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 role of the C-terminal Phe882-Ala883 residues of bacteriophage T7 RNA polymerase in specific transcription has been investigated by means of site-directed mutagenesis. A mutant enzyme that lacks the C-terminal Phe882-Ala883 residues, denoted the "foot" mutant, has been cloned and overproduced, and the effects of the deletion on promoter recognition, initiation, and elongation have been determined. Gel retardation assays and DNase I footprinting show that the foot mutant specifically recognizes and binds to T7 promoters, although this binding appears to be approximately 30-fold weaker than that of the wild-type enzyme. Transcription assays using oligonucleotide templates that contain the consensus T7 promoter show a dramatic decrease in transcriptional activity for the foot mutant. With templates whose coding region begins CCC..., the mutant synthesizes poly(G) products even in the presence of all four nucleotides. The synthesis of poly(G) products from such templates has previously been observed for the wild-type enzyme when GTP is the sole nucleotide present in the reaction and is thought to occur by a novel mechanism involving slippage of the RNA chain 3' to 5' relative to the template [Martin, C.T., Muller, D.K., & Coleman, J.E. (1988) Biochemistry 27, 3966-3974]. These data suggest that the loss in transcriptional activity by the foot mutant results from a severe decrease in processivity as well as catalytic efficiency of the enzyme. Removal of the C-terminal Phe and Ala residues from the wild-type enzyme with carboxypeptidase A generates the phenotype of the mutant precisely, proving that all of the properties of the foot mutant derive from the loss of the Phe-Ala-COOH moiety.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Processivity of T7 RNA polymerase requires the C-terminal Phe882-Ala883-COO- or "foot". 205 36

A phage lambda clone containing a 13.1-kb human DNA fragment was isolated and found to contain a tRNA gene encoding a glycine tRNA. The nucleotide sequence of the gene and its flanking regions has been determined. The gene does not have an intervening sequence nor does it encode the 3'-terminal CCA sequence found in mature tRNAs. Although this tRNA gene has an anticodon sequence of CCC, it has a striking homology (96%) with a human glycine tRNA which has an anticodon of GCC. As in other eukaryotic tRNA genes, the coding region contains a characteristic internal split promoter sequence, and the 3'-flanking region has a typical RNA polymerase III termination site of five consecutive T residues. There is no apparent sequence in the 5'-flanking region which could serve as a regulatory element. This gene is accurately transcribed in vitro by RNA polymerase III using a HeLa cell-free system. During the course of in vitro transcription, larger precursor tRNAGlyCCC transcripts are processed to yield a mature-sized tRNA product. A precursor-product relationship was established by comparing the ribonuclease A fingerprints of the precursor and product tRNA transcripts.
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PMID:Nucleotide sequence and transcription of a gene encoding human tRNAGlyCCC. 298 90

The elongation rate of RNAs synthesized from AI promoters of T7 phage DNA and its deletion mutant delta DIII T7 DNA by E. coli RNA polymerase was analyzed. The distribution of incorporation rates of any definite nucleotides at any definite position along the two RNA chains was studied. The minimal structure which reproducibly forms pauses seems to be trinucleotide. Two main groups of trinucleotides could be distinguished: 1) those mostly associated with pauses and; 2) those usually found in pause free regions. The first group consists of AUG, AUA, AUC, AAU, GUG, GUA, CGU, CGC, UUA, UUU; the second one comprises AAA, CAA, CCC, UCC, CUA, CUG, CUC, GGG, ACU, GAG, GAA, GGA. A model accounting for intermittent elongation has been developed. It is based on the hypothesis that the kinetic constants of each nucleotide incorporation to and pyrophosphorolysis from the 3'-end of the growing RNA chain depend on the nature of the incoming nucleotide as well as on the nature of a nucleotide residue situated at the 3'-end of the growing RNA. A general equation describing the pause distribution along the RNA of a known nucleotide sequence is proposed.
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PMID:[Effect of the primary structure of RNA on the pulse character of RNA elongation in vitro by Escherichia coli RNA polymerase: a model]. 616 4

Staf is a zinc finger protein that we recently identified as the transcriptional activator of the RNA polymerase III-transcribed selenocysteine tRNA gene. In this work we demonstrate that enhanced transcription of the majority of vertebrate snRNA and snRNA-type genes, transcribed by RNA polymerases II and III, also requires Staf. DNA binding assays and microinjection of mutant genes into Xenopus oocytes showed the presence of Staf-responsive elements in the genes for human U4C, U6, Y4 and 7SK, Xenopus U1b1, U2, U5 and MRP and mouse U6 RNAs. Using recombinant Staf, we established that it mediates the activating properties of Staf-responsive elements on RNA polymerase II and III snRNA promoters in vivo. Lastly a 19 bp consensus sequence for the Staf binding site, YY(A/T)CCC(A/G)N(A/C)AT(G/C)C(A/C)YY-RCR, was derived by binding site selection. It enabled us to identify 23 other snRNA and snRNA-type genes carrying potential Staf binding sites. Altogether, our results emphasize the prime importance of Staf as a novel activator for enhanced transcription of snRNA and snRNA-type genes.
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PMID:Staf, a promiscuous activator for enhanced transcription by RNA polymerases II and III. 900 78

Ethanol oxidation in Kupffer cells was investigated by measuring 14C-acetate formation from 14C-ethanol, and the role of aldehyde dehydrogenase 2 (ALDH2) in this process was also examined. Formation of 14C-acetate from 20 mM of 14C-ethanol was significantly increased in medium-containing Kupffer cells (9,003 +/- 2,066 cpm/5 x 10(6) cells), compared with medium containing no cells (1,826 +/- 46 cpm, p < 0.01), or containing acid-killed Kupffer cells (1,629 +/- 210 cpm, p < 0.01). Ethanol formation was significantly attenuated when 20 and 200 microM cyanamide or 2 microM disulfiram were given. Reverse transcriptase-polymerase chain reaction demonstrated that Kupffer cells carry mRNA for ALDH2. These findings indicate that Kupffer cells can oxidize ethanol to acetate. ALDH2 may participate in this process, especially in the conversion of acetaldehyde to acetate.
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PMID:Evidence for ethanol oxidation by Kupffer cells. 1023 87

The minus-strand genome of Sendai virus is an assembly of the nucleocapsid protein (N) and RNA, in which each N subunit is associated with precisely 6 nt. Only genomes that are a multiple of 6 nt long replicate efficiently or are found naturally, and their replication promoters contain sequence elements with hexamer repeats. Paramyxoviruses that are governed by this hexamer rule also edit their P gene mRNA during its synthesis, by G insertions, via a controlled form of viral RNA polymerase "stuttering" (pseudo-templated transcription). This stuttering is directed by a cis-acting sequence (3' UNN UUUUUU CCC), whose hexamer phase is conserved within each virus group. To determine whether the hexamer phase of a given nucleotide sequence within nucleocapsids affected its sensitivity to chemical modification, and whether hexamer phase of the mRNA editing site was important for the editing process, we prepared a matched set of viruses in which a model editing site was displaced 1 nt at a time relative to the genome ends. The relative abilities of these Sendai viruses to edit their mRNAs in cell culture infections were examined, and the ability of DMS to chemically modify the nucleotides of this cis-acting signal within resting viral nucleocapsids was also studied. Cytidines at hexamer phases 1 and 6 were the most accessible to chemical modification, whereas mRNA editing was most extensive when the stutter-site C was in positions 2 to 5. Apparently, the N subunit imprints the nucleotide sequence it is associated with, and affects both the initiation of viral RNA synthesis and mRNA editing. The N-subunit assembly thus appears to superimpose another code upon the genetic code.
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PMID:Chemical modification of nucleotide bases and mRNA editing depend on hexamer or nucleoprotein phase in Sendai virus nucleocapsids. 1221 49

Primase, encoded by dnaG in bacteria, is a specialized DNA-dependent RNA polymerase that synthesizes RNA primers de novo for elongation by DNA polymerase. Genome sequence analysis has revealed two distantly related dnaG genes, TtdnaG and TtdnaG(2), in the thermophilic bacterium Thermoanaerobacter tengcongensis. Both TtDnaG (600 amino acids) and TtDnaG2 (358 amino acids) exhibit primase activities in vitro at a wide range of temperatures. Interestingly, the template recognition specificities of these two primases are quite distinctive. When trinucleotide-specific templates were tested, TtDnaG initiated RNA primer synthesis efficiently only on templates containing the trinucleotide 5'-CCC-3', not on the other 63 possible trinucleotides. When the 5'-CCC-3' sequence was flanked by additional cytosines or guanines, the initiation efficiency of TtDnaG increased remarkably. Significantly, TtDnaG could specifically and efficiently initiate RNA primer synthesis on a limited set of tetranucleotides composed entirely of cytosines and guanines, indicating that TtDnaG initiated RNA primer synthesis more preferably on GC-containing tetranucleotides. In contrast, it seemed that TtDnaG2 had no specific initiation nucleotides, as it could efficiently initiate RNA primer synthesis on all templates tested. The DNA binding affinity of TtDnaG2 was usually 10-fold higher than that of TtDnaG, which might correlate with its high activity but low template specificity. These distinct priming activities and specificities of TtDnaG and TtDnaG2 might shed new light on the diversity in the structure and function of the primases.
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PMID:Two distantly homologous DnaG primases from Thermoanaerobacter tengcongensis exhibit distinct initiation specificities and priming activities. 2034 61

Triple-stranded DNA:RNA helices of unknown function in vertebrate mitochondria associate with replication and transcription. Antiparallel Hoogsteen pairings form triplexes at physiological conditions. Intermolecular antiparallel triplexes require inverted 3'-to-5' RNA polymerization, which was never observed. Three rare, long natural 3'-to-5' inverted GenBank RNAs from mice mitochondria suggest occasional inverted transcription, putatively coding for proteins. BLAST aligns 18 GenBank-stored proteins with hypothetical proteins translated from the 3'-to-5' inverted Mus musculus mitochondrial genome. Three are DNA-binding, five are membrane proteins. 25% of main frame codons contribute to their 3'-to-5' overlap coding. Properties of these codons match those of overlap coding protein genes, as compared to codons not expected involved in inverted coding: a) nucleotide contents at synonymous codon positions in mitochondrial genomes fit replicational deamination gradients (A->G and C->T), but digress from gradients when functioning as nonsynonymous positions in putative 3'-to-5' overlapping genes; b) bias against 'circular code' codons (codon groups creating unambiguity between frames), and favouring homogenous codons (AAA, CCC, GGG, TTT) characterize overlapping genes, including putative 3'-to-5' overlapping genes, as compared to nonoverlapping coding sequences from the same main frame gene. This signature correlates with digression from deamination gradients. Deamination and circular code tests confirm independently alignment-based predictions of overlapping 3'-to-5' protein coding genes. Results indicate varying expression for different 3'-to-5' overlapping genes. Inverted 3'-to-5' RNA is produced, perhaps by an unknown RNA polymerase (invertase) putatively coded by 3'-to-5' inverted RNA.
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PMID:Triplex DNA:RNA, 3'-to-5' inverted RNA and protein coding in mitochondrial genomes. 2384 52

Microsporidia are ubiquitous intracellular pathogens whose opportunistic nature led to their increased recognition with the rise of the AIDS pandemic. As the RNA world was largely unexplored in this parasitic lineage, we developed a dedicated in silico methodology to carry out exhaustive identification of ncRNAs across the Encephalitozoon and Nosema genera. Thus, the previously missing U1 small nuclear RNA (snRNA) and small nucleolar RNAs (snoRNAs) targeting only the LSU rRNA were highlighted and were further validated using 5' and 3'RACE-PCR experiments. Overall, the 15 ncRNAs that were found shared between Encephalitozoon and Nosema spp. may represent the minimal core set required for parasitic life. Interestingly, the systematic presence of a CCC- or GGG-like motif in 5' of all ncRNA and mRNA gene transcripts regardless of the RNA polymerase involved suggests that the RNA polymerase machineries in microsporidia species could use common factors. Our data provide additional insights in accordance with the simplification processes observed in these reduce genomes and underline the usefulness of sequencing closely related species to help identify highly divergent ncRNAs in these parasites.
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PMID:Comparative genomics of microsporidian genomes reveals a minimal non-coding RNA set and new insights for transcription in minimal eukaryotic genomes. 2833 34