Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.1 (chymotrypsin)
 10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Transcription factor IID from Saccharomyces cerevisiae (YIID) binds the TATA box element present in most RNA polymerase II promoters. In this work, partial proteolysis was used as a biochemical probe of YIID structure. YIID consists of a protease-sensitive amino terminus and a highly stable, protease-resistant carboxy-terminal core. The cleavage sites of the predominant chymotrypsin- and trypsin-derived fragments were mapped to amino acid residues 40 to 41 and 48 to 49, respectively, by amino-terminal peptide sequencing. Removal of the amino terminus resulted in a dramatic increase in the ability of YIID to form a stable complex with DNA during gel electrophoresis mobility shift assays and a two- to fourfold increase in DNA-binding affinity, as assayed by DNase I footprinting analysis. The carboxy-terminal 190-amino-acid core was competent for transcription in vitro and was similar in activity to native YIID. DNA containing a TATA element induced hypersensitive sites in the amino-terminal domain and stabilized the core domain to further proteolytic attack. Native YIID did not bind to a TATA box at 0 degrees C, whereas the carboxy-terminal DNA-binding domain did. These results suggest that YIID undergoes a conformational change upon binding to a TATA box. Southern blotting showed that the carboxy-terminal domain is highly conserved, while the amino-terminal domain diverged rapidly in evolution, even between closely related budding yeasts.
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PMID:Two distinct domains in the yeast transcription factor IID and evidence for a TATA box-induced conformational change. 198 53

Transcriptional stimulation by the model activator GAL4-VP16 (a chimeric protein consisting of the DNA-binding domain of the yeast activator GAL4 and the acidic activation domain of the herpes simplex virus protein VP16) involves a series of poorly understood protein-protein interactions between the VP16 activation domain and components of the RNA polymerase II general transcription machinery. One of these interactions is the VP16-mediated binding and recruitment of transcription factor TFIIB. However, TATA box-binding protein (TBP)-associated factors (TAFs), or coactivators, are required for this interaction to culminate in productive transcription complex assembly, and one such TAF, Drosophila TAF40, reportedly forms a ternary complex with VP16 and TFIIB. Due to TFIIB's central role in gene activation, we sought to directly visualize the surfaces of this protein that mediate formation of the ternary complex. We developed an approach called protease footprinting in which the broad-specificity proteases chymotrypsin and alkaline protease were used to probe binding of 32P-end-labeled TFIIB to GAL4-VP16 or TAF40. Analysis of the cleavage products revealed two regions of TFIIB protected by VP16 from protease attack, one of which overlapped with a region protected by TAF40. The close proximity of the VP16 and TAF40 binding sites on the surface of TFIIB suggests that this region could act as a regulatory interface mediating the effects of activators and coactivators on transcription complex assembly.
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PMID:Protease footprinting reveals a surface on transcription factor TFIIB that serves as an interface for activators and coactivators. 759 78

DNA-based replicon expression vector pSMCTA and helper vector pSHCTA were constructed by replacing the SP6 promoter used in the original system pSFV1 and pSFV-helper2 derived from Semliki Forest virus (SFV) with the RNA polymerase II -dependent cytomegalovirus immediate early (CMV IE) enhancer/promoter and T7 promoter, and inserting BGH transcription termination and polyadenylation signal downstream 3'-untranslated region (UTR). The RNA polymerase II -dependent cytomegalovirus immediate early (CMV IE) enhancer/promoter and T7 promoter in pSMCTA and pSHCTA could drive transcription to produce replicon RNA in vivo and ex vivo. High level expression of foreign genes (GFP and LacZ) could be demonstrated by transfecting BHK21 cells with the new replicon expression vectors based on both DNA and RNA, and recombinant virus particles (RVP) be prepared by cotranfecting the expression vectors with the helper vectors. Foreign genes were also highly expressed in cells (BHK21) which were infected with RVP activated by alpha-chymotrypsin. The bifunctional replicon vectors can be used in highly efficient expression of foreign genes and preparation of RVP ex vivo, also in development of replicon vaccines and gene therapy vectors in vivo.
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PMID:[Construction of DNA and RNA based on bifunctional replicon vector derived from Semliki Forest virus]. 1628 10