scholarly journals Sub1 associates with Spt5 and influences RNA polymerase II transcription elongation rate

2012 ◽  
Vol 23 (21) ◽  
pp. 4297-4312 ◽  
Author(s):  
Alicia García ◽  
Alejandro Collin ◽  
Olga Calvo
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
Molecular Data ◽  
Elongation Rate ◽  
Dependent Manner ◽  
Gene Encoding ◽  
Mrna Metabolism ◽  
Carboxy Terminal

The transcriptional coactivator Sub1 has been implicated in several steps of mRNA metabolism in yeast, such as the activation of transcription, termination, and 3′-end formation. In addition, Sub1 globally regulates RNA polymerase II phosphorylation, and most recently it has been shown that it is a functional component of the preinitiation complex. Here we present evidence that Sub1 plays a significant role in transcription elongation by RNA polymerase II (RNAPII). We show that SUB1 genetically interacts with the gene encoding the elongation factor Spt5, that Sub1 influences Spt5 phosphorylation of the carboxy-terminal domain of RNAPII largest subunit by the kinase Bur1, and that both Sub1 and Spt5 copurify in the same complex, likely during early transcription elongation. Indeed, our data indicate that Sub1 influences Spt5–Rpb1 interaction. In addition, biochemical and molecular data show that Sub1 influences transcription elongation of constitutive and inducible genes and associates with coding regions in a transcription-dependent manner. Taken together, our results indicate that Sub1 associates with Spt5 and influences Spt5–Rpb1 complex levels and consequently transcription elongation rate.

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.

Crystal structure of the human transcription elongation factor DSIF hSpt4 subunit in complex with the hSpt5 dimerization interface

2009 ◽  
Vol 425 (2) ◽  
pp. 373-380 ◽  
Author(s):  
Sabine Wenzel ◽  
Berta M. Martins ◽  
Paul Rösch ◽  
Birgitta M. Wöhrl
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
Structural Similarity ◽  
Transcription Elongation Factor

The eukaryotic transcription elongation factor DSIF [DRB (5,6-dichloro-1-β-D-ribofuranosylbenzimidazole) sensitivity-inducing factor] is composed of two subunits, hSpt4 and hSpt5, which are homologous to the yeast factors Spt4 and Spt5. DSIF is involved in regulating the processivity of RNA polymerase II and plays an essential role in transcriptional activation of eukaryotes. At several eukaryotic promoters, DSIF, together with NELF (negative elongation factor), leads to promoter-proximal pausing of RNA polymerase II. In the present paper we describe the crystal structure of hSpt4 in complex with the dimerization region of hSpt5 (amino acids 176–273) at a resolution of 1.55 Å (1 Å=0.1 nm). The heterodimer shows high structural similarity to its homologue from Saccharomyces cerevisiae. Furthermore, hSpt5-NGN is structurally similar to the NTD (N-terminal domain) of the bacterial transcription factor NusG. A homologue for hSpt4 has not yet been found in bacteria. However, the archaeal transcription factor RpoE” appears to be distantly related. Although a comparison of the NusG-NTD of Escherichia coli with hSpt5 revealed a similarity of the three-dimensional structures, interaction of E. coli NusG-NTD with hSpt4 could not be observed by NMR titration experiments. A conserved glutamate residue, which was shown to be crucial for dimerization in yeast, is also involved in the human heterodimer, but is substituted for a glutamine residue in Escherichia coli NusG. However, exchanging the glutamine for glutamate proved not to be sufficient to induce hSpt4 binding.

scholarly journals The Rpb6 Subunit of Fission Yeast RNA Polymerase II Is a Contact Target of the Transcription Elongation Factor TFIIS

2000 ◽  
Vol 20 (4) ◽  
pp. 1263-1270 ◽  
Author(s):  
Akira Ishiguro ◽  
Yasuhisa Nogi ◽  
Koji Hisatake ◽  
Masami Muramatsu ◽  
Akira Ishihama
Keyword(s):  
Rna Polymerase ◽  
Fission Yeast ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
Direct Interaction ◽  
Temperature Sensitive ◽  
Single Mutation ◽  
Transcription Elongation Factor ◽  
Essential Region

ABSTRACT The Rpb6 subunit of RNA polymerase II is one of the five subunits common to three forms of eukaryotic RNA polymerase. Deletion and truncation analyses of the rpb6 gene in the fission yeastSchizosaccharomyces pombe indicated that Rpb6, consisting of 142 amino acid residues, is an essential protein for cell viability, and the essential region is located in the C-terminal half between residues 61 and 139. After random mutagenesis, a total of 14 temperature-sensitive mutants were isolated, each carrying a single (or double in three cases and triple in one) mutation. Four mutants each carrying a single mutation in the essential region were sensitive to 6-azauracil (6AU), which inhibits transcription elongation by depleting the intracellular pool of GTP and UTP. Both 6AU sensitivity and temperature-sensitive phenotypes of these rpb6 mutants were suppressed by overexpression of TFIIS, a transcription elongation factor. In agreement with the genetic studies, the mutant RNA polymerases containing the mutant Rpb6 subunits showed reduced affinity for TFIIS, as measured by a pull-down assay of TFIIS-RNA polymerase II complexes using a fusion form of TFIIS with glutathioneS-transferase. Moreover, the direct interaction between TFIIS and RNA polymerase II was competed by the addition of Rpb6. Taken together, the results lead us to propose that Rpb6 plays a role in the interaction between RNA polymerase II and the transcription elongation factor TFIIS.

Ribavirin-Induced Intracellular GTP Depletion Upregulates Factor VII Expression

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1113-1113
Author(s):  
Atsuo Suzuki ◽  
Yuhri Miyawaki ◽  
Eriko Okuyama ◽  
Moe Murata ◽  
Ando Yumi ◽  
...  
Keyword(s):  
Gene Transcription ◽  
Transcription Initiation ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
Elongation Rate ◽  
The Other ◽  
Factor Vii ◽  
Dependent Manner ◽  
Transcription Elongation Factor ◽  
Impdh Inhibitors

Abstract Abstract 1113 In this study, we investigated the molecular basis of upregulation of factor VII (FVII) gene expression by ribavirin, and found that intracellular GTP depletion induced by ribavirin activated FVII gene transcription and modulated transcription elongation. In 2006, Yamamoto et al. reported that anti-hepatitis C virus (HCV) agent ribavirin elevated the activity of FVII in HCV-infected hemophilia patients; however, the precise mechanisms were still unknown. In addition, the anti-HCV mechanisms of ribavirin were not yet fully elucidated, although the extended studies have been done. We investigated the effects of ribavirin in vitro and confirmed the approximately 4-fold upregulation of FVII mRNA by ribavirin treatment in HepG2 cells. FVII mRNA was increased in a dose-dependent manner up to 100μg/mL of ribavirin at a lower concentration than therapeutic concentration of 150μg/mL. FVII mRNA induction by ribavirin was also observed in a time-dependent manner from 24 h to 72 h after treatment. Ribavirin metabolite ribavirin 5'-monophosphate is one of the IMP dehydrogenase (IMPDH) inhibitors, and the other IMPDH inhibitors mycophenolic acid (MPA) and 6-mercaptupurine (6-MP) also induced FVII upregulation. It is well known that inhibition of IMPDH causes intracellular GTP depletion, and guanosine supplementation to salvage GTP could reverse FVII mRNA increase in ribavirin-treated cells. These results indicated that cellular GTP reduction associated with FVII gene upregulation. The mechanisms of gene upregulation by GTP depletion were not elucidated. The promoter activities and mRNA stability of FVII were analyzed under ribavirin treatment. The FVII gene promoter activity was enhanced up to 1.5-fold by ribavirin treatment; however the activation did not reach 4-fold induction of FVII mRNA increase. There was no significant change of FVII mRNA half-life in ribavirin-treated cells. Since the promoter activation might display transcription initiation capacity, the contribution of transcription elongation stage was further investigated. Transcription elongation was regulated by phosphorylation of carbo-terminal domain (CTD) of RNA polymerase II (PolII). Transcription elongation factor P-TEFb (positive-transcription elongation factor b), which consists as a complex of CDK9 and cyclin T, phosphorylates Ser of PolII CTD. The kinase activity of P-TEFb could be inhibited by 5,6-dichlorobenzimidazole 1-b-D-ribofuranoside (DRB). In FVII gene upregulation, DRB completely canceled ribavirin-induced FVII mRNA increase. We also performed nuclear run-on assay to verify the potential transcription elongation capacity of paused PolII, and observed a dramatic increase of FVII mRNA in ribavirin-treated cells. These results suggested that ribavirin-induced FVII gene upregulation was caused not only by transcription initiation but also by accelerated transcription elongation rate. There are various transcription factor associated with transcription elongation in addition to P-TEFb, such as elongin, ELL (eleven nineteen-lysine rich leukemia). We found that ELL3, a member of ELL family protein, was upregulated by ribavirin treatment. A ELL3 mRNA increase occurred prior to FVII mRNA upregulation, and the ELL3 upregulation was also canceled by guanosine supplementation. These results indicated ELL3 induction by ribavirin was also a response to cellular GTP depletion. To confirm the contribution of ELL3 protein to FVII gene transcription elongation, we used siRNAs specific to ELL3 and as expected, knockdown of ELL3 resulted in diminished FVII upregulation. A chromatin immunoprecipitation (ChIP) revealed ELL3 recruitment to the FVII gene, and the recruitments of PolII and CDK9 were also enhanced by ribavirin treatment. Taken together, FVII gene upregulation by ribavirin was associated with intracellular GTP depletion. The GTP reduction mainly modulates transcription elongation rate rather than transcription initiation, though the relationships between cellular GTP depletion and enhanced transcription elongation must be investigated. This study uncovered candidate mechanisms of ribavirin and the other IMPDH inhibitors and highlights a development of novel pharmaceutical therapies for hemophilia. Disclosures: No relevant conflicts of interest to declare.

scholarly journals FCP1, a Phosphatase Specific for the Heptapeptide Repeat of the Largest Subunit of RNA Polymerase II, Stimulates Transcription Elongation

2002 ◽  
Vol 22 (21) ◽  
pp. 7543-7552 ◽  
Author(s):  
Subhrangsu S. Mandal ◽  
Helen Cho ◽  
Sungjoon Kim ◽  
Kettly Cabane ◽  
Danny Reinberg
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Carboxy Terminal Domain ◽  
Transcription Factor Iif ◽  
Transcript Elongation ◽  
Rnap Ii ◽  
Carboxy Terminal

ABSTRACT FCP1, a phosphatase specific for the carboxy-terminal domain of RNA polymerase II (RNAP II), was found to stimulate transcript elongation by RNAP II in vitro and in vivo. This activity is independent of and distinct from the elongation-stimulatory activity associated with transcription factor IIF (TFIIF), and the elongation effects of TFIIF and FCP1 were found to be additive. Genetic experiments resulted in the isolation of several distinct fcp1 alleles. One of these alleles was found to suppress the slow-growth phenotype associated with either the reduction of intracellular nucleotide concentrations or the inhibition of other transcription elongation factors. Importantly, this allele of fcp1 was found to be lethal when combined individually with two mutations in the second-largest subunit of RNAP II, which had been shown previously to affect transcription elongation.

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 The Recruitment of theSaccharomyces cerevisiaePaf1 Complex to Active Genes Requires a Domain of Rtf1 That Directly Interacts with the Spt4-Spt5 Complex

2013 ◽  
Vol 33 (16) ◽  
pp. 3259-3273 ◽  
Author(s):  
Manasi K. Mayekar ◽  
Richard G. Gardner ◽  
Karen M. Arndt
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
Physical Interaction ◽  
Repeat Domain ◽  
Necessary And Sufficient ◽  
Active Genes ◽  
Interacting Domain

Transcription elongation factors associate with RNA polymerase II and aid its translocation through chromatin. One such factor is the conserved Paf1 complex (Paf1C), which regulates gene expression through several mechanisms, including the stimulation of cotranscriptional histone modifications. Previous studies revealed a prominent role for the Rtf1 subunit in tethering Paf1C to the RNA polymerase II elongation machinery. Here, we investigated the mechanism by which Rtf1 couples Paf1C to active chromatin. We show that a highly conserved domain of Rtf1 is necessary and sufficient for mediating a physical interaction between Rtf1 and the essential transcription elongation factor Spt5. Mutations that alter this Rtf1 domain or delete the Spt5 C-terminal repeat domain (CTR) disrupt the interaction between Rtf1 and Spt5 and release Paf1C from chromatin. When expressed in cells as the only source of Rtf1, the Spt5-interacting domain of Rtf1 can associate independently with active genes in a pattern similar to that of full-length Rtf1 and in a manner dependent on the Spt5 CTR.In vitroexperiments indicate that the interaction between the Rtf1 Spt5-interacting domain and the Spt5 CTR is direct. Collectively, our results provide molecular insight into a key attachment point between Paf1C and the RNA polymerase II elongation machinery.

scholarly journals Characterizing the Interaction between the HTLV-1 Transactivator Tax-1 with Transcription Elongation Factor ELL2 and Its Impact on Viral Transactivation

2021 ◽  
Vol 22 (24) ◽  
pp. 13597
Author(s):  
Stephan Kohrt ◽  
Sarah Strobel ◽  
Melanie Mann ◽  
Heinrich Sticht ◽  
Bernhard Fleckenstein ◽  
...  
Keyword(s):  
Complex Formation ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Point Mutations ◽  
Elongation Factor ◽  
Confocal Imaging ◽  
Cell Leukemia Virus Type ◽  
Transcription Elongation Factor ◽  
Human T Cell

The human T-cell leukemia virus type 1 (HTLV-1)-encoded transactivator and oncoprotein Tax-1 is essential for HTLV-1 replication. We recently found that Tax-1 interacts with transcription elongation factor for RNA polymerase II 2, ELL2, which enhances Tax-1-mediated transactivation of the HTLV-1 promotor. Here, we characterize the Tax-1:ELL2 interaction and its impact on viral transactivation by confocal imaging, co-immunoprecipitation, and luciferase assays. We found that Tax-1 and ELL2 not only co-precipitate, but also co-localize in dot-like structures in the nucleus. Tax-1:ELL2 complex formation occurred independently of Tax-1 point mutations, which are crucial for post translational modifications (PTMs) of Tax-1, suggesting that these PTMs are irrelevant for Tax-1:ELL2 interaction. In contrast, Tax-1 deletion mutants lacking either N-terminal (aa 1–37) or C-terminal regions (aa 150–353) of Tax-1 were impaired in interacting with ELL2. Contrary to Tax-1, the related, non-oncogenic Tax-2B from HTLV-2B did not interact with ELL2. Finally, we found that ELL2-R1 (aa 1–353), which carries an RNA polymerase II binding domain, and ELL2-R3 (aa 515–640) are sufficient to interact with Tax-1; however, only ELL2-truncations expressing R1 could enhance Tax-1-mediated transactivation of the HTLV-1 promoter. Together, this study identifies domains in Tax-1 and ELL2 being required for Tax-1:ELL2 complex formation and for viral transactivation.

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