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Titolo:
WILD-TYPE GAL4 BINDS COOPERATIVELY TO THE GAL1-10 UASG INVITRO
Autore:
KANG T; MARTINS T; SADOWSKI I;
Indirizzi:
UNIV BRITISH COLUMBIA,DEPT BIOCHEM,2146 HLTH SCI MALL VANCOUVER V6T 1Z3 BC CANADA UNIV BRITISH COLUMBIA,DEPT BIOCHEM,2146 HLTH SCI MALL VANCOUVER V6T 1Z3 BC CANADA HARVARD UNIV,DEPT CELLULAR & DEV BIOL CAMBRIDGE MA 02138
Titolo Testata:
The Journal of biological chemistry
fascicolo: 13, volume: 268, anno: 1993,
pagine: 9629 - 9635
SICI:
0021-9258(1993)268:13<9629:WGBCTT>2.0.ZU;2-D
Fonte:
ISI
Lingua:
ENG
Soggetto:
EUKARYOTIC TRANSCRIPTIONAL ACTIVATORS; SACCHAROMYCES-CEREVISIAE; GLUCOSE REPRESSION; REGULATORY PROTEIN; DNA-BINDING; YEAST; GENE; DERIVATIVES; MECHANISM; SITES;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
31
Recensione:
Indirizzi per estratti:
Citazione:
T. Kang et al., "WILD-TYPE GAL4 BINDS COOPERATIVELY TO THE GAL1-10 UASG INVITRO", The Journal of biological chemistry, 268(13), 1993, pp. 9629-9635

Abstract

Transcription of the genes required for utilization of galactose in Saccharomyces cerevisiae is controlled primarily by the transcriptionalactivator protein GAL4. The upstream activating sequences for galactose (UAS(G)) of most GAL genes have multiple sites to which GAL4 can bind. In this report we compare the binding properties of wild type GAL4and derivatives of GAL4 bearing the N-terminal DNA-binding domain to multiple DNA-binding sites in vitro. To produce wild type GAL4, we constructed a recombinant baculovirus for expression in insect cells. Recombinant wild type GAL4 was found to bind efficiently to an oligonucleotide containing a near-consensus 17-mer GAL4 DNA-binding site in electrophoretic mobility shift assays. Footprinting experiments revealed that wild type GAL4 binds cooperatively to the four GAL4 DNA-binding sites of the GAL1-10 UAS(G); however, in contrast an N-terminal fragmentof GAL4 containing only the DNA-binding/dimerization domains binds toeach of these sites with slightly different affinity. With increasingconcentrations of GAL4(1-147), the four sites become filled in the following order: site II, site IV, site I, and site III. In experiments with wild type GAL4, these four sites become fully occupied at approximately the same concentration of protein. In footprints of wild type GAL4 on the UAS(G), enhancements and protections of DNase I-sensitive cleavages are detectable between sites III and IV, indicative of formation of a loop between these distantly spaced sites. Binding of wild type GAL4 to a strong near-consensus binding site assists binding to an adjacent mutant site in both electrophoretic mobility shift and footprinting assays. GAL4(1-147) and GAL4(1-147) fused to portions of GAL4'sactivating region II were incapable of cooperative DNA binding in ourassays. We conclude from these observations that wild type GAL4 has acooperative DNA-binding function that is distinct from the DNA binding and dimerization or transcriptional activation functions, and likelyplays and important role in precise regulation of GAL gene transcription.

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Documento generato il 28/11/20 alle ore 21:58:01