Showing papers by "Phillip A. Sharp published in 1997"
TL;DR: These consensus indicate that the U12-type spliceosome may be more dinucleotides were almost universal, suggesting that inclosely related togroup II introns than the more common trons were of a common origin and excised from preU2- type spliceOSome.
283 citations
TL;DR: It is proposed that regulation of morphogenetic transitions in invertebrates involves binding of JH or JH-like structures to USP.
Abstract: Juvenile hormones (JH), a sesquiterpenoid group of ligands that regulate developmental transitions in insects, bind to the nuclear receptor ultraspiracle (USP) In fluorescence-based binding assays, USP protein binds JH III and JH III acid with specificity, adopting for each ligand a different final conformational state JH III treatment of Saccharomyces cerevisiae expressing a LexA-USP fusion protein stabilizes an oligomeric association containing this protein, as detected by formation of a protein–DNA complex, and induces USP-dependent transcription in a reporter assay We propose that regulation of morphogenetic transitions in invertebrates involves binding of JH or JH-like structures to USP
269 citations
TL;DR: The Tat-Stimulatory factor is involved in the regulation of transcriptional elongation of HIV-1 by Tat as mentioned in this paper, which is a transcriptional activity factor, as well as genes encoding this factor and fragments and biologically functional variants thereof.
Abstract: The invention pertains to a novel transcriptional activity factor, Tat-Stimulatory Factor, as well as genes encoding this factor and fragments and biologically functional variants thereof. The Tat-Stimulatory Factor is involved in the regulation of transcriptional elongation of HIV-1 by Tat. The invention also pertains to therapeutics involving the foregoing proteins and genes, and agents that bind to the foregoing proteins and genes. The invention also relates to methods of screening for a compound which binds to Tat-SF1, Tat-SF1-associated kinase and/or a complex of Tat-SF1 and Tat-SF1-associated kinase, as well as methods of screening for compounds which modulate Tat-SF1-mediated transcriptional activation.
148 citations
TL;DR: The mechanism of coactivation by DNA topoisomerase I (topo I) was examined in a highly defined in vitro transcription system containing Pol II and purified factors and demonstrated a dramatic enhancement in the formation of the TFIID-TFIIA complex by topo I and activator, dependent on the TAF(II)s.
Abstract: The mechanism of coactivation by DNA topoisomerase I (topo I) was examined in a highly defined in vitro transcription system containing Pol II and purified factors. Both stimulation of the basal reaction and coactivation occurred dependent on TAF(II)s. Activation was first observed at the TFIID-TFIIA stage of initiation and maximal activation required the concomitant presence of TFIID, TFIIA, topo I, and activator. Electrophoretic mobility shift assay demonstrated a dramatic enhancement in the formation of the TFIID-TFIIA complex by topo I and activator, dependent on the TAF(II)s. DNase I footprinting confirmed this recruitment. A catalytically inactive topo I, which coactivated transcription, similarly stimulated the rapid formation of the TFIID-TFIIA complex in the presence of activator. A camptothecin-mediated DNA cleavage assay demonstrated the recruitment of topo I to the template by TFIID. Topo I likely functions during activation by enhancing the formation of an active TFIID-TFIIA complex on the promoter.
128 citations
TL;DR: A structure-based design strategy was used to construct a fusion protein, TBP/ZF, in which the three zinc fingers of Zif268 were linked to the COOH terminus of yeast TBP, which formed an extraordinarily stable complex when bound to the appropriate composite DNA site.
Abstract: Fusing the TATA box-binding protein (TBP) to other DNA-binding domains may provide a powerful way of targeting TBP to particular promoters. To explore this possibility, a structure-based design strategy was used to construct a fusion protein, TBP/ZF, in which the three zinc fingers of Zif268 were linked to the COOH terminus of yeast TBP. Gel shift experiments revealed that this fusion protein formed an extraordinarily stable complex when bound to the appropriate composite DNA site (half-life up to 630 h). In vitro transcription experiments and transient cotransfection assays revealed that TBP/ZF could act as a site-specific repressor. Because the DNA-binding specificities of zinc finger domains can be systematically altered by phage display, it may be possible to target such TBP/zinc finger fusions to desired promoters and thus specifically regulate expression of endogenous genes.
101 citations
TL;DR: Surprisingly, the SR protein SC35 can functionally substitute for U2AF65 in the reconstitution of pre-mRNA splicing in U2 AF-depleted extracts, suggesting that there are at least three distinguishable mechanisms for the binding of U2 snRNP to the pre- mRNA, including U2af-dependent and -independent pathways.
Abstract: Assembly of the mammalian spliceosome is known to proceed in an ordered fashion through several discrete complexes, but the mechanism of this assembly process may not be universal. In an early step, pre-mRNAs are committed to the splicing pathway through association with U1 small nuclear ribonucleoprotein (snRNP) and non-snRNP splicing factors, including U2AF and members of the SR protein family. As a means of studying the steps of spliceosome assembly, we have prepared HeLa nuclear extracts specifically depleted of the splicing factor U2AF. Surprisingly, the SR protein SC35 can functionally substitute for U2AF65 in the reconstitution of pre-mRNA splicing in U2AF-depleted extracts. This reconstitution is substrate-specific and is reminiscent of the SC35-mediated reconstitution of splicing in extracts depleted of U1 snRNP. However, SC35 reconstitution of splicing in U2AF-depleted extracts is dependent on the presence of functional U1 snRNP. These observations suggest that there are at least three distinguishable mechanisms for the binding of U2 snRNP to the pre-mRNA, including U2AF-dependent and -independent pathways.
49 citations
TL;DR: Surprisingly, Amin complex formation does not require U1 snRNP or ATP, suggesting that these factors are not necessary for stable binding of U2 snR NP per se, but rather are necessary for accessibility of components on longer RNA substrates.
Abstract: The association of U2 snRNP with the pre-mRNA branch region is a critical step in the assembly of spliceosomal complexes. We describe an assembly process that reveals both minimal requirements for formation of a U2 snRNP-substrate RNA complex, here designated the Amin complex, and specific interactions with the branch site adenosine. The substrate is a minimal RNA oligonucleotide, containing only a branch sequence and polypyrimidine tract. Interactions at the branch site adenosine and requirements for polypyrimidine tract-binding proteins for the Amin complex are the same as those of authentic prespliceosome complex A. Surprisingly, Amin complex formation does not require U1 snRNP or ATP, suggesting that these factors are not necessary for stable binding of U2 snRNP per se, but rather are necessary for accessibility of components on longer RNA substrates. Furthermore, there is an ATP-dependent activity that releases or destabilizes U2 snRNP from branch sequences. The simplicity of the Amin complex will facilitate a detailed understanding of the assembly of prespliceosomes.
42 citations