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)

Rabbit globin complementary DNA made with RNA-dependent DNA polymerase (reverse transcriptase) was used as a template for in vitro synthesis of 32P-labeled RNA and deoxysubstituted RNA. The sequences of the nucleotides in most of the fragments resulting from combined ribonuclease T1 and alkaline phosphatase digestion have been determined. In addition, the 3' nearest neighbor was determined for several fragments resulting from digestion with T1 ribonuclease. The utility of the deoxysubstitution technique was demonstrated by the ease with which the sequences of pyrimidine-rich fragments could be determined. Many sequences thus determined were long enough to fit uniquely with the alpha- or beta-globin amino acid sequences. The positions of these fits were found to be clustered, leading us to believe that only certain regions of the complementary DNA are transcribed by Escherichia coli RNA polymerase. Other unique characteristics of RNA synthesis from a complementary DNA template include a high yield of free poly(A) and the fact that one must use low rather than high salt buffers to obtain transcripts of high molecular weight.
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PMID:Rabbit globin mRNA: analysis of T1 RNAse digestion fragments. 6 35

A transcriptional initiator (Inr) for mammalian RNA polymerase II can be defined as a DNA sequence element that overlaps a transcription start site and is sufficient for (i) determining the start site location in a promoter that lacks a TATA box and (ii) enhancing the strength of a promoter that contains a TATA box. We have prepared synthetic promoters containing random nucleotides downstream of Sp1 binding sites to determine the range of DNA sequences that convey Inr activity. Numerous sequences behaved as functional Inrs in an in vitro transcription assay, but the Inr activities varied dramatically. An examination of the functional elements revealed loose but consistent sequence requirements, with the approximate consensus sequence Py Py A+1 N T/A Py Py. Most importantly, almost every functional Inr that has been described fits into the consensus sequence that we have defined. Although several proteins have been reported to bind to specific Inrs, manipulation of those elements failed to correlate protein binding with Inr activity. The simplest model to explain these results is that all or most Inrs are recognized by a universal binding protein, similar to the functional recognition of all TATA sequences by the same TATA-binding protein. The previously reported proteins that bind near specific Inr elements may augment the strength of an Inr or may impart transcriptional regulation through an Inr.
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PMID:DNA sequence requirements for transcriptional initiator activity in mammalian cells. 826 80

Asp537 and Asp812 are essential in the catalytic mechanism of T7 RNA polymerase. The mutants D537N and D812N have no detectable activity whereas the mutants D537E and D812E have significantly reduced activity relative to the wild-type. The hypothesis that these two amino acids act as metal-binding ligands has been tested using EPR with Mn2+ as the metal ion. Mn2+ is able to substitute for Mg2+ in transcription by T7 RNAP on templates containing the T7 promoter. Mg2+ and Mn2+ compete for binding sites, with the former having lower affinity. Mn2+ binding to the wild-type enzyme and the mutants D537N, D812N, D537E, D812E, and Y649F was measured over the concentration range of 25 microM to 1.5 mM. The data were analyzed by nonlinear least-squares fits to the binding isotherms, and the analysis gave approximately two Mn(2+)-binding sites in all cases and a Kd for the wild-type of approximately 340 microM. The Kd value for the mutant Y639F, in which Asp537 and Asp 812 are not mutated, is comparable to the value for the wild-type. Mn2+ binding to the double mutants, D537N/D812N and D537E/D812E, appears to be nonspecific. The Kd values of the Asp-->Asn mutants are only 2-5 times larger than the value for the wild-type, in contrast to the drastic diminution of enzymatic activity in the mutants. The geometry of metal binding to these Asp residues may be crucial in determining the catalytic competence. Mn2+ binding to the wild-type enzyme in the presence of nucleotides, measured by flow dialysis, is characterized by two Mn(2+)-binding sites with a Kd value of ca. 150 microM. The similarity in values of Kd with and without nucleotide suggests that nucleotides do not have a drastic effect on Mn2+ binding. Our results indicate that monodentate carboxylate oxygens of both conserved Asp residues bridge the two metal ions.
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PMID:Asp537 and Asp812 in bacteriophage T7 RNA polymerase as metal ion-binding sites studied by EPR, flow-dialysis, and transcription. 855 68

A 13 kDa protein from bovine lens was identified and characterized by protein microsequencing and by rapid amplification of cDNA ends (RACE) PCR. Its complete sequence shows that this protein belongs to a family of fatty acid-binding proteins (FABPs), including myelin and adipocyte P2, that are associated with cellular differentiation. The bovine lens protein, designated LP2, shows very close similarity to human epidermal FABP (eFABP) and human eFABP was detected in human lens, suggesting that the two proteins might be orthologous. Reverse transcriptase-PCR (RT-PCR) was used to compare expression patterns of LP2 with those for actin and for the differentiation markers gamma B-crystallin and gamma s-crystallin in lens. Actin was most abundant in the relatively undifferentiated epithelial cells and decreased with lens cell differentiation. In contrast gamma B-crystallin and gamma s-crystallin were detected only in fibres (nuclear and cortical respectively). LP2 transcripts were detected most abundantly in fibre cells and apparently increased with cellular differentiation. Molecular modelling confirms that the sequence of LP2 fits the tertiary template of adipocyte P2 but reveals the presence of two close pairs of cysteine residues that might be susceptible to intramolecular disulphide bond formation under appropriate oxidizing conditions. LP2 is thus another potential target for oxidative stress during cataract formation in lens.
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PMID:LP2, a differentiation-associated lipid-binding protein expressed in bovine lens. 894 66

Bacillus popilliae is an obligate pathogen for larvae of the insect family Scarabaeidae (Coleoptera). It forms parasporal crystals upon sporulation. The gene cry18Aa coding for the parasporal crystal protein and an upstream open reading frame, orf1, were previously isolated from B.popilliae. Here we report an analysis of cry18Aa transcription in Bacillus thuringiensis. The only transcriptional start site of cry18Aa was found 29 bp upstream of the open reading frame orf1, suggesting that orf1 and cry18Aa are transcribed as an operon. lacZ fusion to the cry18Aa promoter was used to follow the time-course of cry18Aa transcription in wild type B.thuringiensis and in various B.thuringiensis sporulation-deficient mutants (spo0A, sigE or sigK). In wild type B.thuringiensis, the cry18Aa promoter was activated 2 h after the end of exponential growth and the expression lasted to the late sporulation phase. The results of promoter activity in Spo+or Spo-backgrounds together with the results of primer extension experiments suggest that the transcription from this promoter can be driven by both sigmaE and sigmaK types of RNA polymerase at a single start site. The promoter region of cry18Aa operon fits the consensus sequences of both sigmaE and sigmaK dependent promoters of Bacillus.
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PMID:Bacillus popilliae cry18Aa operon is transcribed by sigmaE and sigmaK forms of RNA polymerase from a single initiation site. 946 39

The double strand binding protein A (DsbA) of bacteriophage T4 is one of several viral gene products participating in transcriptional regulation. These proteins interact or associate with the host RNA polymerase core enzyme, enabling the enzyme to successively initiate transcription at different classes of viral promoters: early, middle and late. This leads to a temporally controlled expression of the T4 gene products. The DsbA binding site overlaps the late promoter region, and DsbA binding seems to intensify transcription of late genes in vitro, possibly acting as an enhancer protein (Molecular Biology of Phage T4, Karam, 1994). To further investigate the function and structure of DsbA, we overexpressed the protein in E. coli and purified it to homogeneity. Physiological functionality of the recombinant protein was shown by gel retardation experiments and by circular dichroism (CD) spectroscopy. DsbA shows strong bands in the near UV-CD spectra. The far UV-CD spectroscopy analysis shows alpha-helices to be the main secondary structure elements. This is in agreement with secondary structure predictions. A possible helix-turn-helix motif in the center of the protein could be identified. Results from crosslinking and sedimentation analyses show that DsbA forms a dimer in solution. The thermal unfolding curve fits a dimer-two-state-folding-model, and the unfolding temperature was concentration dependent. Therefore, dimerization should supply the main portion of the free energy of stabilization of deltaG0 = 42 kJ/mol.
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PMID:Overexpression and structural characterization of the phage T4 protein DsbA. 950 17

A bacterially expressed single chain antibody (scFv215) directed against the largest subunit of drosophila RNA polymerase II was analysed. Structure and function of the antigen binding site in scFv215 were probed by chain shuffling and by site-specific mutagenesis. The entire variable region of either the heavy or light chain was replaced by an unrelated heavy or light chain. Both replacements resulted in a total loss of binding activity suggesting that the antigen binding site is contributed by both chains. The functional contributions of each complementarity determining region (CDR) were investigated by site specific mutagenesis of each CDR separately. Mutations in two of the CDRs, CDR1 of light chain and CDR2 of heavy chain, reduced the binding activity significantly. Each of the amino acids in these two CDRs was replaced individually by alanine (alanine walking). Seven amino acid substitutions in the two CDRs were found to reduce the binding activity by more than 50%. The data support a computer model of scFv215 which fits an epitope model based on a mutational analysis of the epitope suggesting an alpha-helical structure for the main contact area.
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PMID:Fine mapping of the antigen-antibody interaction of scFv215, a recombinant antibody inhibiting RNA polymerase II from Drosophila melanogaster. 1039 1

Promoter sequences of Escherichia coli were compiled and their transcribed regions characterized by site-specific cluster analysis. Here we report that transcribed regions contain a non-random distribution of A/T tracts with strongly preferred positions at 6 +/- 3, 23 +/- 3, 40 +/- 2 and 56 +/- 2. The maxima of this distribution follow an unusual periodicity (approximately 17 bp) and are in phase with important promoter elements involved in interaction with RNA polymerase, while the value of periodicity numerically fits the spacer length between the canonical -35 and -10 elements. The possible functional significance of this newly described feature is discussed in the context of promoter clearance and transcription pausing.
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PMID:Proximal transcribed regions of bacterial promoters have a non-random distribution of A/T tracts. 1057 77

The potent anticancer drug actinomycin D (ActD) acts by binding to DNA, thereby interfering with replication and transcription. ActD inhibits RNA polymerase far more specifically than DNA polymerase. Such discrimination is not easily understood by the conventional DNA binding mode of ActD. We have solved and refined at 1.7 A resolution the crystal structure of ActD complexed to CGATCGATCG, which contains no canonical GpC binding sequence. The crystal data are space group P4(3)2(1)2, a = b = 47.01 A, and c = 160.37 A. The structure was solved by the multiple wavelength anomalous diffraction method using a 5-bromo-U DNA. The asymmetric unit of the unit cell contains two independent dimers of a novel slipped duplex complex consisting of two decamer DNA strands bound with two ActD drug molecules. (The DNA in one dimer is numbered C1 to G10 in one strand and C11 to G20 in the complementary strand and in the second dimer, C101 to G110 and C111 to G120, respectively.) The structure reveals a highly unusual ActD binding mode in which the DNA adopts a slipped duplex with the A3-T4/A13-T14 dinucleotides looped out. ActD intercalates between G2-C11* (C11* being from a symmetry-related molecule) and C5-G20 base pairs. Two such slipped duplex-ActD complexes bound to each other by mutually intercalating their T4/T14 bases into the helix cavities (located between C5-G20 and G6-C19 base pairs) of neighboring complexes, forming a dimer of drug-DNA complexes. The binding site mimics the drug binding at the elongation point during transcription. Modeling studies show that the ActD-DNA complex fits snugly in the active site cavity in RNA polymerase but not in DNA polymerase. This may explain the strong preference of ActD inhibition toward transcription.
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PMID:Crystallographic analysis of a novel complex of actinomycin D bound to the DNA decamer CGATCGATCG. 1134 23

Elongin is a transcription elongation factor that stimulates the rate of elongation by suppressing transient pausing by RNA polymerase II at many sites along the DNA. It is heterotrimeric in mammals, consisting of elongins A, B and C subunits, and bears overall similarity to a class of E3 ubiquitin ligases known as SCF (Skp1-Cdc53 (cullin)-F-box) complexes. A subcomplex of elongins B and C is a target for negative regulation by the von Hippel-Lindau (VHL) tumor-suppressor protein. Elongin C from Saccharomyces cerevisiae, Elc1, exhibits high sequence similarity to mammalian elongin C. Using NMR spectroscopy we have determined the three-dimensional structure of Elc1 in complex with a human VHL peptide, VHL(157-171), representing the major Elc1 binding site. The bound VHL peptide is entirely helical. Elc1 utilizes two C-terminal helices and an intervening loop to form a binding groove that fits VHL(157-171). Chemical shift perturbation and dynamics analyses reveal that a global conformational change accompanies Elc1/VHL(157-171) complex formation. Moreover, the disappearance of conformational exchange phenomena on the microsecond to millisecond time scale within Elc1 upon VHL peptide binding suggests a role for slow internal motions in ligand recognition.
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PMID:Solution structure and dynamics of yeast elongin C in complex with a von Hippel-Lindau peptide. 1154 95


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