTD phosphorylation. FF domains are essential for the colocalization to speckles, while WW domains are not required for colocalization. We also performed biochemical assays to understand the role of WW and FF domains in mediating the assembly of transcription and splicing components into higher-order complexes. Transcription and splicing components bound to a region in the amino-terminal part of CA150 that contains the three WW domains; however, we identified a region of the C-terminal FF domains that was also critical. Our results suggest that sequences located at both the amino and carboxyl regions of CA150 are required to assemble transcription/splicing complexes, which may be involved in the coupling of those processes.

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Analysis of Gene Induction and Arrest Site Transcription in Yeast with Mutations in the Transcription Elongation Machinery

Journal of Biological Chemistry ◽

10.1074/jbc.m011322200 ◽

2001 ◽

Vol 276 (15) ◽

pp. 11531-11538 ◽

Cited By ~ 13

Author(s):

Megan Wind-Rotolo ◽

Daniel Reines

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Dna Sequences ◽

Transcription Elongation ◽

Elongation Factor ◽

Mrna Accumulation ◽

Mrna Metabolism ◽

Transcript Elongation

In vitro, transcript elongation by RNA polymerase II is impeded by DNA sequences, DNA-bound proteins, and small ligands. Transcription elongation factor SII (TFIIS) assists RNA polymerase II to transcribe through these obstacles. There is however, little direct evidence that SII-responsive arrest sites function in living cells nor that SII facilitates readthroughin vivo. Saccharomyces cerevisiaestrains lacking elongation factor SII and/or containing a point mutation in the second largest subunit of RNA polymerase II, which slows the enzyme's RNA elongation rate, grow slowly and have defects in mRNA metabolism, particularly in the presence of nucleotide-depleting drugs. Here we have examined transcriptional induction in strains lacking SII or containing the slow polymerase mutation. Both mutants and a combined double mutant were defective in induction ofGAL1andENA1. This was not due to an increase in mRNA degradation and was independent of any drug treatment, although treatment with the nucleotide-depleting drug 6-azauracil exacerbated the effect preferentially in the mutants. These data are consistent with mutants in the Elongator complex, which show slow inductive responses. When a potentin vitroarrest site was transcribed in these strains, there was no perceptible effect upon mRNA accumulation. These data suggest that an alternative elongation surveillance mechanism existsin vivoto overcome arrest.

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