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)

The trypanosomatid mitochondrial genome does not encode tRNA genes at all and experimental evidence obtained with Leishmania tarentolae shows that tRNAs in mitochondria represent a selected set of imported nuclear-encoded tRNAs. In this paper we present the data showing that tRNAs derived from the clustered genomic tRNA genes are invariably imported into mitochondria, while tRNA from the solitary gene is not. By sequencing a cosmid DNA clone of L. tarentolae genomic DNA, we have identified a 1.5-kb subclone encoding a duplicate set of the closely linked tRNA(Tyr) (GTA) and tRNA(Thr) (AGT) genes. Northern analysis shows that these tRNAs are imported into mitochondria. In contrast, when the tRNA gene [tRNA(Gln) (CUG)] located alone in a 40-kb DNA fragment was examined, the corresponding tRNA was not detected in the mitochondrion. This "loner" tRNA gene is highly unusual since the 3'-flanking putative RNA polymerase III transcription termination signal sequence is characterized by a long string of 8 Ts followed by an A and a stretch of 7 Cs, while all other trypanosomatid tRNA genes whose tRNA transcripts are imported are terminated by a possible transcription termination signal of only 4-6 Ts. Whether the correlation found between the gene organization and tRNA-import characteristics is of general significance needs to be investigated further. A simple computer analysis presented in this paper rules out the possibility that tRNAs found in the trypanosomatid mitochondrion are the products of the U-addition type 'RNA editing' of maxicircle DNA.
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PMID:Selective import of nuclear-encoded tRNAs into mitochondria of the protozoan Leishmania tarentolae. 847 48

The largest subunit of mammalian RNA polymerase II (RNAP II) contains at its carboxyl terminus an unusual domain consisting of 52 tandem repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. This domain, designated the COOH-terminal domain (CTD), is essential for viability and is extensively phosphorylated during the transition from preinitiation complex assembly to elongation (1). Indeed, phosphorylation of the CTD may play an important regulatory role in this transition. We show here that the CTD is also modified by a novel form of protein glycosylation, O-GlcNAc. This modification has been found on numerous transcription factors and other nuclear and cytosolic proteins (2). Glycopeptides obtained by proteolytic digestion of the CTD were purified by reverse-phase high performance liquid chromatography and sequenced. Results from such experiments suggest that glycosylation occurs at multiple sites throughout the CTD, similar to the phosphorylation of this domain. The carbohydrate, however, is not detectable on the phosphorylated form of the enzyme. This observation is consistent with the idea that phosphorylation and glycosylation are mutually exclusive modifications. The CTD of RNAP II, therefore, appears to exist in three distinct conformational states: unmodified, phosphorylated, and glycosylated. The differential modification of the CTD may play an important role in the regulated expression of genes transcribed by RNA polymerase II.
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PMID:RNA polymerase II is a glycoprotein. Modification of the COOH-terminal domain by O-GlcNAc. 848 97

Random mutagenesis of the gene for bacteriophage T7 RNA polymerase was used to identify functionally essential amino acid residues of the enzyme. A two-plasmid system was developed that permits the straightforward isolation of T7 RNA polymerase mutants that had lost almost all catalytic activity. It was shown that substitutions of Thr and Ala for Pro at the position 563, Ser for Tyr571, Pro for Thr636, Asp for Tyr639 and of Cys for Phe646 resulted in inactivation of the enzyme. It is noteworthy that all these mutations are limited to two short regions that are highly conservative in sequences of monomeric RNA polymerases.
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PMID:Random mutagenesis of the gene for bacteriophage T7 RNA polymerase. 849 13

We have investigated the mechanism of transcription termination in vitro by spinach chloroplast RNA polymerase using templates encoding variants of the transcription-termination structure (attenuator) of the regulatory region of the threonine (thr) operon of Escherichia coli. Fourteen sequence variants located within its d(G+C) stem-loop and d(A+T)-rich regions were studied. We found that the helix integrity in the stem-loop structure is necessary for termination but that its stability is not directly correlated with termination efficiency. The sequence of the G+C stem-loop itself also influences termination. Moreover, the dA template stretch at the 3' end of the terminator plays a major role in termination efficiency, but base pairing between the A and U tract of the transcript does not. From the studies using deletion variants and a series of mutants that alter the sequences immediately downstream from the transcription termination site, we found that termination of transcription by spinach chloroplast RNA polymerase was also modulated by downstream DNA sequences in a sequence-specific manner. The second base immediately following the poly(T) tract is crucial for determining the termination efficiency by chloroplast RNA polymerase, but not of the T7 or E.coli enzymes.
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PMID:Transcription termination at the Escherichia coli thra terminator by spinach chloroplast RNA polymerase in vitro is influenced by downstream DNA sequences. 852 62

The largest subunit of RNA polymerase II contains an essential carboxyl-terminal domain (CTD) that consists of highly conserved heptapeptide repeats with the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. Yeast cells with a partially truncated CTD grow slowly, are temperature- and cold-sensitive, and are unable to fully activate transcription of some genes. Screening a yeast wild-type cDNA library by means of comparative hybridization we find that CTD truncation preferentially reduces transcription of genes encoding glycolytic enzymes. Using a newly developed dual reporter assay we demonstrate that sensitivity to CTD truncation is conferred by the glycolytic gene promoters. Expression driven by glycolytic gene promoters is reduced, on average, about 3-fold in strains with the shortest CTD growing on either fermentable or nonfermentable carbon sources. Sensitivity to CTD truncation is particularly acute for the constitutively expressed ENO1 gene, which is reduced 10-fold in a strain with only eight CTD repeats. The sensitivity of constitutive ENO1 expression argues that CTD truncation can cause defects in uninduced as well as induced transcription.
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PMID:Partial truncation of the yeast RNA polymerase II carboxyl-terminal domain preferentially reduces expression of glycolytic genes. 853 92

A newly identified temperature-sensitive mutant whose defect was mapped to the reovirus M1 gene (minor core protein mu2) was studied to better understand the functions of this virion protein. Sequence determination of the Ml gene of this mutant (tsH11.2) revealed a predicted methionine-to-threonine alteration at amino acid 399 and a change from proline to histidine at amino acid 414. The mutant made normal amounts of single-stranded RNA, both in in vitro transcriptase assays and in infected cells, and normal amounts of progeny viral protein at early times in a restrictive infection. However, tsH11.2 produced neither detectable progeny protein nor double-stranded RNA at late times in a restrictive infection. These studies indicate that mu2 plays a role in the conversion of reovirus mRNA to progeny double-stranded RNA.
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PMID:Identification and characterization of a double-stranded RNA- reovirus temperature-sensitive mutant defective in minor core protein mu2. 867 44

We have examined transcription in an early diverging eukaryote by analyzing the effect of the fungus-derived toxin alpha-amanitin on the transcription of protein-coding genes of the protist Trichomonas vaginalis. In contrast to that typical in eukaryotes, the RNA polymerase that transcribes T. vaginalis protein-coding genes is relatively resistant to alpha-amanitin (50% inhibition = 250 microg alpha-amanitin/ml). We have also characterized the gene encoding the largest subunit of RNA polymerase II, the subunit that binds alpha-amanitin. This protein is 41% identical to the mouse RNA polymerase II. Sequence analysis of the 50-amino-acid region thought to bind alpha-amanitin shows that this region of the trichomonad RNA polymerase II lacks many of the conserved amino acids present in the putative binding site, in agreement with the observed insensitivity to this inhibitor. Similar to other RNA polymerase IIs analyzed from ancient eukaryotes, the T. vaginalis RNA polymerase II lacks the typical heptapeptide (Tyr-Ser-Pro-Thr-Ser-Pro-Ser) repeat carboxyl-terminal domain (CTD) that is a hallmark of higher eukaryotic RNA polymerase IIs. The trichomonad enzyme, however, does contain a short modified CTD that is rich in the amino acid residues that compose the repeat. These data suggest that T. vaginalis protein-coding genes are transcribed by a RNA polymerase II that is relatively insensitive to alpha-amanitin and that differs from typical eukaryotic RNA polymerase IIs as it lacks a heptapeptide repeated CTD.
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PMID:Transcription in the early diverging eukaryote Trichomonas vaginalis: an unusual RNA polymerase II and alpha-amanitin-resistant transcription of protein-coding genes. 870 91

Peptides representing single repeat units of the carboxy-terminal domain (CTD) of RNA polymerase II (Tyr-Ser-Pro-Thr-Ser-Pro-Ser-Tyr-NH2, 1) contain overlapping Ser-Pro-Xaa-Xaa beta-turn forming sites which permit their overall structure to closely resemble members of the quinoxaline class of antitumor DNA bisintercalators. We have modified this native sequence at the i+2 positions of each beta-turn unit by substituting Gly or D-Ala in an attempt to preorganize this structure in aqueous solution. CD and NMR spectroscopic investigations confirmed the presence of type II beta-turns within each of the substituted peptides in contrast to the native sequence which contains a relatively low population of turn structure. In addition, an examination of singly substituted peptides suggests that an increase in the population of beta-turn structure within the amino-terminal Ser-Pro-Xaa-Xaa site also increased the formation of beta-turn structure in the carboxy-terminal (unmodified) Ser-Pro-Xaa-Xaa site; in comparison, substitution in the carboxy-terminal site did not influence structure in the remaining portion of the peptide. Overall, these results suggest that the structures formed could provide unique, preorganized linkers for the construction of novel DNA-interactive bisintercalators.
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PMID:Structural redesign and stabilization of the overlapping tandem beta-turns of RNA polymerase II. 873 51

Escherichia coli RNA polymerase is composed of four different subunits, alpha (present in two copies), beta, beta' and sigma. Among these, the beta' polypeptide shares nine conserved regions with the largest subunits of eukaryotic RNA polymerases, but its role is poorly understood. We isolated novel mutations in a plasmid-borne copy of rpoC, which encodes beta', as dominant suppressors of two temperature-sensitive nusA alleles. All 20 suppressors of nusA11 (single missense mutation) isolated had either of two specific substitutions: Lys for Glu-402 (rpoC10) and Thr for Ala-904 (rpoC111) in the beta' subunit. In vivo and in vitro transcription assays revealed that the rpoC10 allele of beta' participates in Rho-dependent transcription termination. On the other hand, of 20 suppressors of nusA134 (deletion of C-terminal one-third) scattered at 18 distinct sites, 16 were assigned to one of six conserved regions C-I. These results suggested that the conserved domains of the beta' subunit of E. coli RNA polymerase are involved in transcript termination or interaction with termination factor(s).
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PMID:Localization of nusA-suppressing amino acid substitutions in the conserved regions of the beta' subunit of Escherichia coli RNA polymerase. 875 1

The crystal structure of the DNA-actinomycin D (AMD) complex and a simple molecular modeling study indicated that AMD analogues derivatized at N-methyl-L-valine residues (fifth amino acid residue in the cyclic depsipeptide of AMD) could bind to DNA as strongly as the parent AMD. The analogues in which N-methyl-L-valine residues were replaced with L- and D-forms of N-methylvalines, N-methylthreonines, N-methylphenylalanies, N-methyltyrosines, and N-methyl-O-methyltyrosines have been totally synthesized. The characteristics of binding of the analogues to various DNAs including DNA-1 [d(TATATATGCATATATA)], DNA-2 [d(TATATACGCGTATATA)], DNA-3 [d(ATATATAGCTATATAT)], and DNA-4 [d(ATATATGGCCATATAT)] have been examined by using visible absorption spectrum methods. The association constants calculated from the absorption spectra indicate that the modifications of the N-methyl-L-valine residues in the AMD molecule do affect the DNA binding characteristics of the analogues. The L-aromatic analogues bind slightly better than the L-aliphatic analogues except for binding to DNA-1 (-TGCA-), whereas the D-aliphatic analogues bind consistently better than the D-aromatic analogues. In the L-form analogues, the L-Tyr analogue has the highest overall association constant, whereas the D-Val analogue has the highest association constant among the D-form analogues. In spite of substitution of bulky aromatic groups, the D-aromatic analogues bind to the DNA-1 quite well. However, D-aromatic analogues have significantly reduced their binding capacities to the other DNAs, indicating that the substitution of the D-aromatic residues creates a unique four-base sequence preference (-TGCA-). The RNA polymerase inhibitory activities of the AMD analogues in vivo have been examined using human cells (HeLa). All AMD analogues except for the L-Thr analogues severely inhibit RNA synthesis at relatively low drug concentrations. The D-Val, L-OMT, L-Phe, and D-Phe analogues inhibit RNA synthesis more strongly than the natural antibiotic (AMD itself).
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PMID:Physical and biological characteristics of the antitumor drug actinomycin D analogues derivatized at N-methyl-L-valine residues. 885 63


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