scholarly journals Spt5 phosphorylation and the Rtf1 Plus3 domain promote Rtf1 function through distinct mechanisms

2020 ◽  
Author(s):  
Jennifer J. Chen ◽  
Jean Mbogning ◽  
Mark A. Hancock ◽  
Dorsa Majdpour ◽  
Manan Madhok ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
Cyclin Dependent Kinase ◽  
Positive Transcription Elongation Factor ◽  
Transcription Elongation Factor ◽  
Binding Factor ◽  
Transcript Elongation ◽  
Processivity Factor

AbstractRtf1 is a conserved RNA polymerase II (RNAPII) elongation factor that promotes co-transcriptional histone modification, RNAPII transcript elongation, and mRNA processing. Rtf1 function requires phosphorylation of Spt5, an essential RNAPII processivity factor. Spt5 is phosphorylated within its C-terminal domain (CTD) by cyclin-dependent kinase 9 (Cdk9), catalytic component of positive transcription elongation factor b (P-TEFb). Rtf1 recognizes phosphorylated Spt5 (pSpt5) through its Plus3 domain. Since Spt5 is a unique target of Cdk9, and Rtf1 is the only known pSpt5-binding factor, the Plus3/pSpt5 interaction is thought to be a key Cdk9-dependent event regulating RNAPII elongation. Here we dissect Rtf1 regulation by pSpt5 in the fission yeast Schizosaccharomyces pombe. We demonstrate that the Plus3 domain of Rtf1 (Prf1 in S. pombe) and pSpt5 are functionally distinct, and that they act in parallel to promote Prf1 function. This alternate Plus3 domain function involves an interface that overlaps with the pSpt5 binding site and that can interact with single-stranded nucleic acid or with the Polymerase Associated Factor (PAF) Complex in vitro. We further show that the C-terminal region of Prf1, which also interacts with PAF, has a similar parallel function with pSpt5. Our results elucidate unexpected complexity underlying Cdk9-dependent pathways that regulate transcription elongation.

scholarly journals Spt5 Phosphorylation and the Rtf1 Plus3 Domain Promote Rtf1 Function through Distinct Mechanisms

2020 ◽  
Vol 40 (15) ◽  
Author(s):  
Jennifer J. Chen ◽  
Jean Mbogning ◽  
Mark A. Hancock ◽  
Dorsa Majdpour ◽  
Manan Madhok ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
Cyclin Dependent Kinase ◽  
Content Type ◽  
Positive Transcription Elongation Factor ◽  
Binding Factor ◽  
Transcript Elongation ◽  
Processivity Factor

ABSTRACT Rtf1 is a conserved RNA polymerase II (RNAPII) elongation factor that promotes cotranscriptional histone modification, RNAPII transcript elongation, and mRNA processing. Rtf1 function requires the phosphorylation of Spt5, an essential RNAPII processivity factor. Spt5 is phosphorylated within its C-terminal domain (CTD) by cyclin-dependent kinase 9 (Cdk9), the catalytic component of positive transcription elongation factor b (P-TEFb). Rtf1 recognizes phosphorylated Spt5 (pSpt5) through its Plus3 domain. Since Spt5 is a unique target of Cdk9 and Rtf1 is the only known pSpt5-binding factor, the Plus3/pSpt5 interaction is thought to be a key Cdk9-dependent event regulating RNAPII elongation. Here, we dissect Rtf1 regulation by pSpt5 in the fission yeast Schizosaccharomyces pombe. We demonstrate that the Plus3 domain of Rtf1 (Prf1 in S. pombe) and pSpt5 are functionally distinct and that they act in parallel to promote Prf1 function. This alternate Plus3 domain function involves an interface that overlaps the pSpt5-binding site and that can interact with single-stranded nucleic acid or with the polymerase-associated factor (PAF) complex in vitro. We further show that the C-terminal region of Prf1, which also interacts with PAF, has a similar parallel function with pSpt5. Our results elucidate unexpected complexity underlying Cdk9-dependent pathways that regulate transcription elongation.

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

eLife ◽  
2020 ◽  
Vol 9 ◽  
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.

scholarly journals Transcription elongation factor SII interacts with a domain of the large subunit of human RNA polymerase II.

1988 ◽  
Vol 8 (8) ◽  
pp. 3136-3142 ◽  
Author(s):  
J Rappaport ◽  
K Cho ◽  
A Saltzman ◽  
J Prenger ◽  
M Golomb ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Fusion Protein ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Large Subunit ◽  
Elongation Factor ◽  
Transcription Elongation Factor ◽  
Beta Galactosidase

Genomic sequences for the large subunit of human RNA polymerase II corresponding to a part of the fifth exon were inserted into an expression vector at the carboxy-terminal end of the beta-galactosidase gene. The in-frame construct produced a 125-kilodalton fusion protein, containing approximately 10 kilodaltons of the large subunit of RNA polymerase II and 116 kilodaltons of beta-galactosidase. The purified bacterially produced fusion protein inhibited specific transcription from the adenovirus type 2 major late promoter, while beta-galactosidase had no effect. This effect of the fusion protein was during RNA elongation, not at the level of initiation, resembling the faithfully initiated but incomplete transcripts produced with purified factors in the absence of SII. Similarly, monoclonal antibody 2-7B, which reacts with the RNA polymerase II region represented in the fusion protein, inhibited specific transcription at the level of elongation in a whole-cell extract. Both monoclonal antibody 2-7B and the fusion protein, although unable to inhibit purified RNA polymerase II in a nonspecific transcription assay, selectively blocked the stimulation elicited by transcription elongation factor SII on the activity of the purified enzyme in vitro. This suggests that the fusion protein traps the SII in nonstimulatory interactions and that antibody 2-7B inhibits SII binding to RNA polymerase II. Thus, this suggests that an SII-binding contact required for specific RNA elongation resides within the fifth exon region of the largest RNA polymerase II subunit.

scholarly journals HEXIM2, a HEXIM1-related Protein, Regulates Positive Transcription Elongation Factor b through Association with 7SK

2005 ◽  
Vol 280 (16) ◽  
pp. 16360-16367 ◽  
Author(s):  
Sarah A. Byers ◽  
Jason P. Price ◽  
Jeffrey J. Cooper ◽  
Qintong Li ◽  
David H. Price
Keyword(s):  
Rna Polymerase Ii ◽  
Kinase Activity ◽  
Sequence Similarity ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
Jurkat Cells ◽  
Mobility Shift ◽  
Factor B ◽  
Positive Transcription Elongation Factor ◽  
Transcription Elongation Factor

The kinase activity of positive transcription elongation factor b (P-TEFb), composed of cyclin-dependent kinase 9 and cyclin T1 or T2, is required for the transition of RNA polymerase II into productive elongation. P-TEFb activity has been shown to be negatively regulated by association with the small nuclear RNA 7SK and the HEXIM1 protein. Here, we characterize HEXIM2, a previously predicted protein with sequence similarity to HEXIM1. HEXIM2 is expressed in HeLa and Jurkat cells, and glycerol gradient analysis and immunoprecipitations indicate that HEXIM2, like HEXIM1, has a regulated association with P-TEFb. As HEXIM1 is knocked down, HEXIM2 functionally compensates for its association with P-TEFb. Electrophoretic mobility shift assays andin vitrokinase assays demonstrate that HEXIM2 forms complexes containing 7SK and P-TEFb and, in conjunction with 7SK, inhibits P-TEFb kinase activity. Our results provide strong evidence that HEXIM2 is a regulator of P-TEFb function. Furthermore, our results support the idea that the utilization of HEXIM1 or HEXIM2 to bind and inhibit P-TEFb can be differentially regulatedin vivo.

Transcription elongation factor SII interacts with a domain of the large subunit of human RNA polymerase II

1988 ◽  
Vol 8 (8) ◽  
pp. 3136-3142
Author(s):  
J Rappaport ◽  
K Cho ◽  
A Saltzman ◽  
J Prenger ◽  
M Golomb ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Fusion Protein ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Large Subunit ◽  
Elongation Factor ◽  
Transcription Elongation Factor ◽  
Beta Galactosidase

Genomic sequences for the large subunit of human RNA polymerase II corresponding to a part of the fifth exon were inserted into an expression vector at the carboxy-terminal end of the beta-galactosidase gene. The in-frame construct produced a 125-kilodalton fusion protein, containing approximately 10 kilodaltons of the large subunit of RNA polymerase II and 116 kilodaltons of beta-galactosidase. The purified bacterially produced fusion protein inhibited specific transcription from the adenovirus type 2 major late promoter, while beta-galactosidase had no effect. This effect of the fusion protein was during RNA elongation, not at the level of initiation, resembling the faithfully initiated but incomplete transcripts produced with purified factors in the absence of SII. Similarly, monoclonal antibody 2-7B, which reacts with the RNA polymerase II region represented in the fusion protein, inhibited specific transcription at the level of elongation in a whole-cell extract. Both monoclonal antibody 2-7B and the fusion protein, although unable to inhibit purified RNA polymerase II in a nonspecific transcription assay, selectively blocked the stimulation elicited by transcription elongation factor SII on the activity of the purified enzyme in vitro. This suggests that the fusion protein traps the SII in nonstimulatory interactions and that antibody 2-7B inhibits SII binding to RNA polymerase II. Thus, this suggests that an SII-binding contact required for specific RNA elongation resides within the fifth exon region of the largest RNA polymerase II subunit.

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

2001 ◽  
Vol 276 (15) ◽  
pp. 11531-11538 ◽  
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.

scholarly journals Cockayne syndrome B protein acts as an ATP-dependent processivity factor that helps RNA polymerase II overcome nucleosome barriers

2020 ◽  
Vol 117 (41) ◽  
pp. 25486-25493 ◽  
Author(s):  
Jun Xu ◽  
Wei Wang ◽  
Liang Xu ◽  
Jia-Yu Chen ◽  
Jenny Chong ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Neurological Diseases ◽  
Transcription Elongation ◽  
Cockayne Syndrome ◽  
Stable Complex ◽  
Loss Of Function ◽  
Pol Ii ◽  
Chromatin Remodelers ◽  
Processivity Factor

While loss-of-function mutations in Cockayne syndrome group B protein (CSB) cause neurological diseases, this unique member of the SWI2/SNF2 family of chromatin remodelers has been broadly implicated in transcription elongation and transcription-coupled DNA damage repair, yet its mechanism remains largely elusive. Here, we use a reconstituted in vitro transcription system with purified polymerase II (Pol II) and Rad26, a yeast ortholog of CSB, to study the role of CSB in transcription elongation through nucleosome barriers. We show that CSB forms a stable complex with Pol II and acts as an ATP-dependent processivity factor that helps Pol II across a nucleosome barrier. This noncanonical mechanism is distinct from the canonical modes of chromatin remodelers that directly engage and remodel nucleosomes or transcription elongation factors that facilitate Pol II nucleosome bypass without hydrolyzing ATP. We propose a model where CSB facilitates gene expression by helping Pol II bypass chromatin obstacles while maintaining their structures.

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