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

---

Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A cDNA clone encoding the 3CD proteinase (3CDpro) of poliovirus type 2 (Sabin), the precursor to proteinase 3Cpro and RNA polymerase 3Dpol, was expressed in bacteria by using a T7 expression system. Site-specific mutagenesis of the 3C/3D cleavage site was performed to generate active proteolytic precursors impaired in their ability to process themselves to 3Cpro and 3Dpol. Of these mutations, the exchange of the Thr residue at the P4 position of the 3C/3D cleavage site for a Lys residue (3CDpro T181K) resulted in a mutant polypeptide exhibiting the smallest amount of autoprocessing. This mutant was purified to 86% homogeneity and used for subsequent proteolytic studies. Purified 3CDproM (M designates the cleavage site mutant 3CDpro T181K) was capable of cleaving the P1 capsid precursor, a peptide representing the 2BC cleavage site, and the 2BC precursor polypeptide. Purified 3CDproM demonstrated the same detergent sensitivity in processing experiments with the capsid precursor as was observed by using P1 and crude extracts of poliovirus-infected HeLa cell lysates. Purified 3CDproM did not have any detectable RNA polymerase activity, whereas 3Dpol, separated from 3CDproM by gel filtration in the last step of purification, did. We conclude that 3CDproM can process both structural and nonstructural precursors of the poliovirus polyprotein and that it is active against a synthetic peptide substrate. Moreover, cleavage of 3CD to 3Dpol is needed to activate the 3D RNA polymerase.
...
PMID:Purification and characterization of poliovirus polypeptide 3CD, a proteinase and a precursor for RNA polymerase. 133 32

A molecular cDNA clone (P1 KIN) was isolated that encodes the human RNA-dependent P1/eIF-2 alpha protein kinase. The complete cDNA sequence of the P1 KIN cDNA was determined; the longest open reading frame (ORF) encoded a 551 amino acid protein with a deduced molecular weight of 62055 Da. Transcripts prepared from the P1 KIN cDNA by transcription in vitro with T7 RNA polymerase programmed the cell-free synthesis of a protein indistinguishable by immunoprecipitation and immunoblot gel analyses from the authentic 67-kDa P1 protein synthesized in human U cells treated with interferon (IFN). Furthermore, by use of a sensitive primer extension assay with T7 DNA polymerase, the major site of translation initiation within the deduced ORF of the P1 KIN cDNA was directly identified. Northern RNA gel-blot analysis revealed that the P1 KIN cDNA strongly hybridized to two IFN-induced mRNAs present in both human amnion U cells and HeLa cells; their sizes were 2.5 and 6 kb. Both transcripts were efficiently induced by IFN-alpha, but poorly by IFN-gamma. Polyclonal antibody was prepared against the product of the P1 KIN cDNA expressed in Escherichia coli. In Western blot analysis the antibody recognized a 67-kDa protein induced in human cells by IFN-alpha and, in addition, a 90-kDa protein whose level was not greatly altered by IFN treatment. The IFN-induced 67-kDa protein was found associated with the ribosomal salt-wash fraction of IFN-treated human cells, whereas the 90-kDa protein was predominantly in the S100 soluble fraction. The time course for the induction by IFN-alpha of RNA-dependent protein P1 kinase activity measured by immunoprecipitation was comparable to the time course for protein P1 induction measured by Western immunoblot analysis. The amino acid sequence of P1/eIF-2 alpha protein kinase deduced from the cDNA was 62% identical with the 518-residue murine TIK kinase and contained, within the carboxy-terminal half of the protein, the motifs commonly conserved among protein-serine/threonine kinases. The amino-terminal half of the P1 protein did not possess conserved kinase motifs, but did show extensive homology with vaccinia virus-predicted protein E3L.
...
PMID:Mechanism of interferon action: cDNA structure, expression, and regulation of the interferon-induced, RNA-dependent P1/eIF-2 alpha protein kinase from human cells. 137 53

The high affinity branched-chain amino acid transport system (LIV-I) in Pseudomonas aeruginosa is composed of five components: BraC, a periplasmic binding protein for branched-chain amino acids; BraD and BraE, integral membrane proteins; BraF and BraG, putative nucleotide-binding proteins. By using a T7 RNA polymerase/promoter system we overproduced the BraD, BraE, BraF, and BraG proteins in Escherichia coli. The proteins were found to form a complex in the E. coli membrane and solubilized from the membrane with octyl glucoside. The LIV-I transport system was reconstituted into proteoliposomes from solubilized proteins by a detergent dilution procedure. In this reconstituted system, leucine transport was completely dependent on the presence of all five Bra components and on ATP loaded internally to the proteoliposomes. Alanine and threonine in addition to branched-chain amino acids were transported by the proteoliposomes, reflecting the substrate specificity of the BraC protein. GTP replaced ATP well as an energy source, and CTP and UTP also replaced ATP partially. Consumption of loaded ATP and concomitant production of orthophosphate were observed only when BraC and leucine, a substrate for LIV-I, were added together to the proteoliposomes, indicating that the LIV-I transport system has an ATPase activity coupled to translocation of branched-chain amino acids across the membrane.
...
PMID:Solubilization and reconstitution of the Pseudomonas aeruginosa high affinity branched-chain amino acid transport system. 140 Apr 43

We scrutinized the possible existence of human prepro-THR messenger RNA (mRNA) and of its posttranslational processing products in the human placenta. Human placental mRNA of preproTRH was found to have a single species identical to that predicted from the hypothalamic mRNA, and could be reverse transcribed to complementary DNA (cDNA) encoding preproTRH in a size similar to the hypothalamic counterpart by means of reverse-transcriptase-polymerase chain reaction. Five different intervening peptides, designated human TRH-associated peptide (hTAP) [hTAP-1, preproTRH(90-111); hTAP-2, preproTRH(120-132); hTAP-3, preproTRH(141-149); hTAP-4, preproTRH(158-183); hTAP-5, preproTRH(192-224)], and a TRH precursor comprising the TRH progenitor sequence (octa-TRH) were synthesized, and six different antisera raised against individual peptides were used to develop specific RIA systems. Significant concentrations of hTAP-5 and octa-TRH-like immunoreactivities were quantitated in acid extracts of human placentae. In human hypothalamic extracts, immunoreactivities of hTAP-3, hTAP-4, hTAP-5, and octa-TRH, were apparently detected. Chromotographic analysis showed a single peak corresponding to each authentic peptide in RIA systems of hTAPs detected. In placentae, a single peak of octa-TRH-like substance was observed, and two octa-TRH-like substances with different molecular weights detected in hypothalami. The present data indicate that unique posttranslational processing steps of human preproTRH differ in human placentae and hypothalami, and that the human tissues involve profound amounts of several preproTRH-related peptides which do not comprise the TRH progenitor sequence.
...
PMID:Different posttranslational processing of human preprothyrotropin-releasing hormone in the human placenta and hypothalamus. 146 61

cDNAs encoding the largest subunit of RNA polymerase II were isolated from a Dictyostelium cDNA library. A total of 2.9 kilobases (kb) of cDNA was sequenced and the amino acid sequence of the carboxyl-terminal half of the protein was deduced. Similar to other eukaryotic RNA polymerases II, the largest subunit of Dictyostelium RNA polymerase II contains a unique repetitive tail domain at its carboxyl-terminal region. It consists of 24 highly conserved heptapeptide repeats, with a consensus sequence of Tyr-Ser-Pro-Thr-Ser-Pro-Ser. In addition to the tail domain, five segments of the deduced primary structure show > 50% sequence identity with either yeast or mouse protein. RNA blots show that cDNA probes hybridized with a single mRNA species of approximately 6 kb and immunoblots using a monoclonal antibody raised against the tail domain lighted up a single protein band of 200 kilodaltons. Interestingly, expression of the largest subunit of RNA polymerase II appears to be under developmental regulation. The accumulation of its mRNA showed a 60% increase during the first 3 h of development, followed by a steady decrease during the next 6 h. Cells began to accumulate a higher level of the RNA polymerase II mRNA after 9 h of development. When cells were treated with low concentrations of cAMP pulses to stimulate the developmental process, the pattern of mRNA accumulation moved 3 h ahead, but otherwise remained similar to that of control cells.
...
PMID:The largest subunit of RNA polymerase II in Dictyostelium: conservation of the unique tail domain and gene expression. 148 55

rho-Independent transcription terminators in Escherichia coli contain a dG+dC-rich dyad-symmetrical structure that encodes an RNA hairpin structure and an adjacent, downstream dA+dT-rich region which encodes uridines at the 3'-end of the transcript. In the threonine (thr) attenuator, there are at least six sequence segments in the DNA that might affect termination: the sequence upstream of the attenuator, the deoxythymidine-rich stretch immediately preceding the G+C-rich region, the G+C-rich region itself and its hairpin loop-encoding region, the deoxyadenosine tract following the G+C-rich region, and the following downstream sequence. Our previous studies (Jeng, S.-T., Gardner, J.F., and Gumport, R.I. (1990) J. Biol. Chem. 265, 3823-3830) indicate that both the stability and sequence of the RNA hairpin formed by the G+C-rich region and the length of the uridine tract encoded by the deoxyadenosine stretch influence the termination of T7 RNA polymerase in vitro. In this report, we demonstrate that the template deoxythymidine run upstream of the G+C-rich region, the loop-encoding segment, and the sequences upstream and downstream of the thr attenuator also affect termination. These results indicate that: 1) a deoxythymidine tract is not absolutely required for termination, but increasing the number of deoxythymidines from one to nine base pairs causes T7 RNA polymerase to terminate more efficiently; 2) a template with the natural loop sequence reversed results in a higher termination efficiency than one encoded by the the wild-type attenuator; 3) the termination of T7 RNA polymerase is affected by sequences both proximal and distal to the thr attenuator.
...
PMID:Transcription termination in vitro by bacteriophage T7 RNA polymerase. The role of sequence elements within and surrounding a rho-independent transcription terminator. 152 50

To investigate the identity determinants of E. coli threonine tRNA, various transcripts were prepared by in vitro transcription system with T7 RNA polymerase. Substitutions of the anticodon second letter G35 and the third letter U36 to other nucleotides led to a remarkable decrease of threonine charging activity. Charging experiments with a series of anticodon-deletion transcripts also suggest the importance of the G35U36 sequence. A mutation at either the G1-C72 or C2-G71 base pair in the acceptor stem seriously affected the threonine charging activity. These results indicate that the second and third positions of the anticodon and the first and second base pairs in the acceptor stem are the recognition sites of E. coli tRNA(THR) for threonyl-tRNA synthetase. Discriminator base, A73, is not involved in threonine charging activity.
...
PMID:Identity determinants of E. coli threonine tRNA. 156 50

CRP is resistant to attack by carboxypeptidase Y at 37 degrees C, whereas cAMP-CRP is digested yielding a core terminating at Thr-202 and lacking the seven carboxyl-terminal amino acid residues. A similar core (CRPCY) is formed when CRP is incubated with carboxypeptidase Y at 47 degrees C in the absence of cAMP. CRPCY has a more open conformation than CRP at 37 degrees C. While unliganded CRP is resistant to trypsin, CRPCY is sensitive to tryptic attack. Dithionitrobenzoic acid-mediated intersubunit disulfide crosslinking of CRP requires cAMP, CRPCY subunits are crosslinked in the absence of cAMP. The carboxyl-terminal region of unliganded CRP is conformationally restricted at 37 degrees C. The CRPCY retains cAMP binding activity. The CRPCY which terminates at Thr-202, no longer binds lac P+ DNA nor stimulates abortive initiation by RNA polymerase from the lac P+ promoter. The results indicate that the C-terminal region of CRP participates in the conformational stability of the closed form of CRP and indirectly in DNA binding by the open cAMP-CRP conformer.
...
PMID:Characterization of the CRPCY core formed after treatment with carboxypeptidase Y. 165 82

In this study we describe the activation of a protein kinase which phosphorylates a peptide, T669, comprising amino acids 663-681 of the epidermal growth factor receptor and containing the phosphate acceptor site Pro-Leu-Thr669-Pro. In the human epidermoid carcinoma cell line KB, T669 kinase activity in cytosolic extracts peaked (up to 15-fold compared with basal levels) 15-30 min after addition of interleukin-1 (IL-1) and closely paralleled receptor occupancy with a half-maximally effective concentration of approximately 100 pM IL-1 alpha. IL-1 treatment elevated T669 kinase activity to a variable extent in selected fibroblast lines, the hepatoma cell line HepG2, and the murine thymoma EL4 6.1. An IL-1 receptor-negative EL4 variant and the B cell lines 70Z/3, CB23, and RPMI 1788 did not respond in this way. All of the cell lines except 70Z/3 showed increased levels of T669 kinase when treated with the protein kinase C activator phorbol myristate acetate and/or with epidermal growth factor. This finding is in agreement with a previous study (Countaway, J. L., Northwood, I. C., and Davis, R. J. (1989) J. Biol. Chem. 264, 10828-10835). Activators of protein kinase A did not mimic the ability of IL-1 to stimulate T669 kinase activity, nor did the protein kinase C inhibitor staurosporine abrogate the effect of IL-1. T669 kinase activity from IL-1-stimulated KB cells was partially purified by ion exchange, hydrophobic interaction, and size exclusion chromatography. The partially purified enzyme phosphorylated myelin basic protein, a characteristic substrate of microtubule-associated protein-2 kinase (MAP-2 kinase) and the peptide Arg-Arg-Arg-(Tyr-Ser-Pro-Thr-Ser-Pro-Ser)4 from RNA polymerase II. Western blotting of chromatographic fractions revealed that T669 kinase activity corresponded with two proteins of 43 and 45 kilodaltons which cross-reacted with antibodies raised against peptide sequences of rat extracellular signal-regulated kinase-1/microtubule-associated protein-2 kinase. T669 kinase activity was critically dependent on the presence of phosphatase inhibitors. Since both the 43- and 45-kDa proteins, immunoprecipitated from [32P]phosphate-labeled cells, demonstrated a dramatic increase in their levels of serine, threonine, and tyrosine phosphorylation after brief treatment with IL-1, we conclude that IL-1 modulates the activity of these extracellular signal-regulated kinase/microtubule-associated protein-2 kinases by altering the level of their phosphorylation.
...
PMID:Interleukin-1 represents a new modality for the activation of extracellular signal-regulated kinases/microtubule-associated protein-2 kinases. 165 5

Structural resemblance of the human Alu family with a subset of vertebrate tRNAs was detected. Of four tRNAs, tRNA(Lys), tRNA(Ile), tRNA(Thr), and tRNA(Tyr), which comprise a structurally related family, tRNA(Lys) is the most similar to the human Alu family. Of the 76 nucleotides in lysine tRNA (including the CCA tail), 47 are similar to the human Alu family (60% identity). The secondary structure of the human Alu family corresponding to the D-stem and anticodon stem regions of the tRNA appears to be very stable. The 7SL RNA, which is a progenitor of the human Alu family, is less similar to lysine tRNA (55% identity), and the secondary structure of the 7SL RNA folded like a tRNA is less stable than that of the human Alu family folded likewise. Insertion of the tetranucleotide GAGA, which is an important region of the second promoter for RNA polymerase III in the Alu sequence, occurred during the deletion and ligation process to generate the Alu sequence from the parental 7SL RNA. These results suggest that the human Alu family was generated from the 7SL RNA by deletion, insertion, and mutations, which thus modified the ancestral 7SL sequence so that it could form a structure more closely resembling lysine tRNA. The similarities of several short interspersed sequences to the lysine tRNA were also examined. The Galago type 2 family, which was reported to be derived from a methionine initiator tRNA, was also found to be similar to the lysine tRNA. Thus lysine tRNA-like structures are widespread in genomes in the animal kingdom. The implications of these findings in relation to the mechanism of generation of the human Alu family and its possible functions are discussed.
...
PMID:Transfer RNA-like structure of the human Alu family: implications of its generation mechanism and possible functions. 170 38


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---