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Query: UNIPROT:P23193 (transcription elongation factor)
 739 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Prior phenotypic analysis of a vaccinia virus gene A18R mutant, Cts23, showed the synthesis of longer than wild type (Wt) length viral transcripts during the intermediate stage of infection, indicating that the A18R protein may act as a negative transcription elongation factor. The purpose of the work described here was to determine a biochemical activity for the A18R protein. Pulse-labeled transcription complexes established from intermediate virus promoters on bead-bound DNA templates were assayed for transcript release during an elongation step that contained nucleotides and various proteins. Pulse-labeled transcription complexes elongated in the presence of only nucleotides were unable to release nascent RNA. The addition of Wt extract during the elongation phase resulted in release of the nascent transcript, indicating that additional factors present in the Wt extract are capable of inducing transcript release. Extract from Cts23 or mock-infected cells was unable to induce release. The lack of release upon addition of Cts23 extract suggests that A18R is involved in release of nascent RNA. By itself, purified polyhistidine-tagged A18R protein (His-A18R) was unable to induce release; however, release did occur in the presence of purified His-A18R protein plus extract from either Cts23 or mock-infected cells. These data taken together indicate that A18R is necessary but not sufficient for release of nascent transcripts. We have also demonstrated that the combination of A18R protein and mock extract induces transcript release in an ATP-dependent manner, consistent with the fact that the A18R protein is an ATP-dependent helicase. Further analysis revealed that the release activity is not restricted to a vaccinia intermediate promoter but is observed using pulse-labeled transcription complexes initiated from all three viral gene class promoters. Therefore, we conclude that A18R and an as yet unidentified cellular factor(s) are required for the in vitro release of nascent RNA from a vaccinia virus transcription elongation complex.
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PMID:Vaccinia virus gene A18R DNA helicase is a transcript release factor. 1062 2

We have determined the solution NMR structure of the ribosomal protein L36 from Thermus thermophilus. L36 is the smallest protein in the large subunit of the prokaryotic ribosome. The sequence contains three completely conserved cysteine residues and one conserved histidine residue in a C-X(2)-C-X(12)-C-X(4)-H motif. Extended X-ray absorption fine structure spectroscopy was used to confirm that a purified L36 sample contains an equimolar amount of zinc. The structure of L36 was determined using simulated annealing based on NOE distance restraints, dihedral angle restraints and hydrogen bond distance restraints derived from NMR spectra of (15)N-labeled and non-labeled L36 samples at pH 7 and 12 degrees C, and by imposing tetrahedral zinc ion coordination geometry. The L36 fold is characterized by a triple-stranded antiparallel beta-sheet with the zinc-binding site at one end. The structure of the zinc site is well-determined and shows that the three cysteine sulphur atoms are supported by hydrogen bonds to backbone amide protons. The conserved histidine residue is located in a short 3(10)-helix and coordinates zinc by the N(delta1) atom. The electrostatic surface potential and location of conserved Arg, Lys and His side-chains suggest a large continuous L36-rRNA interaction interface. The folding topology as well as position and conformation of many conserved side-chains in L36 are very similar to those of zinc-ribbon domains found in the archaeal transcription factor TFIIB N terminus and the eukaryal transcription elongation factor hTFIIS C terminus. Given the relative antiquity of the ribosome it is possible that L36 reflects the parent of transcription-related zinc ribbons.
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PMID:The solution structure of ribosomal protein L36 from Thermus thermophilus reveals a zinc-ribbon-like fold. 1065 25

Transcriptional elongation by RNA polymerase II (RNAPII) is regulated by the positive transcription elongation factor b (P-TEFb). P-TEFb is composed of Cdk9 and C-type cyclin T1 (CycT1), CycT2a, CycT2b, or CycK. The role of the C-terminal region of CycT1 and CycT2 remains unknown. In this report, we demonstrate that these sequences are essential for the activation of transcription by P-TEFb via DNA, i.e., when CycT1 is tethered upstream or downstream of promoters and coding sequences. A histidine-rich stretch, which is conserved between CycT1 and CycT2 in this region, bound the C-terminal domain of RNAPII. This binding was required for the subsequent expression of full-length transcripts from target genes. Thus, P-TEFb could mediate effects of enhancers on the elongation of transcription.
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PMID:Interaction between P-TEFb and the C-terminal domain of RNA polymerase II activates transcriptional elongation from sites upstream or downstream of target genes. 1173 44

Different positive transcription elongation factor b (P-TEFb) complexes isolated from mammalian cells contain a common catalytic subunit (Cdk9) and the unique regulatory cyclins CycT1, CycT2a, CycT2b, or CycK. The role of CycK as a transcriptional cyclin was demonstrated in this study. First, CycK activated transcription when tethered heterologously to RNA, which required the kinase activity of Cdk9. Although this P-TEFb could phosphorylate the C-terminal domain (CTD) of RNA polymerase II (RNAPII) in vitro, in contrast to CycT1 and CycT2, CycK did not activate transcription when tethered to DNA. Interestingly, when the C termini of CycT1 and CycT2 or only the histidine-rich stretch from positions 481 to 551 in CycT1 were added to CycK, the extended chimeras activated transcription equivalently via DNA. Moreover, these transcriptional effects required the CTD of RNAPII in cells. Thus, CycK functions as P-TEFb only via RNA, which suggests the presence of cellular RNA-bound activators that require CycK for their transcriptional activity.
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PMID:P-TEFb containing cyclin K and Cdk9 can activate transcription via RNA. 1188 99

The transcriptional elongation of human immunodeficiency virus type 1 (HIV-1) is mediated by the virally encoded transactivator Tat and its cellular cofactor, positive transcription elongation factor b (P-TEFb). The human cyclin T1 (hCycT1) subunit of P-TEFb forms a stable complex with Tat and the transactivation response element (TAR) RNA located at the 5' end of all viral transcripts. Previous studies have demonstrated that hCycT1 binds Tat in a Zn(2+)-dependent manner via the cysteine at position 261, which is a tyrosine in murine cyclin T1. In the present study, we mutated all other cysteines and histidines that could be involved in this Zn(2+)-dependent interaction. Because all of these mutant proteins except hCycT1(C261Y) activated viral transcription in murine cells, no other cysteine or histidine in hCycT1 is responsible for this interaction. Next, we fused the N-terminal 280 residues in hCycT1 with Tat. Not only the full-length chimera but also the mutant hCycT1 with an N-terminal deletion to position 249, which retained the Tat-TAR recognition motif, activated HIV-1 transcription in murine cells. This minimal hybrid mutant hCycT1-Tat protein bound TAR RNA as well as human and murine P-TEFb in vitro. We conclude that this minimal chimera not only reproduces the high-affinity binding among P-TEFb, Tat, and TAR but also will be invaluable for determining the three-dimensional structure of this RNA-protein complex.
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PMID:A minimal chimera of human cyclin T1 and tat binds TAR and activates human immunodeficiency virus transcription in murine cells. 1243 19

Cyclin T1, together with the kinase CDK9, is a component of the transcription elongation factor P-TEFb which binds the human immunodeficiency virus type 1 (HIV-1) transactivator Tat. P-TEFb facilitates transcription by phosphorylating the carboxy-terminal domain (CTD) of RNA polymerase II. Cyclin T1 is an exceptionally large cyclin and is therefore a candidate for interactions with regulatory proteins. We identified granulin as a cyclin T1-interacting protein that represses expression from the HIV-1 promoter in transfected cells. The granulins, mitogenic growth factors containing repeats of a cysteine-rich motif, were reported previously to interact with Tat. We show that granulin formed stable complexes in vivo and in vitro with cyclin T1 and Tat. Granulin bound to the histidine-rich domain of cyclin T1, which was recently found to bind to the CTD, but not to cyclin T2. Binding of granulin to P-TEFb inhibited the phosphorylation of a CTD peptide. Granulin expression inhibited Tat transactivation, and tethering experiments showed that this effect was due, at least in part, to a direct action on cyclin T1 in the absence of Tat. In addition, granulin was a substrate for CDK9 but not for the other transcription-related kinases CDK7 and CDK8. Thus, granulin is a cellular protein that interacts with cyclin T1 to inhibit transcription.
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PMID:The growth factor granulin interacts with cyclin T1 and modulates P-TEFb-dependent transcription. 1258 88

The positive transcription elongation factor b (P-TEFb) contains cyclin T1 (CycT1) and cyclin-dependent kinase 9 (Cdk9). For activating the expression of eukaryotic genes, the histidine-rich sequence in CycT1 binds the heptapeptide repeats in the C-terminal domain (CTD) of RNA polymerase II (RNAPII), whereupon Cdk9 phosphorylates the CTD. We found that alanine-substituted heptapeptide repeats that cannot be phosphorylated also bind CycT1. When placed near transcription units, these CTD analogs block effects of P-TEFb. Remarkably, the transcriptional repressor PIE-1 from Caenorhabditis elegans behaves analogously. It binds CycT1 via an alanine-containing heptapeptide repeat and inhibits transcriptional elongation. Thus, our findings reveal a new mechanism by which repressors inhibit eukaryotic transcription.
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PMID:A model of repression: CTD analogs and PIE-1 inhibit transcriptional elongation by P-TEFb. 1265 93

Positive transcription elongation factor b (P-TEFb) hyperphosphorylates the carboxy-terminal domain of RNA polymerase II, permitting productive transcriptional elongation. The cyclin T1 subunit of P-TEFb engages cellular transcription factors as well as the human immunodeficiency virus type 1 (HIV-1) transactivator Tat. To identify potential P-TEFb regulators, we conducted a yeast two-hybrid screen with cyclin T1 as bait. Among the proteins isolated was the human I-mfa domain-containing protein (HIC). HIC has been reported to modulate expression from both cellular and viral promoters via its C-terminal cysteine-rich domain, which is similar to the inhibitor of MyoD family a (I-mfa) protein. We show that HIC binds cyclin T1 in yeast and mammalian cells and that it interacts with intact P-TEFb in mammalian cell extracts. The interaction involves the I-mfa domain of HIC and the regulatory histidine-rich region of cyclin T1. HIC also binds Tat via its I-mfa domain, although the sequence requirements are different. HIC colocalizes with cyclin T1 in nuclear speckle regions and with Tat in the nucleolus. Expression of the HIC cDNA modulates Tat transactivation of the HIV-1 long terminal repeat (LTR) in a cell type-specific fashion. It is mildly inhibitory in CEM cells but stimulates gene expression in HeLa, COS, and NIH 3T3 cells. The isolated I-mfa domain acts as a dominant negative inhibitor. Activation of the HIV-1 LTR by HIC in NIH 3T3 cells occurs at the RNA level and is mediated by direct interactions with P-TEFb.
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PMID:The human I-mfa domain-containing protein, HIC, interacts with cyclin T1 and modulates P-TEFb-dependent transcription. 1294 66

Transcriptional elongation by RNA polymerase II (RNAPII) is regulated by the positive transcription elongation factor b (P-TEFb), which contains Cdk9 and a C-type cyclin (CycT1, CycT2a, CycT2b, or CycK). Whereas their N-terminal cylin boxes are almost identical, the C-terminal sequences of CycT1 and CycT2 are divergent. Previously, a histidine-rich stretch in CycT1 was found to bind the CTD of RNAPII and direct the transcriptional activity of this P-TEFb complex when tethered artificially to DNA. The global repressor PIE-1 from C. elegans blocked its effects. In this study, C-terminal truncations of CycT2 past its histidine-rich stretch, to a leucine-rich region next to its cyclin boxes, still maintained appreciable transcriptional activity. Moreover, this domain bound RNAPII via its CTD and PIE-1 blocked its effects. Thus, CycT2 not only contains two domains that target RNAPII but this substrate recognition is necessary for its transcriptional activity via DNA.
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PMID:Transcriptional activity and substrate recognition of cyclin T2 from P-TEFb. 1556 43

The cellular positive transcription elongation factor b (P-TEFb), containing cyclin T1 and cyclin-dependent kinase 9 (CDK9), interacts with the human immunodeficiency virus, type 1 (HIV-1) regulatory protein Tat to enable viral transcription and replication. Cyclin T1 is an unusually long cyclin and is engaged by cellular regulatory proteins. Previous studies showed that the granulin/epithelin precursor (GEP) binds the histidine-rich region of cyclin T1 and inhibits P-TEFb function. GEP is composed of repeats that vary in sequence and properties. GEP also binds directly to Tat. Here we show that GEP and some of its constituent granulin repeats can inhibit HIV-1 transcription via Tat without directly binding to cyclin T1. The interactions of granulins with Tat and cyclin T1 differ with respect to their binding sites and divalent cation requirements, and we identified granulin repeats that bind differentially to Tat and cyclin T1. Granulins DE and E bind Tat but do not interact directly with cyclin T1. These granulins are present in complexes with Tat and P-TEFb in which Tat forms a bridge between the cellular proteins. Granulins DE and E repress transcription from the HIV-1 LTR and gene expression from the viral genome, raising the possibility of developing granulin-based inhibitors of viral infection.
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PMID:Granulin and granulin repeats interact with the Tat.P-TEFb complex and inhibit Tat transactivation. 1565 95


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