Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.5.1.18 (glutathione S-transferase)
 22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The bipartite POU domain of transcription factor Oct-1 stimulates adenovirus DNA replication through an interaction with the octamer sequence present in the auxiliary origin. Employing an immobilized in vitro DNA replication system, we show that the POU domain enhances the formation of a pre-initiation complex composed of the viral precursor terminal protein-DNA polymerase (pTP-pol) complex and the origin. To investigate the mechanism of stimulation we have explored protein-protein interactions between the POU domain and the pTP-pol complex. Such an interaction could be detected using a GST-POU fusion protein bound to glutathione-agarose beads. Binding was also observed with the POU homeodomain (POUHD), albeit weaker than with the intact POU domain, but not with the POU specific subdomain. Four point mutations localized in the POUHD were analyzed for pTP-pol binding. Two of these, E22A and E30A, bound pTP-pol equally as well as the wild-type, while the other two, Q24A and E29A, were able to bind 2- to 4-fold better. These mutations are localized in the same region where the HSV transactivator VP16 binds, but did not coincide with the VP16 contacts. A direct correlation between pTP-pol binding and stimulation of DNA replication in vitro was observed for all mutants, suggesting that stimulation by the POU domain is caused by an interaction with the viral pTP-pol complex.
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PMID:The Oct-1 POU domain stimulates adenovirus DNA replication by a direct interaction between the viral precursor terminal protein-DNA polymerase complex and the POU homeodomain. 795 6

The frameshift protein p6* encoded directly upstream of the protease in the human immunodeficiency virus type 1 (HIV-1) pol reading frame is thought to be a natural inhibitor of protease activation and to play a role in the polyprotein processing of Gag and Gag-Pol precursors. To allow structural characterization of the p6* transframe protein, the p6* coding region was cloned into the vector pGEX-KG and expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST) under the control of the tac promoter. Thrombin cleavage of the construct resulted in a 70-amino-acid polypeptide which is extended by two additional residues at the N-terminus compared to the natural p6* sequence. The native purification procedure including an affinity and a size-exclusion chromatography step yielded sufficient amounts of highly pure protein suitable for NMR spectroscopy. Fluorescence, circular dichroism and 1H-NMR spectroscopy were applied to characterize the structure of protein. Two-dimensional NMR spectra provided essentially complete sequence-specific resonance assignments at pH 5.9. Although there is evidence for a helix-forming tendency in the N-terminus of the protein, the experiments indicate that p6* has no overall stable secondary or tertiary structure with the single tryptophan exposed in aqueous solution. However, the results reported herein open the way to characterize further the interaction of p6* with the HIV-1 protease in structural and functional in vitro studies.
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PMID:Sequence-specific resonance assignments of the 1H-NMR spectra and structural characterization in solution of the HIV-1 transframe protein p6. 864 76

A new gene, RRN11, has been defined by certain rrn mutants of Saccharomyces cerevisiae which are defective specifically in the transcription of 35 S rRNA gene by RNA polymerase I (pol I). We have cloned the gene and found that it encodes a protein of 507 amino acids. We have used a strain with the chromosomal RRN11 deleted and carrying HA1 epitope-tagged RRN11 on a plasmid to isolate a protein complex containing the protein encoded by RRN11. This protein complex complemented rrn6 mutant extracts, which were previously shown to be deficient in the essential pol I transcription factor called Rrn6/7 complex or core factor (CF). The CF complex was previously shown to consist of three proteins, the 102- and 60-kDa subunits encoded by RRN6 and RRN7, respectively, and the 66-kDa subunit. The results of the above complementation experiments combined with mobility of Rrn11p in SDS-polyacrylamide gel electrophoresis analysis relative to Rrn6p and Rrn7p led to the conclusion that RRN11 encodes the 66-kDa subunit of CF. Glutathione S-transferase-Rrn11p fusion protein was found to bind strongly to 35S-labeled Rrn6p and Rrn7p but only weakly to 35S-labeled TATA-binding protein. Similarly, glutathione S-transferase-Rrn7p fusion protein bound strongly to 35S-labeled Rrn6p and Rrn11p but only weakly to 35S-labeled TATA-binding protein. These results are consistent with the fact that one can purify CF consisting of Rrn6p, Rrn7p, and Rrn11p from yeast cell extracts, but the purified complex does not contain TATA-binding protein. RRN11 was shown to be an essential gene, and [3H]uridine pulse experiments demonstrated directly that RRN11 is essential for rDNA transcription by pol I in vivo. Thus all three subunits of CF are essential for rDNA transcription. Because of the resemblance of CF to mammalian essential pol I transcription factor SL1, the amino acid sequences of Rrn11p and the other two subunits of CF were compared with those of the three TATA-binding protein-associated factors (TAFs) in the human SL1, TAFI48, TAFI63, and TAFI110. No significant similarity was detected between two sets of the proteins. Similarity as well as differences between CF and SL1 are discussed.
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PMID:RRN11 encodes the third subunit of the complex containing Rrn6p and Rrn7p that is essential for the initiation of rDNA transcription by yeast RNA polymerase I. 870 72

Simian and human foamy viruses (SFV and HFV) encode a transcriptional transactivator, Tas, which governs the levels of viral transcripts initiated by both the promoter in the long terminal repeat (LTR) and the internal promoter (IP) located within the env gene of these viruses. Tas-responsive target elements,(TRE) LTR in the LTR and (TRE) IP in the env gene, are located 5' of the TATA box in both viral promoters and function as orientation- and position-independent enhancers. We have identified a strong Tas-responsive element, designated TRE (GP), near the 3' end of the gag gene and preceding the pol gene of SFV-1. In transient-expression assays with plasmids containing reporter genes, a 59-bp DNA fragment containing TRE (GP) (nucleotides 2224 to 2282) functioned as an enhancer element, dependent on Tas, in several cell types and in the context of a heterologous basal promoter. DNase footprinting revealed that the fusion protein glutathione S-transferase-Tas, purified from genetically engineered bacteria, interacts with about 40 hp (nucleotides 2237 to 2279) in the TRE (GP). A low degree of sequence homology was noted between TRE (GP) and TRE (IP). In virus-infected cells, novel transcripts with 5' ends immediately upstream from the reverse transcriptase translation frame (nucleotides 2611 to 5778) were identified. Upstream of the start site for these transcripts is a TATA box (nucleotides 2575 to 2579), which was required for transcription in transient-expression assays. Although a spliced mRNA initiated in the viral LTR is implicated in the synthesis of the HFV Pol polyprotein which encodes protease, reverse transcriptase, and integrase, it is possible that SFV-1 contains a promoter within the pol gene for initiating a reverse transcriptase transcript. Taken together, these studies define a novel Tas-responsive enhancer element, which binds the viral transactivator, and a potential promoter within the pol gene.
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PMID:The simian foamy virus type 1 transcriptional transactivator (Tas) binds and activates an enhancer element in the gag gene. 879 26

Initiation of adenovirus DNA replication is strongly enhanced by two cellular transcription factors, NFI and Oct-1, which bind to the auxiliary origin and tether the viral precursor terminal protein-DNA polymerase (pTP.pol) complex to the core origin. NFI acts through a direct contact with the DNA polymerase, but the mode of action of Oct 1 is unknown. Employing glutathione S-transferase-POU pull-down assays and protein affinity chromatography, we have established that the POU domain contacts pTP rather than pol. The POU homeodomain is responsible for this interaction. The protein-protein contacts lead to increased binding of pTP-pol to the core origin, which is caused by a reduced off-rate. The enhanced formation of a pTP.pol.POU complex on the origin correlates with stimulation of replication. Using an immobilized replication system, we have studied the kinetics of dissociation of the Oct-1 POU domain during replication. In contrast to NFI, which dissociates very early in initiation, Oct-1 dissociates only when the binding site is rendered single-stranded upon translocation of the replication fork. Our data indicate that NFI and Oct-1 enhance initiation synergistically by touching different targets in the preinitiation complex and dissociate independently after initiation.
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PMID:The Oct-1 POU homeodomain stabilizes the adenovirus preinitiation complex via a direct interaction with the priming protein and is displaced when the replication fork passes. 901 82

The Srb/Mediator, a multisubunit subcomplex of the RNA polymerase II (RNA pol II) holoenzyme has been proposed to function as a control panel regulating transcription in response to gene-specific activator proteins. In this report, we identify the Mediator subunit Hrs1/Med3 as a physical target for Cyc8-Tup1, a yeast transcriptional corepressor. Two-hybrid and glutathione S-transferase interaction assays show that Hrs1 can associate directly with Cyc8-Tup1. Moreover, affinity chromatography experiments, using yeast protein extracts, reveal that Cyc8-Tup1 co-purifies with Hrs1 and with additional Mediator subunits in a Hrs1-dependent manner. These observations suggest that Cyc8-Tup1 contacts the Mediator complex via its interaction with the Hrs1 subunit. Further on, genetic analysis indicates that increased Hrs1 dosage can alleviate Cyc8-Tup1-mediated repression, suggesting that Hrs1/Mediator's function is inhibited upon its interaction with Cyc8-Tup1. Finally, artificial holoenzyme recruitment assays support a model by which the contact between the corepressor and the Hrs1/Mediator may prevent pol II holoenzyme recruitment to the core promoter. These data, together with previous genetic evidence, establish a functional and physical interaction between the Cyc8-Tup1 corepressor and the RNA pol II holoenzyme and support a central role of the Mediator complex in transcriptional repression.
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PMID:Hrs1/Med3 is a Cyc8-Tup1 corepressor target in the RNA polymerase II holoenzyme. 1072 72

In transcriptional regulation, RNA polymerase II (pol II) interacts and forms complexes with a number of protein factors. To isolate and identify the pol II-associated proteins, we constructed a Schizosaccharomyces pombe strain carrying a FLAG tag sequence fused to the rpb3 gene encoding the pol II subunit Rpb3. By immunoaffinity purification with anti-FLAG antibody-resin, a pol II complex containing the Rpb1 subunit with a nonphosphorylated carboxyl-terminal domain (CTD) was isolated. In addition to the pol II subunits, the complex was found to contain three subunits of a transcription factor TFIIF (TFIIF alpha, TFIIF beta, and Tfg3) and TFIIF-interacting CTD-phosphatase Fcp1. The same type of pol II complex could also be purified from an Fcp1-tagged strain. The isolated Fcp1 showed CTD-phosphatase activity in vitro. The fcp1 gene is essential for cell viability. Fcp1 and pol II interacted directly in vitro. Furthermore, by chemical cross-linking, glutathione S-transferase pulldown, and affinity chromatography, the Fcp1-interacting subunit of pol II was identified as Rpb4, which plays regulatory roles in transcription. We also constructed an S. pombe thiamine-dependent rpb4 shut-off system. On repression of rpb4 expression, the cell produced more of the nonphosphorylated form of Rpb1, but the pol II complex isolated with the anti-FLAG antibody contained less Fcp1 and more of the phosphorylated form of Rpb1 with a concomitant reduction in Rpb4. This result indicates the importance of Fcp1-Rpb4 interaction for formation of the Fcp1/TFIIF/pol II complex in vivo.
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PMID:Formation of a carboxy-terminal domain phosphatase (Fcp1)/TFIIF/RNA polymerase II (pol II) complex in Schizosaccharomyces pombe involves direct interaction between Fcp1 and the Rpb4 subunit of pol II. 1183 23

The yeast two-hybrid screening method was used to identify novel proteins that associate with human DNA polymerase delta (pol delta). Two baits were used in this study. These were the large (p125) and small (p50) subunits of the core pol delta heterodimer. p50 was the only positive isolated with p125 as the bait. Two novel protein partners, named PDIP38 and PDIP46, were identified from the p50 screen. In this study, the interaction of PDIP38 with pol delta was further characterized. PDIP38 encodes a protein of 368 amino acids whose C terminus is conserved with the bacterial APAG protein and with the F box A protein. It was found that PDIP38 also interacts with proliferating cell nuclear antigen (PCNA). The ability of PDIP38 to interact with both the p50 subunit of pol delta and with PCNA was confirmed by pull-down assays using glutathione S-transferase (GST)-PDIP38 fusion proteins. The PCNA-PDIP38 interaction was also demonstrated by PCNA overlay experiments. The association of PDIP38 with pol delta was shown to occur in calf thymus tissue and mammalian cell extracts by GST-PDIP38 pull-down and coimmunoprecipitation experiments. PDIP38 was associated with pol delta isolated by immunoaffinity chromatography. The association of PDIP38 with pol delta could also be demonstrated by native gel electrophoresis.
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PMID:Identification of a novel protein, PDIP38, that interacts with the p50 subunit of DNA polymerase delta and proliferating cell nuclear antigen. 1252 11

We analyzed the composition and abundance of two forms of RNA polymerase II (pol II) holoenzyme in synchronized HeLa cells. We did not detect significant changes in pol II holoenzyme composition, but we noticed differences in the abundance of the two complexes at different stages of the cell cycle. Summarized data from several independent experiments demonstrate that pol II holoenzyme, which is purified by GST-TFIIS affinity chromatography, is more abundant during G1/S and S phases. Another form of pol II holoenzyme, which is purified by anti-CDK7 antibodies, shows relatively higher amounts in G2/M and early G1 phases.
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PMID:Two forms of RNA polymerase II holoenzyme display different abundance during the cell cycle. 1261 59

Proliferating cell nuclear antigen (PCNA) plays an essential role in DNA replication, repair, and cell cycle control. PCNA is a homotrimeric ring that, when encircling DNA, is not easily extractable. Consequently, the dynamics of protein-protein interactions established by PCNA at DNA replication sites is not well understood. We have used DNase I to release DNA-bound PCNA together with replication proteins including the p125-catalytic subunit of DNA polymerase delta (p125-pol delta), DNA ligase I, cyclin A, and cyclin-dependent kinase 2 (CDK2). Interaction with these proteins was investigated by immunoprecipitation with antibodies binding near the interdomain connector loop or to the C-terminal domain of PCNA, respectively, or with antibodies to p125-pol delta or DNA ligase I. PCNA interaction with p125-pol delta or DNA ligase I was detected only by the latter antibodies, and found to be mutually exclusive. In contrast, antibodies to PCNA co-immunoprecipitated only CDK2. A GST-p21(waf1/cip1) C-terminal peptide displaced p125-pol delta and DNA ligase I, but not CDK2, from PCNA. These results suggest that PCNA trimers bound to DNA during the S phase are organized as distinct pools able to bind selectively different partners. Among them, p125-pol delta and DNA ligase I interact with PCNA in a mutually exclusive manner.
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PMID:Distinct pools of proliferating cell nuclear antigen associated to DNA replication sites interact with the p125 subunit of DNA polymerase delta or DNA ligase I. 1472 73


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