Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

P-TEFb is required for the transition from abortive elongation into productive elongation and is capable of phosphorylating the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II. We cloned a cDNA encoding the large subunit of Drosophila P-TEFb and found the predicted protein contained a cyclin motif. We now name the large subunit cyclin T and the previously cloned small subunit (Zhu, Y. R., Peery, T., Peng, J. M., Ramanathan, Y., Marshall, N., Marshall, T., Amendt, B., Mathews, M. B., and Price, D. H. (1997) Genes Dev. 11, 2622-2632) cyclin-dependent kinase 9 (CDK9). Recombinant P-TEFb produced in baculovirus-transfected Sf9 cells exhibited 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole-sensitive kinase activity similar to native P-TEFb. Kc cell nuclear extract depleted of P-TEFb failed to generate long DRB-sensitive transcripts, but this activity was restored upon addition of either native or recombinant P-TEFb. Like other CDKs, CDK9 is essentially inactive in the absence of its cyclin partner. P-TEFb containing a CDK9 mutation that knocked out the kinase activity did not function in transcription. Deletion of the carboxyl-terminal domain of cyclin T in P-TEFb reduced both the kinase and transcription activity to about 10%. The CDK-activating kinase in TFIIH was unable to activate the CTD kinase activity of P-TEFb.
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PMID:Identification of a cyclin subunit required for the function of Drosophila P-TEFb. 959 31

Two cyclin-dependent kinases have been identified in yeast and mammalian RNA polymerase II transcription initiation complexes. We find that the two yeast kinases are indistinguishable in their ability to phosphorylate the RNA polymerase II CTD, and yet in living cells one kinase is a positive regulator and the other a negative regulator. This paradox is resolved by the observation that the negative regulator, Srb10, is uniquely capable of phosphorylating the CTD prior to formation of the initiation complex on promoter DNA, with consequent inhibition of transcription. In contrast, the TFIIH kinase phosphorylates the CTD only after the transcription apparatus is associated with promoter DNA. These results reveal that the timing of CTD phosphorylation can account for the positive and negative functions of the two kinases and provide a model for Srb10-dependent repression of genes involved in cell type specificity, meiosis, and sugar utilization.
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PMID:Temporal regulation of RNA polymerase II by Srb10 and Kin28 cyclin-dependent kinases. 970 90

Cell cycle progression is controlled by the sequential functions of cyclin-dependent kinases (cdks). Cdk activation requires phosphorylation of a key residue (on sites equivalent to Thr-160 in human cdk2) carried out by the cdk-activating kinase (CAK). Human CAK has been identified as a p40(MO15)/cyclin H/MAT1 complex that also functions as part of transcription factor IIH (TFIIH) where it phosphorylates multiple transcriptional components including the C-terminal domain (CTD) of the large subunit of RNA polymerase II. In contrast, CAK from budding yeast consists of a single polypeptide (Cak1p), is not a component of TFIIH, and lacks CTD kinase activity. Here we report that Cak1p and p40(MO15) have strikingly different substrate specificities. Cak1p preferentially phosphorylated monomeric cdks, whereas p40(MO15) preferentially phosphorylated cdk/cyclin complexes. Furthermore, p40(MO15) only phosphorylated cdk6 bound to cyclin D3, whereas Cak1p recognized monomeric cdk6 and cdk6 bound to cyclin D1, D2, or D3. We also found that cdk inhibitors, including p21(CIP1), p27(KIP1), p57(KIP2), p16(INK4a), and p18(INK4c), could block phosphorylation by p40(MO15) but not phosphorylation by Cak1p. Our results demonstrate that although both Cak1p and p40(MO15) activate cdks by phosphorylating the same residue, the structural mechanisms underlying the enzyme-substrate recognition differ greatly. Structural and physiological implications of these findings will be discussed.
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PMID:Human and yeast cdk-activating kinases (CAKs) display distinct substrate specificities. 972 11

The interferon-inducible, double-stranded (ds) RNA-dependent protein kinase (PKR) regulates protein synthesis initiation by phosphorylating the alpha-subunit of eukaryotic translation initiation factor 2 (eIF-2). The amino-terminal half of PKR contains two dsRNA binding domains, and the kinase domain resides in the carboxy-terminal half of the protein. PKR is a ribosomal-associated protein. In this report, we provide evidence that PKR contains three ribosome interaction sites, two that are localized in each of the dsRNA binding domains and one that is localized in the kinase domain. All three domains can associate with polysomes independently. The ribosome association of the dsRNA binding domains requires dsRNA binding activity. Ribosome interaction of either the individual or the combined dsRNA binding domains was disrupted by 0.1 M KCl. In contrast, the ribosome interaction of intact PKR and the isolated kinase domain was largely resistant to 0.5 M KCl. These results indicate that all three domains of PKR contribute to the high-affinity ribosomal association. After dissociation of polysomes with EDTA, both intact PKR and the isolated kinase domain were primarily associated with the 60S ribosomal subunit. Coexpression of the adenovirus VAI RNA, an RNA polymerase III gene product that binds and inactivates PKR, disrupted ribosomal association of intact PKR, but not of the isolated PKR kinase domain. The results support a model where VAI RNA induces a major conformational change in PKR to prohibit ribosome association of all interaction sites. In contrast, other inhibitors of PKR including vaccinia virus E3L and K3L gene products, and the HIV trans-activating response (TAR) element binding protein TRBP, did not disrupt ribosome association of PKR. The results suggest a novel mechanism by which viral RNAs may inactivate PKR through disrupting ribosome association.
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PMID:Identification and requirement of three ribosome binding domains in dsRNA-dependent protein kinase (PKR). 975 71

Tat stimulation of HIV-1 transcriptional elongation is species-specific and is believed to require a specific cellular cofactor present in many human and primate cells but not in nonpermissive rodent cells. Human P-TEFb, composed of Cdk9 and cyclin T1, is a general transcription elongation factor that phosphorylates the C-terminal domain of RNA polymerase II. Previous studies have also implicated P-TEFb as a Tat-specific cellular cofactor and, in particular, human cyclin T1 as responsible for the species-specific Tat activation. To obtain functional evidence in support of these hypotheses, we generated and examined the activities of human-rodent "hybrid" P-TEFb complexes. We found that P-TEFb complexes containing human cyclin T1 complexed with either human or rodent Cdk9 supported Tat transactivation and interacted with the Tat activation domain and the HIV-1 TAR RNA element to form TAR loop-dependent ribonucleoprotein complexes. Although a stable complex containing rodent cyclin T1 and human Cdk9 was capable of phosphorylating CTD and mediating basal HIV-1 elongation, it failed to interact with Tat and to mediate Tat transactivation, indicating that the abilities of P-TEFb to support basal elongation and Tat activation can be separated. Together, our data indicated that the specific interaction of human P-TEFb with Tat/TAR, mostly through cyclin T1, is crucial for P-TEFb to mediate a Tat-specific and species-restricted activation of HIV-1 transcription. Amino acid residues unique to human Cdk9 also contributed partially to the formation of the P-TEFb-Tat-TAR complex. Moreover, the cyclin box of cyclin T1 and its immediate flanking region are largely responsible for the specific P-TEFb-Tat interaction.
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PMID:Specific interaction of Tat with the human but not rodent P-TEFb complex mediates the species-specific Tat activation of HIV-1 transcription. 1007 79

Phosphorylation of the yeast transcription factor GAL4 at S699 is required for efficient galactose-inducible transcription. We demonstrate that this site is a substrate for the RNA polymerase holoenzyme-associated CDK SRB10. S699 phosphorylation requires SRB10 in vivo, and this site is phosphorylated by purified SRB10/ SRB11 CDK/cyclin in vitro. RNA Pol II holoenzymes purified from WT yeast phosphorylate GAL4 at sites observed in vivo whereas holoenzymes from srb10 yeast are incapable of phosphorylating GAL4 at S699. Mutations at GAL4 S699 and srb10 are epistatic for GAL induction, demonstrating that SRB10 regulates GAL4 activity through this phosphorylation in vivo. These results demonstrate a function for the SRB10/ CDK8 holoenzyme-associated CDK that involves regulation of transactivators by phosphorylation during transcriptional activation.
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PMID:GAL4 is regulated by the RNA polymerase II holoenzyme-associated cyclin-dependent protein kinase SRB10/CDK8. 1036 Jan 83

The CDK9 kinase in association with Cyclin T is a component of the transcription positive-acting complex pTEFb which facilitates the transition from abortive to productive transcription elongation by phosphorylating the carboxyl-terminal domain of RNA polymerase II. The Cyclin T1/CDK9 complex is implicated in Tat transactivation, and it has been suggested that Tat functions by recruiting this complex to RNAPII through cooperative binding to RNA. Here, we demonstrate that targeted recruitment of Cyclin T1/CDK9 kinase complex to specific promoters, through fusion to a DNA-binding domain of either Cyclin T1 or CDK9 kinase, stimulates transcription in vivo. Transcriptional enhancement was dependent on active CDK9, as a catalytically inactive form had no transcriptional effect. We determined that, unlike conventional activators, DNA-bound CDK9 does not activate enhancerless TATA-promoters unless TBP is overexpressed, suggesting that CDK9 acts in vivo at a step subsequent to TFIID recruitment DNA-bound. Finally, we determined that CDK9-mediated transcriptional activation is mediated by preferentially stimulating productive transcription elongation.
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PMID:Transcriptional regulation by targeted recruitment of cyclin-dependent CDK9 kinase in vivo. 1046 4

The metazoan cyclin-dependent kinase Cdk7 was purified originally as part of a biochemical activity called CAK (Cdk-activating kinase) capable of phosphorylating and activating in vitro the Cdks that promote the different cell cycle transitions. Cdk7 is also found in the transcription factor complex TFIIH, suggesting that it participates in vivo in the control of RNA polymerase II. We have examined the physiological role of Cdk7 during the course of Drosophila development. By expressing dominant-negative forms of the kinase, we were able to alter Cdk7 function at given developmental stages. Expression of Cdk7 mutants severely delayed the onset of zygotic transcription in the early embryo, but did not alter the timing of the first 13 embryonic nuclear cycles. These results implicate Cdk7 in the control of transcriptional machinery in vivo. While cell cycle regulation is not sensitive to our manipulations of Cdk7 activity, it suggests that a distinct pool of CAK activity that is unaffected by expression of the cdk7(DN) mutants is present in these embryos.
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PMID:Dominant-negative mutants reveal a role for the Cdk7 kinase at the mid-blastula transition in Drosophila embryos. 1074 25

The cdk-activating kinase (CAK) activates cyclin-dependent kinases (cdks) that control cell-cycle progression by phosphorylating a threonine residue conserved in cdks. CAK from humans contains p40MO15 (cdk7), cyclin H and MAT1, which are also subunits of transcription factor IIH where they phosphorylate the C-terminal domain of the large subunit of RNA polymerase II. In contrast, budding yeast Cak1p is a monomeric enzyme without C-terminal domain kinase activity. Here, we analyze CAK activities in HeLa cells using cdk2-affinity chromatography. In addition to MO15, a second CAK activity was detected that runs on gel filtration at 30-40 kDa. This activity phosphorylated and activated cdk2 and cdk6. Furthermore, this 'small CAK' activity resembled Cak1p rather than MO15 in terms of substrate specificity, reactivity to antibodies against MO15 and Cak1p, and sensitivity to 5'-fluorosulfonylbenzoyladenosine, an irreversible inhibitory ATP analog. Our findings suggest the presence of at least two different CAK activities in human cells.
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PMID:Analysis of CAK activities from human cells. 1086 26

Contacts of phosphate groups at positions -12, -15, and -18 in relation to the transcription initiation site in the non-template strand of lac UV5 promoter with lysines or histidines of E. coli RNA polymerase in the open complex model were studied. A number of synthetic oligonucleotides from the -10-area of the non-template strand containing activated 5'-terminal phosphate group were cross-linked with holo- or core-enzyme of RNA polymerase. 5'-N-Hydroxybenzotriazole phosphodiesters of oligonucleotides were used as phosphate activated derivatives. They are capable of phosphorylating amino groups of lysines and histidines in the enzyme molecule that are brought into proximity with activated phosphate in the complex, resulting in the formation of a covalent bond between the oligonucleotide and the protein. The analysis of the products of cross-linking allowed the protein subunit and the amino acid residue taking part in the formation of the covalent bond for each oligonucleotide to be identified. It was found that all oligonucleotides from the non-template strand of promoter in the complex with the holo-enzyme are bound with the sigma70-subunit. When analyzing the products of partial cleavage of the complexes cross-linked at cysteines and methionines using SDS-PAGE, it was shown that phosphate at position -12 made contacts with His180 or His242 of the sigma70-subunit, the reactive amino acid residue being located between the first and second conservative regions. Phosphate at position -15 is located near lysines from two different areas--between Met413 and Met456 (regions 2.3 and 2.4) and between Met470 and Met507 (region 3.1). Phosphate at position -18 makes preferential contacts with a lysine situated between Met470 and Met507 (region 3.1). Based on the analysis of contacts of phosphate groups and the structure of the isolated sigma70-subunit established previously, a scheme of the mutual arrangement of the oligonucleotide and the sigma70-subunit possessed by the holo-enzyme has been proposed.
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PMID:Probing contacts of phosphate groups of oligonucleotides from the non-template strand of lac UV5 promoter with E. coli RNA polymerase using regioselective cross-linking. 1088 81


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