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)

During mRNA elongation, the SRI domain of the histone H3 methyltransferase Set2 binds to the phosphorylated carboxyl-terminal domain (CTD) of RNA polymerase II. The solution structure of the yeast Set2 SRI domain reveals a novel CTD-binding fold consisting of a left-handed three-helix bundle. NMR titration shows that the SRI domain binds an Ser2/Ser5-phosphorylated CTD peptide comprising two heptapeptide repeats and three flanking NH2-terminal residues, whereas a single CTD repeat is insufficient for binding. Residues that show strong chemical shift perturbations upon CTD binding cluster in two regions. Both CTD tyrosine side chains contact the SRI domain. One of the tyrosines binds in the region with the strongest chemical shift perturbations, formed by the two NH2-terminal helices. Unexpectedly, the SRI domain fold resembles the structure of an RNA polymerase-interacting domain in bacterial sigma factors (domain sigma2 in sigma70).
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PMID:Structure and carboxyl-terminal domain (CTD) binding of the Set2 SRI domain that couples histone H3 Lys36 methylation to transcription. 1628 74

1-Methyladenosine modification at position 58 of tRNA is catalyzed by a two-subunit methyltransferase composed of Trm6p and Trm61p in Saccharomyces cerevisiae. Initiator tRNA (tRNAi(Met)) lacking m1A58 (hypomethylated) is rendered unstable through the cooperative function of the poly(A) polymerases, Trf4p/Trf5p, and the nuclear exosome. We provide evidence that a catalytically active Trf4p poly(A) polymerase is required for polyadenylation of hypomethylated tRNAi(Met) in vivo. DNA sequence analysis of tRNAi(Met) cDNAs and Northern hybridizations of poly(A)+ RNA provide evidence that nascent pre-tRNAi(Met) transcripts are targeted for polyadenylation and degradation. We determined that a mutant U6 snRNA and an aberrant form of 5S rRNA are stabilized in the absence of Trf4p, supporting that Trf4p facilitated RNA surveillance is a global process that stretches beyond hypomethylated tRNAi(Met). We conclude that an array of RNA polymerase III transcripts are targeted for Trf4p/ Trf5p-dependent polyadenylation and turnover to eliminate mutant and variant forms of normally stable RNAs.
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PMID:Nuclear RNA surveillance in Saccharomyces cerevisiae: Trf4p-dependent polyadenylation of nascent hypomethylated tRNA and an aberrant form of 5S rRNA. 1643 88

In Arabidopsis thaliana, small interfering RNAs (siRNAs) direct cytosine methylation at endogenous DNA repeats in a pathway involving two forms of nuclear RNA polymerase IV (Pol IVa and Pol IVb), RNA-DEPENDENT RNA POLYMERASE 2 (RDR2), DICER-LIKE 3 (DCL3), ARGONAUTE4 (AGO4), the chromatin remodeler DRD1, and the de novo cytosine methyltransferase DRM2. We show that RDR2, DCL3, AGO4, and NRPD1b (the largest subunit of Pol IVb) colocalize with siRNAs within the nucleolus. By contrast, Pol IVa and DRD1 are external to the nucleolus and colocalize with endogenous repeat loci. Mutation-induced loss of pathway proteins causes downstream proteins to mislocalize, revealing their order of action. Pol IVa acts first, and its localization is RNA dependent, suggesting an RNA template. We hypothesize that maintenance of the heterochromatic state involves locus-specific Pol IVa transcription followed by siRNA production and assembly of AGO4- and NRPD1b-containing silencing complexes within nucleolar processing centers.
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PMID:The Arabidopsis chromatin-modifying nuclear siRNA pathway involves a nucleolar RNA processing center. 1683 78

Flexibility and dynamics of DNA are important for DNA-binding and recognition by proteins. Here the flexibility of DNA is calculated from the distribution of DNA-bending angles of single DNA molecules as observed by scanning force microscopy by applying an equation that links the force constant of DNA-bending (f) to the variance of the distribution of bending angles (sigma): f=RT/sigma(2). Using published data, f is calculated to be 3-5 J/degree(2) for free DNA. Thus, bending DNA by 20 degrees requires approx. 0.5-1 kJ/mol. This result shows that DNA is very flexible and readily can be bent by thermal motion. DNA-flexibility is not altered in some protein-DNA complexes (HhaI methyltransferase, EcoRV restriction endonuclease). In contrast, DNA-binding by EcoRI endonuclease increases DNA-flexibility and binding by EcoRI methyltransferase restricts the flexibility of DNA. During the transition of the RNA polymerase-sigma(54)-DNA complex from the closed to the open form and of cro repressor from a non-specific to a specific binding mode the flexibility of the DNA is strongly reduced.
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PMID:Flexibility of DNA in complex with proteins deduced from the distribution of bending angles observed by scanning force microscopy. 1702 32

Capping of nascent pre-mRNAs is thought to be a prerequisite for productive elongation and associated serine 2 phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (PolII). The mechanism mediating this link is unknown, but is likely to include the capping machinery and P-TEPb. We report that the fission yeast P-TEFb (Cdk9-Pch1) forms a complex with the cap-methyltransferase Pcm1 and these proteins colocalise on chromatin. Ablation of Cdk9 function through chemical genetics causes growth arrest and abolishes serine 2 phosphorylation on the PolII CTD. Strikingly, depletion of Pcm1 also leads to a dramatic decrease of phospho-serine 2. Chromatin immunoprecipitations show a severe decrease of chromatin-bound Cdk9-Pch1 when Pcm1 is depleted. On the contrary, Cdk9 is not required for association of Pcm1 with chromatin. Furthermore, compromising Cdk9 activity leads to a promoter-proximal PolII stalling and sensitivity to 6-azauracil, reflecting elongation defects. The in vivo data presented here strongly support the existence of a molecular mechanism where the cap-methyltransferase recruits P-TEFb to chromatin, thereby ensuring that only properly capped transcripts are elongated.
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PMID:Recruitment of P-TEFb (Cdk9-Pch1) to chromatin by the cap-methyl transferase Pcm1 in fission yeast. 1733 44

The core human mitochondrial transcription machinery comprises a single subunit bacteriophage-related RNA polymerase, POLRMT, the high mobility group box DNA-binding protein h-mtTFA/TFAM, and two transcriptional co-activator proteins, h-mtTFB1 and h-mtTFB2 that also have rRNA methyltransferase activity. Recapitulation of specific initiation of transcription in vitro can be achieved by a complex of POL-RMT, h-mtTFA, and either h-mtTFB1 or h-mtTFB2. However, the nature of mitochondrial transcription complexes in vivo and the potential involvement of additional proteins in the transcription process in human mitochondria have not been extensively investigated. In Saccharomyces cerevisiae, transcription and translation are physically coupled via the formation of a multiprotein complex nucleated by the binding of Nam1p to the amino-terminal domain of mtRNA polymerase (Rpo41p). This model system paradigm led us to search for proteins that interact with POLRMT to regulate mitochondrial gene expression in humans. Using an affinity capture strategy to identify POL-RMT-binding proteins, we identified mitochondrial ribosomal protein L7/L12 (MRPL12) as a protein in HeLa mitochondrial extracts that interacts specifically with POLRMT in vitro. Purified recombinant MRPL12 binds to POLRMT and stimulates mitochondrial transcription activity in vitro, demonstrating that this interaction is both direct and functional. Finally, from HeLa cells that overexpress FLAG epitope-tagged MRPL12, increased steady-state levels of mtDNA-encoded transcripts are observed and MRPL12-POLRMT complexes can be co-immunoprecipitated, providing strong evidence that this interaction enhances mitochondrial transcription or RNA stability in vivo. We speculate that the MRPL12 interaction with POLRMT is likely part of a novel regulatory mechanism that coordinates mitochondrial transcription with translation and/or ribosome biogenesis during human mitochondrial gene expression.
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PMID:Human mitochondrial ribosomal protein MRPL12 interacts directly with mitochondrial RNA polymerase to modulate mitochondrial gene expression. 1733 45

In Arabidopsis thaliana, the pathway for transcriptional silencing via RNA-directed DNA methylation and chromatin modification involves two forms of nuclear RNA polymerase IV (pol IVa and pol IVb), RNA-DEPENDENT RNA POLYMERASE2 (RDR2), DICER-LIKE3 (DCL3), ARGONAUTE4 (AGO4), the chromatin remodeler, DRD1, and the de novo cytosine methyltransferase, DRM2. New insight into the order of events, as well as the spatial organization of this pathway within the nucleus, has come from the combined use of protein immunolocalization, RNA fluorescence in situ hybridization (RNA-FISH), DNA-FISH, and genetic analysis. New findings show that pol IVa, pol IVb, and DRD1 colocalize with DNA loci that are both the sources and targets of small interfering RNAs (siRNAs). However, RDR2-dependent doublestranded RNA production, dicing by DCL3, and loading of siRNAs into AGO4-containing RNA-induced silencing complexes (RISCs) appear to take place at a distant site, in an siRNA processing center located in the nucleolus. This siRNA processing center shares features of Cajal bodies, which are nucleolus-associated entities involved in the processing and trafficking of RNAs found in ribonucleoprotein (RNP) complexes that splice or modify mRNA, rRNA, or telomeres. Therefore, assembly and trafficking of chromatin-modifying RISCs may share similarities with other nuclear RNPs.
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PMID:Cell biology of the Arabidopsis nuclear siRNA pathway for RNA-directed chromatin modification. 1738 29

Lysine methylation has important functions in biological processes that range from heterochromatin formation to transcription regulation. Here, we demonstrate that Drosophila dSet2 encodes a developmentally essential histone H3 lysine 36 (K36) methyltransferase. Larvae subjected to RNA interference-mediated (RNAi) suppression of dSet2 lack dSet2 expression and H3-K36 methylation, indicating that dSet2 is the sole enzyme responsible for this modification in Drosophila melanogaster. dSet2 RNAi blocks puparium formation and adult development, and causes partial (blister) separation of the dorsal and ventral wing epithelia, defects suggesting a failure of the ecdysone-controlled genetic program. A transheterozygous EcR null mutation/dSet2 RNAi combination produces a complete (balloon) separation of the wing surfaces, revealing a genetic interaction between EcR and dSet2. Using immunoprecipitation, we demonstrate that dSet2 associates with the hyperphosphorylated form of RNA polymerase II (RNAPII).
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PMID:Drosophila dSet2 functions in H3-K36 methylation and is required for development. 1756 May 46

Histone modifications play an important role in transcription. We previously studied histone H2B ubiquitylation on lysine 123 and subsequent deubiquitylation by SAGA-associated Ubp8. Unlike other histone modifications, both the addition and removal of ubiquitin are required for optimal transcription. Here we report that deubiquitylation of H2B is important for recruitment of a complex containing the kinase Ctk1, resulting in phosphorylation of the RNA polymerase II (Pol II) C-terminal domain (CTD), and for subsequent recruitment of the Set2 methyltransferase. We find that Ctk1 interacts with histones H2A and H2B, and that persistent H2B ubiquitylation disrupts these interactions. We further show that Ubp8 enters the GAL1 coding region through an interaction with Pol II. These findings reveal a mechanism by which H2B ubiquitylation acts as a barrier to Ctk1 association with active genes, while subsequent deubiquitylation by Ubp8 triggers Ctk1 recruitment at the appropriate point in activation.
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PMID:H2B ubiquitylation acts as a barrier to Ctk1 nucleosomal recruitment prior to removal by Ubp8 within a SAGA-related complex. 1764 76

Chimeric proteins joining the histone methyltransferase MLL with various fusion partners trigger distinctive lymphoid and myeloid leukemias. Here, we immunopurified proteins associated with ENL, a protein commonly fused to MLL. Identification of these ENL-associated proteins (EAPs) by mass spectrometry revealed enzymes with a known role in transcriptional elongation (RNA polymerase II C-terminal domain kinase [RNAPolII CTD] positive transcription elongation factor b [pTEFb]), and in chromatin modification (histone-H3 methyltransferase DOT1L) as well as other frequent MLL partners (AF4, AF5q31, and LAF4), and polycomb group members (RING1, CBX8, and BCoR). The composition of EAP was further verified by coimmunoprecipitation, 2-hybrid analysis, pull-down, and colocalization experiments. Purified EAP showed a histone H3 lysine 79-specific methylase activity, displayed a robust RNAPolII CTD kinase function, and counteracted the effect of the pTEFb inhibitor 5,6-dichloro-benzimidazole-riboside. In vivo, an ENL knock-down diminished genome-wide as well as gene-specific H3K79 dimethylation, reduced global run-on elongation, and inhibited transient transcriptional reporter activity. According to structure-function data, DOT1L recruitment was important for transformation by the MLL-ENL fusion derivative. These results suggest a function of ENL in histone modification and transcriptional elongation.
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PMID:A role for the MLL fusion partner ENL in transcriptional elongation and chromatin modification. 1785 33


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