Showing papers by "Phillip A. Sharp published in 1995"

Structure-based design of transcription factors

Abstract: Computer modeling suggested that transcription factors with novel sequence specificities could be designed by combining known DNA binding domains. This structure-based strategy was tested by construction of a fusion protein, ZFHD1, that contained zinc fingers 1 and 2 from Zif268, a short polypeptide linker, and the homeodomain from Oct-1. The fusion protein bound optimally to a sequence containing adjacent homeodomain (TAATTA) and zinc finger (NGGGNG) subsites. When fused to an activation domain, ZFHD1 regulated promoter activity in vivo in a sequence-specific manner. Analysis of known protein-DNA complexes suggests that many other DNA binding proteins could be designed in a similar fashion.

Pre-bending of a promoter sequence enhances affinity for the TATA-binding factor

Abstract: TATA-binding protein (TBP) binds the minor groove of the TATA element with the DNA bent 80° towards the major groove1–4. A constrained minicircle strategy5 has been used to test the effect of DNA topology on the affinity of TBP for the TATA element. We report here that TBP bound to DNA which was slightly pre-bent towards the major groove with 100-fold higher affinity than unbent (linear) DNA of identical sequence and 300-fold higher affinity than DNA pre-bent towards the minor groove. Similar discrimination was observed with the holo-TFIID transcription complex. DNA topology, particularly bending, is determined by many factors including chromatin in cells and may, through changes in the affinity of the TATA factor, be important in the control of transcription.

Activation of the TFIID-TFIIA complex with HMG-2.

Abstract: The nonhistone chromosomal protein HMG-2 was identified as a factor necessary for activation in a defined transcription reaction in vitro containing RNA polymerase II and purified factors. Activation occurred on all promoters assayed except that of the immunoglobulin IgH gene. TFIIA was required for stimulated levels of transcription. The activation process depended on the presence of TAFs in the TFIID complex and generated a preinitiation complex from which TFIIB dissociated more slowly. However, titration of TFIIB over three orders of magnitude did not obviate the requirement of activator and HMG-2 to achieve stimulated levels of transcription. Analysis of the activated reaction identified the TFIID-TFIIA complex as the first stage of modification during activation. These results suggest that activation can occur solely in the presence of the basal factors, activator protein, and an "architectural" HMG factor, which probably stabilizes an activated conformation of the TFllD-TFIIA-promoter complex.

Novel mechanism and factor for regulation by HIV-1 Tat.

Abstract: Tat regulation of human immunodeficiency virus (HIV) transcription is unique because of its specificity for an RNA target, TAR, and its ability to increase the efficiency of elongation by polymerase. A reconstituted reaction that is Tat-specific and TAR-dependent for activation of HIV transcription has been used to identify and partially purify a cellular activity that is required for trans-activation by Tat, but not by other activators. In the reaction, Tat stimulates the efficiency of elongation by polymerase, whereas Sp1 and other DNA sequence-specific transcription factors activate the rate of initiation. Furthermore, while TATA binding protein (TBP)-associated factors (TAFs) in the TFIID complex are required for activation by transcription factors, they are dispensable for Tat function. Thus, Tat acts through a novel mechanism, which is mediated by a specific host cellular factor, to stimulate HIV-1 gene expression.

Nuclear factors associated with transcriptional regulation

Abstract: Constitutive and tissue-specific protein factors which bind to transcriptional regulatory elements of Ig genes (promoter and enhancer) are described. The factors were identified and isolated by an improved assay for protein-DNA binding. Genes encoding factors which positively regulate transcription can be isolated and employed to enhance transription of Ig genes. In particular, NF-kB, the gene encoding NF-kB, IkB and the gene encoding IkB and uses therefor.

A kinase-deficient transcription factor TFIIH is functional in basal and activated transcription.

Abstract: Phosphorylation of the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II has been suggested to be critical for transcription initiation, activation, or elongation. A kinase activity specific for CTD is a component of the general transcription factor TFIIH. Recently, a cyclin-dependent kinase-activator kinase (MO15 and cyclin H) was found to be associated with TFIIH preparations and was suggested to be the CTD kinase. TFIIH preparations containing mutant, kinase-deficient MO15 lack CTD kinase activity, indicating that MO15 is critical for polymerase phosphorylation. Nonetheless, these mutant TFIIH preparations were fully functional (in vitro) in both basal and activated transcription. These results indicate that CTD phosphorylation is not required for transcription with a highly purified system.

Chimeric DNA-binding proteins

Abstract: Chimeric proteins containing composite DNA-binding regions are disclosed together with DNA constructs encoding them, compositions containing them and applications in which they are useful.

Analysis of homeodomain function by structure-based design of a transcription factor

Abstract: The homeodomain is a 60-amino acid module which mediates critical protein-DNA and protein-protein interactions for a large family of regulatory proteins. We have used structure-based design to analyze the ability of the Oct-1 homeodomain to nucleate an enhancer complex. The Oct-1 protein regulates herpes simplex virus (HSV) gene expression by participating in the formation of a multiprotein complex (C1 complex) which regulates alpha (immediate early) genes. We recently described the design of ZFHD1, a chimeric transcription factor containing zinc fingers 1 and 2 of Zif268, a four-residue linker, and the Oct-1 homeodomain. In the presence of alpha-transinduction factor and C1 factor, ZFHD1 efficiently nucleates formation of the C1 complex in vitro and specifically activates gene expression in vivo. The sequence specificity of ZFHD1 recruits C1 complex formation to an enhancer element which is not efficiently recognized by Oct-1. ZFHD1 function depends on the recognition of the Oct-1 homeodomain surface. These results prove that the Oct-1 homeodomain mediates all the protein-protein interactions that are required to efficiently recruit alpha-transinduction factor and C1 factor into a C1 complex. The structure-based design of transcription factors should provide valuable tools for dissecting the interactions of DNA-bound domains in other regulatory circuits.

A U6 snRNA:pre-mRNA interaction can be rate-limiting for U1-independent splicing.

Abstract: The full set of consensus sequences at the 5' splice site is recognized during splicing of pre-mRNA in extracts depleted of U1 snRNP. High concentrations of HeLa SR proteins or purified SC35 alone promote the splicing of specific RNA substrates, bypassing the requirement for U1 snRNP in formation of the U2 snRNP-pre-mRNA complex. Under these conditions, mutations in the substrate that increase the sequence complementarity between U6 snRNA and the 5' splice site region can facilitate splicing. This provides additional strong evidence that U1 snRNP is not essential for splicing. Thus, the consensus sequence at the 5' splice site is probably recognized twice during splicing of most introns; however, some pre-mRNAs could potentially be processed in the absence of interactions with U1 snRNP in regions of the nucleus containing high concentrations of SR protein.

New proteins related to the Ser-Arg family of splicing factors.

Abstract: A family of six highly conserved proteins that contain domains rich in alternating serine/arginine residues (SR proteins) function in the regulation of splice site selection and are required for splicing. Using a selective precipitation method, more than 35 proteins were detected in nuclear extracts of HeLa cells that co-fractionate with the defined SR family. Many of these proteins were recognized by three monoclonal antibodies that bind to distinct phosphoepitopes on SR proteins. Two of these SR-related proteins were identified as the nuclear matrix antigens B1C8 and B4A11, which previously have been implicated in splicing. A subset of SR proteins, in their phosphorylated state, are associated with spliceosome complexes through both steps of the splicing reaction, remaining preferentially bound to complexes containing the exon-product. In contrast, other SR-related proteins appear to remain specifically associated with the intron-Iariat complex. The results indicate the existence of a potentially large group of SR-related proteins, and also suggest possible additional functions of SR proteins at a post-splicing level.

The branch site adenosine is recognized differently for the two steps of pre-mRNA splicing.

Abstract: The functional groups responsible for branch site identity in the two steps of pre-mRNA splicing as well as for spliceosome assembly were tested by incorporation of site-specific modifications at the branch site of a pre-mRNA. These results show that recognition of the adenosine occurs early in complex formation and that the branch site adenosine is recognized differently before the first step and for the second step. Further, direct UV cross-linking with these modified RNAs was used to identify a factor whose interaction was dependent upon the identity of the branch site nucleotide.

Proteines chimeres de liaison d'adn

Abstract: Proteines chimeres contenant des regions de liaison d'ADN composites, ADN de recombinaison codant ces proteines, compositions les contenant et applications dans lesquelles lesdites proteines peuvent etre utilisees.