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)

In most eukaryotic organisms the U2 small nuclear RNA (snRNA) gene is transcribed by RNA polymerase II to generate a primary transcript with a 5' terminal 7-methylguanosine cap structure. Following nuclear export, the U2 snRNA is assembled into a core ribonucleoprotein particle (RNP). This involves binding a set of proteins that are shared by spliceosomal snRNPs to the highly conserved Sm site. Prior to nuclear import, the snRNA-(guanosine-N:2)-methyltransferase appears to interact with the core RNP and hypermethylates the cap structure to 2,2, 7-trimethylguanosine (m(3)G). In the protist parasite Trypanosoma brucei, U-snRNAs are complexed with a set of common proteins that are analogous to eukaryotic Sm antigens but do not have a highly conserved Sm sequence motif, and most U-snRNAs are synthesised by RNA polymerase III. Here, we examined the determinants for m(3)G cap formation in T.brucei by expressing mutant U2 snRNAs in vivo and assaying trimethylation and RNP assembly by immunoprecipitation. Surprisingly, these studies revealed that the Sm-analogous region is not required either for binding of the common proteins or for cap trimethylation. Furthermore, except for the first 24 nt which are part of the U2 promoter, the U2 coding region could be substituted or deleted without affecting cap trimethylation.
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PMID:Determinants for cap trimethylation of the U2 small nuclear RNA are not conserved between Trypanosoma brucei and higher eukaryotic organisms. 1100 Feb 61

The mRNA capping apparatus of the pathogenic fungus Candida albicans consists of three components: a 520- amino acid RNA triphosphatase (CaCet1p), a 449-amino acid RNA guanylyltransferase (Cgt1p), and a 474-amino acid RNA (guanine-N7-)-methyltransferase (Ccm1p). The fungal guanylyltransferase and methyltransferase are structurally similar to their mammalian counterparts, whereas the fungal triphosphatase is mechanistically and structurally unrelated to the triphosphatase of mammals. Hence, the triphosphatase is an attractive antifungal target. Here we identify a biologically active C-terminal domain of CaCet1p from residues 202 to 520. We find that CaCet1p function in vivo requires the segment from residues 202 to 256 immediately flanking the catalytic domain from 257 to 520. Genetic suppression data implicate the essential flanking segment in the binding of CaCet1p to the fungal guanylyltransferase. Deletion analysis of the Candida guanylyltransferase demarcates an N-terminal domain, Cgt1(1-387)p, that suffices for catalytic activity in vitro and for cell growth. An even smaller domain, Cgt1(1-367)p, suffices for binding to the guanylyltransferase docking site on yeast RNA triphosphatase. Deletion analysis of the cap methyltransferase identifies a C-terminal domain, Ccm1(137-474)p, as being sufficient for cap methyltransferase function in vivo and in vitro. Ccm1(137-474)p binds in vitro to synthetic peptides comprising the phosphorylated C-terminal domain of the largest subunit of RNA polymerase II. Binding is enhanced when the C-terminal domain is phosphorylated on both Ser-2 and Ser-5 of the YSPTSPS heptad repeat. We show that the entire three-component Saccharomyces cerevisiae capping apparatus can be replaced by C. albicans enzymes. Isogenic yeast cells expressing "all-Candida" versus "all-mammalian" capping components can be used to screen for cytotoxic agents that specifically target the fungal capping enzymes.
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PMID:Characterization of the mRNA capping apparatus of Candida albicans. 1103 9

J3R, the 39-kDa subunit of vaccinia virus poly(A) polymerase, is a multifunctional protein that catalyzes (nucleoside-2'-O-)-methyltransferase activity, serves as a poly(A) polymerase stimulatory factor, and acts as a postreplicative positive transcription elongation factor. Prior results support an association between poly(A) polymerase and the virion RNA polymerase. A possible direct interaction between J3R and H4L subunit of virion RNA polymerase was evaluated. J3R was shown to specifically bind to H4L amino acids 235-256, C terminal to NPH I binding site on H4L. H4L binds to the C-terminal region of J3R between amino acids 169 and 333. The presence of a J3R binding site near to the NPH I binding region on H4L led us to evaluate a physical interaction between NPH I and J3R. The NPH I binding site was located on J3R between amino acids 169 and 249, and J3R was shown to bind to NPH I between amino acids 457 and 524. To evaluate a role for J3R in early gene mRNA synthesis, transcription termination, and/or release, a transcription-competent extract prepared from cells infected with mutant virus lacking J3R, J3-7. Analysis of transcription activity demonstrated that J3R is not required for early mRNA synthesis and is not an essential factor in early gene transcription termination or transcript release in vitro. J3R interaction with NPH I and H4L may serve as a docking site for J3R on the virion RNA polymerase, linking transcription to mRNA cap formation and poly(A) addition.
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PMID:Interaction between the J3R subunit of vaccinia virus poly(A) polymerase and the H4L subunit of the viral RNA polymerase. 1116 28

RNA replication of all positive-strand RNA viruses is closely associated with intracellular membranes. Brome mosaic virus (BMV) RNA replication occurs on the perinuclear region of the endoplasmic reticulum (ER), both in its natural plant host and in the yeast Saccharomyces cerevisiae. The only viral component in the BMV RNA replication complex that localizes independently to the ER is 1a, a multifunctional protein with an N-terminal RNA capping domain and a C-terminal helicase-like domain. The other viral replication components, the RNA polymerase-like protein 2a and the RNA template, depend on 1a for recruitment to the ER. We show here that, in membrane extracts, 1a is fully susceptible to proteolytic digestion in the absence of detergent and thus, a finding consistent with its roles in RNA replication, is wholly or predominantly on the cytoplasmic face of the ER with no detectable lumenal protrusions. Nevertheless, 1a association with membranes is resistant to high-salt and high-pH treatments that release most peripheral membrane proteins. Membrane flotation gradient analysis of 1a deletion variants and 1a segments fused to green fluorescent protein (GFP) showed that sequences in the N-terminal RNA capping module of 1a mediate membrane association. In particular, a region C-terminal to the core methyltransferase homology was sufficient for high-affinity ER membrane association. Confocal immunofluorescence microscopy showed that even though these determinants mediate ER localization, they fail to localize GFP to the narrow region of the perinuclear ER, where full-length 1a normally resides. Instead, they mediate a more globular or convoluted distribution of ER markers. Thus, additional sequences in 1a that are distinct from the primary membrane association determinants contribute to 1a's normal subcellular distribution, possibly through effects on 1a conformation, orientation, or multimerization on the membrane.
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PMID:Identification of sequences in Brome mosaic virus replicase protein 1a that mediate association with endoplasmic reticulum membranes. 1171 27

Activation of gene transcription involves chromatin remodeling by coactivator proteins that are recruited by DNA-bound transcription factors. Local modification of chromatin structure at specific gene promoters by ATP-dependent processes and by posttranslational modifications of histone N-terminal tails provides access to RNA polymerase II and its accompanying transcription initiation complex. While the roles of lysine acetylation, serine phosphorylation, and lysine methylation of histones in chromatin remodeling are beginning to emerge, low levels of arginine methylation of histones have only recently been documented, and its physiological role is unknown. The coactivator CARM1 methylates histone H3 at Arg17 and Arg26 in vitro and cooperates synergistically with p160-type coactivators (e.g., GRIP1, SRC-1, ACTR) and coactivators with histone acetyltransferase activity (e.g., p300, CBP) to enhance gene activation by steroid and nuclear hormone receptors (NR) in transient transfection assays. In the current study, CARM1 cooperated with GRIP1 to enhance steroid hormone-dependent activation of stably integrated mouse mammary tumor virus (MMTV) promoters, and this coactivator function required the methyltransferase activity of CARM1. Chromatin immunoprecipitation assays and immunofluorescence studies indicated that CARM1 and the CARM1-methylated form of histone H3 specifically associated with a large tandem array of MMTV promoters in a hormone-dependent manner. Thus, arginine-specific histone methylation by CARM1 is an important part of the transcriptional activation process.
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PMID:Hormone-dependent, CARM1-directed, arginine-specific methylation of histone H3 on a steroid-regulated promoter. 1174 26

Histone methylation is known to be associated with both transcriptionally active and repressive chromatin states. Recent studies have identified SET domain-containing proteins such as SUV39H1 and Clr4 as mediators of H3 lysine 9 (Lys9) methylation and heterochromatin formation. Interestingly, H3 Lys9 methylation is not observed from bulk histones isolated from asynchronous populations of Saccharomyces cerevisiae or Tetrahymena thermophila. In contrast, H3 lysine 4 (Lys4) methylation is a predominant modification in these smaller eukaryotes. To identify the responsible methyltransferase(s) and to gain insight into the function of H3 Lys4 methylation, we have developed a histone H3 Lys4 methyl-specific antiserum. With this antiserum, we show that deletion of SET1, but not of other putative SET domain-containing genes, in S. cerevisiae, results in the complete abolishment of H3 Lys4 methylation in vivo. Furthermore, loss of H3 Lys4 methylation in a set1 Delta strain can be rescued by SET1. Analysis of histone H3 mutations at Lys4 revealed a slow-growth defect similar to a set1 Delta strain. Chromatin immunoprecipitation assays show that H3 Lys4 methylation is present at the rDNA locus and that Set1-mediated H3 Lys4 methylation is required for repression of RNA polymerase II transcription within rDNA. Taken together, these data suggest that Set1-mediated H3 Lys4 methylation is required for normal cell growth and transcriptional silencing.
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PMID:Histone H3 lysine 4 methylation is mediated by Set1 and required for cell growth and rDNA silencing in Saccharomyces cerevisiae. 1175 34

Hormone-activated nuclear receptors (NR) bind to specific regulatory DNA elements associated with their target genes and recruit coactivator proteins to remodel chromatin structure, recruit RNA polymerase, and activate transcription. The p160 coactivators (e.g., SRC-1, GRIP1, and ACTR) bind directly to activated NR and can recruit a variety of secondary coactivators. We have established a transient-transfection assay system under which the activity of various NR is highly or completely dependent on synergistic cooperation among three classes of coactivators: a p160 coactivator, the protein methyltransferase CARM1, and any of the three protein acetyltransferases, p300, CBP, or p/CAF. The three-coactivator functional synergy was only observed when low levels of NR were expressed and was highly or completely dependent on the methyltransferase activity of CARM1 and the acetyltransferase activity of p/CAF, but not the acetyltransferase activity of p300. Other members of the protein arginine methyltransferase family, which methylate different protein substrates than CARM1, could not substitute for CARM1 to act synergistically with p300 or p/CAF. A ternary complex of GRIP1, CARM1, and p300 or CBP was demonstrated in cultured mammalian cells, supporting a physiological role for the observed synergy. The transfection assay described here is a valuable new tool for investigating the mechanism of coactivator function and demonstrates the importance of multiple coactivators, including CARM1 and its specific protein methyltransferase activity, in transcriptional activation.
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PMID:Synergy among nuclear receptor coactivators: selective requirement for protein methyltransferase and acetyltransferase activities. 1199 99

A 5-methyluridine (m(5)U) residue at position 54 is a conserved feature of bacterial and eukaryotic tRNAs. The methylation of U54 is catalyzed by the tRNA(m5U54)methyltransferase, which in Saccharomyces cerevisiae is encoded by the nonessential TRM2 gene. In this study, we identified four different strains with mutant forms of tRNA(Ser)CGA. The absence of the TRM2 gene in these strains decreased the stability of tRNA(Ser)CGA and induced lethality. Two alleles of TRM2 encoding catalytically inactive tRNA(m5U54)methyltransferases were able to stabilize tRNA(Ser)CGA in one of the mutants, revealing a role for the Trm2 protein per se in tRNA maturation. Other tRNA modification enzymes interacting with tRNA(Ser)CGA in the maturation process, such as Pus4p, Trm1 p, and Trm3p were essential or important for growth of the tRNA(Ser)CGA mutants. Moreover, Lhp1p, a protein binding RNA polymerase III transcripts, was required to stabilize the mutant tRNAs. Based on our results, we suggest that tRNA modification enzymes might have a role in tRNA maturation not necessarily linked to their known catalytic activity.
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PMID:Dual function of the tRNA(m(5)U54)methyltransferase in tRNA maturation. 1200 92

S-adenosylhomocysteine hydrolase (SAHH) is the only enzyme known to cleave S-adenosylhomocysteine (SAH), a product and an inhibitor of all S-adenosylmethionine-dependent transmethylation reactions. Xenopus SAHH is a nuclear enzyme in transcriptionally active cells and inhibition of xSAHH prevents cap methylation of hnRNA [Mol. Biol. Cell 10 (1999) 4283]. Here, we demonstrate that inhibition of xSAHH in Xenopus XTC cells results in a cytoplasmic accumulation of the shuttling hnRNPs, while xSAHH itself remains in the nucleus. The functional link between xSAHH and mRNA cap methylation is further supported by a physical association between xSAHH and mRNA(guanine-7-)methyltransferase (CMT). We show by co-immunoprecipitation of tagged proteins that both enzymes interact in vivo. Direct interaction in vitro is shown by pull-down experiments that further demonstrate that the N-terminal 55 amino acids of xSAHH are sufficient for binding to CMT. Since CMT is known to bind to the hyperphoshorylated C-terminal domain (CTD) of its large subunit of RNA polymerase II, we have studied the co-localisation of RNA polymerase II and xSAHH in oocyte nuclei. Immunolocalisation on spreads of lampbrush chromosomes shows xSAHH on the loops of the transcriptionally active lampbrush chromosomes, in Cajal bodies and in B-snurposomes, the nuclear compartments that are most likely engaged in storage and recycling of RNA polymerase II and its cofactors. We therefore suggest that a subfraction of the nuclear xSAHH remains associated with the RNA polymerase holoenzyme complexes, also while these are not actively engaged in transcription.
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PMID:Interaction of S-adenosylhomocysteine hydrolase of Xenopus laevis with mRNA(guanine-7-)methyltransferase: implication on its nuclear compartmentalisation and on cap methylation of hnRNA. 1206 72

The p160 coactivator complex plays a critical role in transcriptional activation by nuclear receptors and possibly other classes of DNA-binding transcriptional activators. The complex contains at least one of three p160 coactivators (SRC-1, GRIP1/TIF2, or pCIP/RAC3/ACTR/AIB1/TRAM1), a histone acetyltransferase such as CBP or p300, and the histone methyltransferase CARM1 (coactivator-associated arginine methyltransferase 1). Methylation of histone H3 and possibly other proteins in the transcription initiation complex by CARM1 occurs along with acetylation of histones and other proteins by CBP and p300 to help remodel chromatin structure and recruit RNA polymerase II. Here we show that other domains of CARM1 are required for the coactivator function of CARM1 in addition to the methyltransferase activity. The methyltransferase GRIP1, binding, and homo-oligomerization activities all reside in the central region of CARM1, which is highly conserved among the entire protein arginine methyltransferase family. In addition to this conserved domain, the unique N- and C-terminal regions of CARM1 were also required for enhancement of transcriptional activation by nuclear receptors. While the N-terminal region has no known activity at present, the C-terminal part of CARM1 contains an autonomous activation domain, suggesting that it interacts with other proteins that help to mediate CARM1 coactivator function.
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PMID:Requirement for multiple domains of the protein arginine methyltransferase CARM1 in its transcriptional coactivator function. 1235 36


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