scholarly journals Negative Elongation Factor-Mediated Suppression of RNA Polymerase II Elongation of Kaposi's Sarcoma-Associated Herpesvirus Lytic Gene Expression

2012 ◽  
Vol 86 (18) ◽  
pp. 9696-9707 ◽  
Author(s):  
Zsolt Toth ◽  
Kevin F. Brulois ◽  
Lai-Yee Wong ◽  
Hye-Ra Lee ◽  
Brian Chung ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
Lytic Gene ◽  
Histone Marks ◽  
Lytic Gene Expression ◽  
External Stimuli ◽  
Lytic Genes

Genome-wide chromatin immunoprecipitation assays indicate that the promoter-proximal pausing of RNA polymerase II (RNAPII) is an important postinitiation step for gene regulation. During latent infection, the majority of Kaposi's sarcoma-associated herpesvirus (KSHV) genes is silenced via repressive histone marks on their promoters. Despite the absence of their expression during latency, however, several lytic promoters are enriched with activating histone marks, suggesting that mechanisms other than heterochromatin-mediated suppression contribute to preventing lytic gene expression. Here, we show that the RNAPII-mediated transcription of the KSHV OriLytL, K5, K6, and K7 (OriLytL-K7) lytic genes is paused at the elongation step during latency. Specifically, the RNAPII-mediated transcription is stalled by the host's negative elongation factor (NELF) at the promoter regions of OriLytL-K7 lytic genes during latency, leading to the hyperphosphorylation of the serine 5 residue and the hypophosphorylation of the serine 2 of the C-terminal domain of the RNAPII large subunit, a hallmark of stalled RNAPII. Consequently, depletion of NELF expression induced transition of stalled RNAPII into a productive transcription elongation at the promoter-proximal regions of OriLytL-K7 lytic genes, leading to their RTA-independent expression. Using an RTA-deficient recombinant KSHV, we also showed that expression of the K5, K6, and K7 lytic genes was highly inducible upon external stimuli compared to other lytic genes that lack RNAPII on their promoters during latency. These results indicate that the transcription elongation of KSHV OriLytL-K7 lytic genes is inhibited by NELF during latency, but can also be promptly reactivated in an RTA-independent manner upon external stimuli.

scholarly journals Acute perturbation strategies in interrogating RNA polymerase II elongation factor function in gene expression

2021 ◽  
Vol 35 (3-4) ◽  
pp. 273-285
Author(s):  
Bin Zheng ◽  
Yuki Aoi ◽  
Avani P. Shah ◽  
Marta Iwanaszko ◽  
Siddhartha Das ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Factor Function

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.

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 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 Concurrent Expression of Latent and a Limited Number of Lytic Genes with Immune Modulation and Antiapoptotic Function by Kaposi's Sarcoma-Associated Herpesvirus Early during Infection of Primary Endothelial and Fibroblast Cells and Subsequent Decline of Lytic Gene Expression

2004 ◽  
Vol 78 (7) ◽  
pp. 3601-3620 ◽  
Author(s):  
Harinivas H. Krishnan ◽  
Pramod P. Naranatt ◽  
Marilyn S. Smith ◽  
Ling Zeng ◽  
Clark Bloomer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Immune Modulation ◽  
Kaposi's Sarcoma ◽  
Lytic Cycle ◽  
Lytic Gene ◽  
Kshv Infection ◽  
Lytic Gene Expression ◽  
Genome Array ◽  
Lytic Genes

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) infection of in vitro target cells is characterized by the expression of the latency-associated open reading frame (ORF) 73 gene (LANA-1) and the absence of progeny virus production. This default latent infection can be switched into lytic cycle by phorbol ester and by the lytic cycle ORF 50 (RTA) protein. In this study, the kinetics of latent and lytic gene expression immediately following KSHV infection of primary human dermal microvascular endothelial (HMVEC-d) and foreskin fibroblast (HFF) cells were examined by real-time reverse transcriptase PCR and whole-genome array. Within 2 h postinfection (p.i.), high levels of ORF 50 transcripts were detected in both cell types, which declined sharply by 24 h p.i. In contrast, comparatively low levels of ORF 73 expression were detected within 2 h p.i., increased subsequently, were maintained at a steady state, and declined slowly by 120 h p.i. The RTA and LANA-1 proteins were detected in the majority of infected cells by immunoperoxidase assays. In genome array, only 29 of 94 (31%) KSHV genes were expressed, which included 11 immediate-early/early, 8 early, and 5 late lytic genes and 4 latency-associated genes. While the expression of latent ORF 72, 73, and K13 genes continued, nearly all of the lytic genes declined or were undetectable by 8 and 24 h p.i. in HMVEC-d and HFF cells, respectively. Only a limited number of RTA-activated KSHV genes were expressed briefly, and the majority of KSHV genes involved in viral DNA synthesis and structural proteins were not expressed. However, early during infection, the lytic K2, K4, K5, K6, and vIRF2 genes with immune modulation functions and the K7 gene with antiapoptotic function were expressed. Expression of K5 was detected for up to 5 days of observation, and vIRF2 was expressed up to 24 h p.i. The full complement of lytic cycle genes were expressed when 12-O-tetradecanoylphorbol-13-acetate was added to the HMVEC-d cells after 48 h p.i. These data suggest that in contrast to alpha- and betaherpesviruses and some members of gammaherpesviruses, gamma-2 KSHV in vitro infection is characterized by the concurrent expression of latent and a limited number of lytic genes immediately following infection and a subsequent decline and/or absence of lytic gene expression with the persistence of latent genes. Expression of its limited lytic cycle genes could be a “strategy” that evolved in KSHV allowing it to evade the immune system and to provide the necessary factors and time to establish and/or maintain latency during the initial phases of infection. These are unique observations among in vitro herpesvirus infections and may have important implications in KSHV biology and pathogenesis.

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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