DNA-binding properties of the yeast transcriptional activator, Gcr1p

✍ Scribed by Huie, Michael A.; Baker, Henry V.

• Publisher: John Wiley and Sons
• Year: 1996
• Tongue: English
• Weight: 901 KB
• Volume: 12
• Category: Article
• ISSN: 0749-503X
• DOI: 10.1002/(sici)1097-0061(19960330)12:4<307::aid-yea912>3.0.co;2-c

✦ Synopsis

In Succharomyces cerevisiae the GCRl gene product is required for high-level expression o f genes encoding glycolytic enzymes. In this communication, we extend our analysis of the DNA binding properties of Gcrlp. The DNA-binding domain of Gcrlp binds DNA with high affinity. The apparent dissociation constant of the Gcrlp DNA-binding domain for one of its specific binding sites (TTTCAGCTTCCTCTAT) is 2.9 x 10l o M. However, competition experiments showed that Gcrlp binds this site in vitro with a low degree of specificity. We measured a 33-fold difference between the ability of specific competitor and DNA o f random sequence to inhibit the formation o f nucleoprotein complexes between Gcrlp and a radiolabeled DNA probe containing its binding site. DNA band-shift experiments, utilizing probes of constant length in which the positions of Gcrlp-binding sites are varied relative to the ends, indicated that Gcrlp-DNA nucleoprotein complexes contain bent DNA. The implications of these findings in terms of the combinatorial interactions that occur at the upstream activating sequence elements of genes encoding glycolytic enzymes are discussed.