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 S2 gene nucleotide sequences of prototype strains of the three reovirus serotypes were determined to gain insight into the structure and function of the S2 translation product, virion core protein sigma 2. The S2 sequences of the type 1 Lang, type 2 Jones, and type 3 Dearing strains are 1,331 nucleotides in length and contain a single large open reading frame that could encode a protein of 418 amino acids, corresponding to sigma 2. The deduced sigma 2 amino acid sequences of these strains are very conserved, being identical at 94% of the sequence positions. Predictions of sigma 2 secondary structure and hydrophobicity suggest that the protein has a two-domain structure. A larger domain is suggested to be formed from the amino-terminal three-fourths of sigma 2 sequence, which is separated from a smaller carboxy-terminal domain by a turn-rich hinge region. The carboxy-terminal domain includes sequences that are more hydrophilic than those in the rest of the protein and contains sequences which are predicted to form an alpha-helix. A region of striking similarity was found between amino acids 354 and 374 of sigma 2 and amino acids 1008 and 1031 of the beta subunit of the Escherichia coli DNA-dependent RNA polymerase. We suggest that the regions with similar sequence in sigma 2 and the beta subunit form amphipathic alpha-helices which may play a related role in the function of each protein. We have also performed experiments to further characterize the double-stranded RNA-binding activity of sigma 2 and found that the capacity to bind double-stranded RNA is a property of the sigma 2 protein of prototype strains and of the S2 mutant tsC447.
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PMID:The S2 gene nucleotide sequences of prototype strains of the three reovirus serotypes: characterization of reovirus core protein sigma 2. 192 Jun 14

Both NMR and nuclease mapping have been used to probe the structure of an unmodified yeast tRNA(phe) precursor synthesized in vitro by T7 RNA polymerase. A comparison of the NMR data of the precursor and of the mature tRNA transcript shows that the mature tRNA domain structure is similar in both molecules. In the tRNA precursor, the intron consists of a stem of at least four base-pairs, identified by NMR, and two single-stranded loops, identified by nuclease mapping. This is in agreement with the structure previously proposed for the native tRNA(phe) precursor (1). However, our data also show the intron structure to be less stable than the mature tRNA domain, suggesting that the precursor may best be described as having two domains with a hinge at the junction of the anticodon and intron stems.
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PMID:Structural investigation of the in vitro transcript of the yeast tRNA(phe) precursor by NMR and nuclease mapping. 212 75

Structures of the 31-kilodalton catalytic domain of rat DNA polymerase beta (pol beta) and the whole 39-kilodalton enzyme were determined at 2.3 and 3.6 angstrom resolution, respectively. The 31-kilodalton domain is composed of fingers, palm, and thumb subdomains arranged to form a DNA binding channel reminiscent of the polymerase domains of the Klenow fragment of Escherichia coli DNA polymerase I, HIV-1 reverse transcriptase, and bacteriophage T7 RNA polymerase. The amino-terminal 8-kilodalton domain is attached to the fingers subdomain by a flexible hinge. The two invariant aspartates found in all polymerase sequences and implicated in catalytic activity have the same geometric arrangement within structurally similar but topologically distinct palms, indicating that the polymerases have maintained, or possibly re-evolved, a common nucleotidyl transfer mechanism. The location of Mn2+ and deoxyadenosine triphosphate in pol beta confirms the role of the invariant aspartates in metal ion and deoxynucleoside triphosphate binding.
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PMID:Crystal structure of rat DNA polymerase beta: evidence for a common polymerase mechanism. 751 81

Ewing tumours (ET), including Ewing's sarcoma and peripheral primitive neuroectodermal tumour, are well characterised at the molecular level by a unique chromosomal rearrangement which fuses the EWS gene to one of two closely related ETS proto-oncogenes, FLI-1 or ERG. Expression of the resulting chimaeric transcripts can be readily detected by reversed transcriptase polymerase chain reaction (RT-PCR). This approach led to the identification of a number of different exon combinations at the junction site of coding sequences. The physiological consequences of the observed variability in the hinge region of EWS chimaeric proteins are not known. We have analysed tumour-derived material from 30 ET patients with well-documented clinical course (18 with localised and 12 with metastatic disease at diagnosis) for the presence of EWS/FLI-1 or EWS/ERG RNA. Karyotypes were obtained in 21 out of 27 cases and analysed by routine cytogenetics. A chromosome 22 rearrangement was demonstrated in 18 cases (67%). In contrast, RT-PCR revealed the presence of chimaeric transcripts in 28 tumours (93%), with fusions of EWS exon 7 to FLI-1 exons 6 (19/28), 5 (4/28) and 7 (1/28). In addition, EWS/FLI-1 exon combinations 10/5 and 9/4 were observed in one case each. In the last tumour, the presence of at least four additional splicing variants corresponding to fusion of EWS exon 7 to FLI-1 exons 4, 6, 8 and 9 was demonstrated. Two tumours expressed EWS/ERG fusion transcripts involving EWS exon 7 and ERG exon 6. In this study, EWS/FLI-1 exon combinations 7/6 (type I) predominated over 7/5 (type II) in localised ET (14 versus 1) and were more abundant in tumours affecting the long bones (9 versus 0), whereas in central axis tumours and metastatic disease there was only little difference in the frequency of the two types. So far, no correlations between different chimaeric EWS transcripts and any other clinical parameters have been identified.
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PMID:Variability of EWS chimaeric transcripts in Ewing tumours: a comparison of clinical and molecular data. 752 4

The ada gene of Escherichia coli K-12 encodes the 39-kDa Ada protein, which consists of two domains joined by a hinge region that is sensitive to proteolytic cleavage in vitro. The amino-terminal domain has a DNA methyltransferase activity that repairs the S-diastereoisomer of methylphosphotriesters while the carboxyl-terminal domain has a DNA methyltransferase activity that repairs O6-methylguanine and O4-methylthymine lesions. Transfer of a methyl group to Cys-69 by repair of a methylphosphotriester lesion converts Ada into a transcriptional activator of the ada and alkA genes. Activation of ada, but not alkA, requires elements contained within the carboxyl-terminal domain of Ada. In addition, physiologically relevant concentrations of the unmethylated form of Ada specifically inhibit methylated Ada-promoted ada transcription both in vitro and in vivo and it has been suggested that this phenomenon plays a pivotal role in the down-regulation of the adaptive response. A set of site-directed mutations were generated within the hinge region, changing the lysine residue at position 178 to leucine, valine, glycine, tyrosine, arginine, cysteine, proline, and serine. All eight mutant proteins have deficiencies in their ability to activate ada transcription in the presence or absence of a methylating agent but are proficient in alkA activation. AdaK178P (lysine 178 changed to proline) is completely defective for the transcriptional activation function of ada while it is completely proficient for transcriptional activation of alkA. In addition, AdaK178P possesses both classes of DNA repair activities both in vitro and in vivo. Transcriptional activation of ada does not occur if both the amino- and carboxyl-terminal domains are produced separately within the same cell. The mutation at position 178 might interfere with activation of ada transcription by changing a critical contact with RNA polymerase, by causing a conformational change of Ada, or by interfering with the communication of conformational information between the amino- and the carboxyl-terminal domains. These results indicate that the hinge region of Ada is important for ada but not alkA transcription and further support the notion that the mechanism(s) by which Ada activates ada transcription differs from that by which it activates transcription at alkA.
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PMID:Alteration of lysine 178 in the hinge region of the Escherichia coli ada protein interferes with activation of ada, but not alkA, transcription. 786 1

An expression system for alpha 1-antitrypsin in Escherichia coli was developed using a T7 RNA polymerase promoter. Addition of rifampicin to inhibit the E. coli RNA polymerase after induction of the T7 RNA polymerase gene resulted in about 30% of newly synthesized protein being alpha 1-antitrypsin. This expression system was then used to examine the effect of mutations in the hinge region of alpha 1-antitrypsin on its activity. The mutations were based on ones in antithrombin III that had previously been shown to have adverse effects on activity. Mutation of Ala347 to threonine in alpha 1-antitrypsin did not affect the kinetic behavior of the protein with trypsin or human leukocyte elastase. In contrast, mutation of Gly349 to proline converted the majority of the protein into a substrate for both proteinases. The small fraction of this mutant that was active, however, had kinetic parameters that were indistinguishable from wild-type alpha 1-antitrypsin. Cleavage within the reactive-site loop of wild-type alpha 1-antitrypsin causes a conformational change in the molecules (the S-to-R transition) and results in a marked increase in heat stability. This increase in heat stability was also seen upon cleavage within the reactive-site loops of both of the alpha 1-antitrypsin mutants. The results are discussed in terms of a kinetic mechanism for serpin-proteinase interactions, in which after the formation of an initial complex the serpin partitions between the formation of a stable complex and a cleavage reaction.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effects of mutations in the hinge region of serpins. 834 75

Site-specific post-transcriptional conversion of uridines to pseudouridine in ribosomal RNAs and small nuclear RNAs (snRNAs) is directed by guide RNAs which possess the conserved box H and ACA sequence elements and fold into the consensus 'hairpin-hinge-hairpin-tail' secondary structure. Here, we describe an unusual mammalian pseudouridylation guide RNA, called U93, that is composed of two tandemly arranged box H/ACA RNA domains. The U93 RNA therefore carries two H and two ACA box motifs, all of which are essential for accumulation of the full-length RNA. The human U93 RNA accumulates in Cajal (coiled) bodies and it is predicted to function in pseudouridylation of the U2 spliceosomal snRNA. Our results lend further support to the notion that modification of the RNA polymerase II-transcribed spliceosomal snRNAs takes place in Cajal bodies.
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PMID:A Cajal body-specific pseudouridylation guide RNA is composed of two box H/ACA snoRNA-like domains. 1240 54

We report the crystal structure of N-utilizing substance A protein (NusA) from Thermotoga maritima (TmNusA), a protein involved in transcriptional pausing, termination, and antitermination. TmNusA has an elongated rod-shaped structure consisting of an N-terminal domain (NTD, residues 1-132) and three RNA binding domains (RBD). The NTD consists of two subdomains, the globular head and the helical body domains, that comprise a unique three-dimensional structure that may be important for interacting with RNA polymerase. The globular head domain possesses a high content of negatively charged residues that may interact with the positively charged flaplike domain of RNA polymerase. The helical body domain is composed of a three-helix bundle that forms a hydrophobic core with the aid of two neighboring beta-strands. This domain shows structural similarity with one of the helical domains of sigma(70) factor from Escherichia coli. One side of the molecular surface shows positive electrostatic potential suitable for nonspecific RNA interaction. The RBD is composed of one S1 domain and two K-homology (KH) domains forming an elongated RNA binding surface. Structural comparison between TmNusA and Mycobacterium tuberculosis NusA reveals a possible hinge motion between NTD and RBD. In addition, a functional implication of the NTD in its interaction with RNA polymerase is discussed.
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PMID:Crystal structure of NusA from Thermotoga maritima and functional implication of the N-terminal domain. 1462 88

Sinorhizobium meliloti DctD is an activator of sigma(54)-RNA polymerase holoenzyme and member of the AAA+ superfamily of ATPases. DctD uses energy released from ATP hydrolysis to stimulate the isomerization of a closed promoter complex to an open complex. DctD binds to upstream activation sequences (UAS) and contacts the closed complex through DNA looping to activate transcription, but the UAS is not essential for activation if DctD is expressed at higher than normal levels. Introduction of specific substitutions within or near the conserved ESELFG motif in the C3 region of a truncated, constitutively active form of DctD produced several mutant forms of the protein that had increased dependence on the UAS for activation. Removing the DNA-binding domain from one UAS-dependent mutant and from one activation-deficient mutant significantly increased transcriptional activation, indicating that the DNA-binding domain interfered with the activities of these mutant proteins. A UAS-dependent mutant with a P315L substitution in the C6 region was identified from a genetic screen. Alanine scanning mutagenesis of conserved amino acid residues around Pro-315 produced two additional UAS-dependent mutants as well as several mutants that failed to activate transcription but retained ATPase activity. In contrast to the two mutant proteins with substitutions in the C3 region, removal of the DNA-binding domain from the mutant proteins with substitutions in the C6 region did not stimulate their activity. The residues in the C6 region that were altered are in a probable hinge region between the alpha/beta and alpha-helical subdomains of the AAA+ domain. The alpha-helical subdomain contains the sensor II helix that has been implicated in other AAA+ proteins as sensing changes in the nucleotide during the hydrolysis cycle. Substitutions in the hinge region may have abolished nucleotide sensing by interfering with subdomain interactions, altering the relative orientation of the sensor II helix or interfering with oligomerization of the protein.
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PMID:Novel substitutions in the sigma54-dependent activator DctD that increase dependence on upstream activation sequences or uncouple ATP hydrolysis from transcriptional activation. 1545 3

The Mediator of transcriptional regulation is the central coactivator that enables a response of RNA polymerase II (Pol II) to activators and repressors. We present the 3.0-A crystal structure of a highly conserved part of the Mediator, the MED7.MED21 (Med7.Srb7) heterodimer. The structure is very extended, spanning one-third of the Mediator length and almost the diameter of Pol II. It shows a four-helix bundle domain and a coiled-coil protrusion connected by a flexible hinge. Four putative protein binding sites on the surface allow for assembly of the Mediator middle module and for binding of the conserved subunit MED6, which is shown to bridge to the Mediator head module. A flexible MED6 bridge and the MED7.MED21 hinge could account for changes in overall Mediator structure upon binding to Pol II or activators. Our results support the idea that transcription regulation involves conformational changes within the general machinery.
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PMID:A conserved mediator hinge revealed in the structure of the MED7.MED21 (Med7.Srb7) heterodimer. 1571 Jun 19


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