Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Alt gene product is a component of the T4 phage head. Upon infection of the host cell, approximately 40 copies of the Alt protein enter the cell together with the viral DNA molecule. The Alt protein then ADP-ribosylates one of the two alpha-subunits of host RNA polymerase. A restriction fragment harboring the ADP-ribosyltransferase gene of bacteriophage T4 was cloned into the plasmid vector pBluescript, the nucleotide sequence was determined, and the reading frame was identified. Two M13 clone libraries, established with DNA isolated from bacteriophages T2 and T6, then were screened for the corresponding genes. The nucleotide sequences of the three alt genes and the deduced amino acid sequences were compared. Secondary structure predictions and NAD-binding studies resulted in the location of the substrate-binding site in the NH2-terminal regions of the enzymes.
 ...
PMID:The ADP-ribosyltransferases (gpAlt) of bacteriophages T2, T4, and T6: sequencing of the genes and comparison of their products. 805 53

We have isolated an ADP-ribosylation factor (ARF) gene from the human malarial parasite, Plasmodium falciparum. The gene (P. falciparum arf1) has four introns and the exons encode a protein of 181 amino acids with high similarity to the mammalian class I ARF proteins 1-3 (> or = 74% amino acid identity). Southern hybridization suggests there is at least one additional arf in the P. falciparum genome. Northern analysis identified a single P. falciparum arf1 mRNA of 1.8 kb in the asexual blood stage form of the parasite. The P. falciparum arf1 mRNA levels are developmentally regulated, reaching a maximum during nuclear division towards the end of the intraerythrocytic cycle. P. falciparum arf1 cDNA was isolated by reverse-transcriptase polymerase chain reaction and used to express a recombinant protein in Escherichia coli. Recombinant P. falciparum ARF1 protein was purified with stoichiometric amounts of bound GDP, although intrinsic guanose triphosphatase activity of the protein could not be detected. The protein stimulated cholera-toxin-catalyzed ADP-ribosyltransferase activity in a reaction that was dependent upon the addition of both dimyristoylglycerophosphocholine and cholate. The protein bound GTP with first-order kinetics with an apparent rate constant, k', of 0.0145 (+/- 0.0019) min-1. These results suggest that P. falciparum ARF1 is a member of the class 1 ARF family and provide additional evidence for the existence of a classical secretory pathway in P. falciparum.
 ...
PMID:Isolation, expression and characterization of the gene for an ADP-ribosylation factor from the human malaria parasite, Plasmodium falciparum. 895 60

The consensus sequence of T4 early promoters differs in length, sequence and degree of conservation from that of Escherichia coli sigma(70) promoters. The enzyme interacting with these promoters, and transcribing the T4 genome, is native host RNA polymerase, which is increasingly modified by the phage-encoded ADP-ribosyltransferase, Alt. T4 early transcription is a very active process, possibly out-competing host transcription. The much stronger T4 promoters enhance viral transcription by a factor of at least two and the Alt-catalysed ADP-ribosylation of the host enzyme triggers an additional enhancement, again by a factor of about two. To address the question of which promoter elements contribute to the increasing transcriptional activity directed towards phage genes, the very strong E. coli promoter, Ptac, was sequentially mutated towards the sequence of the T4 early promoter consensus. Second, mutations were introduced into the highly conserved regions of the T4 early promoter, P8.1. The co-occurrence of the promoter-encoding plasmid pKWIII and vector pTKRI, which expresses Alt in E. coli, constitutes a test system that allows comparison of the transcriptional activities of phage and bacterial promoters, in the presence of native, or alternatively ADP-ribosylated RNA polymerase. Results reveal that T4 early promoters exhibit a bipartite structure, capable of strong interaction with both types of RNA polymerase. The -10, -16, -42 and -52 regions are important for transcript initiation with the native polymerase. To facilitate acceleration of transcription, the ADP-ribosylated enzyme requires not only the integrity of the -10, -16 and -35 regions, but also that of position -33, and even more importantly, maintenance of the upstream promoter element at position -42. The latter positions introduced into the E. coli Ptac promoter render this mutant promoter responsive to Alt-ADP-ribosylated RNA polymerase, like T4 early promoters.
 ...
PMID:T4 early promoter strength probed in vivo with unribosylated and ADP-ribosylated Escherichia coli RNA polymerase: a mutation analysis. 1102 39

We recently identified a novel type III secretion system (T3SS) effector, AexU, from a diarrheal isolate SSU of Aeromonas hydrophila, and demonstrated that mice infected with the DeltaaexU mutant were significantly protected from mortality. Although the NH(2)-terminal domain of this toxin exhibits homology to AexT of A. salmonicida, a fish pathogen, and ExoT/S of Pseudomonas aeruginosa, the COOH-terminal domain of AexU is unique, with no homology to any known proteins in the NCBI database. In this study, we purified the full-length AexU and its NH(2)-terminal (amino acid residues 1-231) and COOH-terminal (amino acid residues 232-512) domains after expression of their corresponding genes in Escherichia coli as histidine-tag fusion proteins using the bacteriophage T7 RNA polymerase/promoter-based pET-30a vector system. The full-length and NH(2)- and COOH-terminal domains of AexU exhibited ADP-ribosyltransferase activity, with the former two exhibiting much higher activity than the latter. These different forms of AexU were also successfully expressed and produced in the HeLa Tet-Off cell system using a pBI-EGFP vector, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot analysis, and intracellular staining of the toxin using flow cytometric analysis. Production of AexU in HeLa cells resulted in possible actin reorganization and cell rounding, as determined by phalloidin staining and confocal microscopy. Based on electron microscopy, the toxin also caused chromatin condensation, which is indicative of apoptosis. Apoptosis of HeLa cells expressing and producing AexU was confirmed by 7-amino actinomycin D (7-AAD) and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide] assays, by detection of cytoplasmic histone-associated DNA fragments, and by activation of caspases 3 and 9. These effects were much more pronounced in host cells that expressed and produced the full-length or NH(2)-terminal domain of AexU, compared to those that expressed and produced the COOH-terminal domain or the vector alone. This study represents the first characterization of this novel T3SS effector.
 ...
PMID:Biological characterization of a new type III secretion system effector from a clinical isolate of Aeromonas hydrophila-part II. 1758 31