Use of a selection technique to identify the diversity of binding sites for the yeast RAP1 transcription factor.

Abstract

We have used the technique known as selected and amplified binding (SAAB) to isolate binding sites for the yeast transcription factor RAP1 from a degenerate pool of oligonucleotides. A total of 47 sequences were isolated, of which two were shown to be contaminating non-RAP1 binding sites. After excluding these two sequences the remainder of the sequences were used to derive a new consensus binding site for RAP1. The new consensus 5' A/G T A/G C A C C C A N N C C/A C C 3' is a significant extension of the existing consensus (4). It is longer by two base pairs at the 5' end and is significantly more constrained at the 3' end. An analysis of the combinations of mis-matches in individual SAAB sequences, compared to the consensus RAP1 binding site, has allowed us to analyse the structure of the RAP1 binding site in some detail. The binding site can be sub-divided into three regions; a core binding site, a 5' flanking region and a 3' flanking region. The core binding site, consisting of the sequence 5'CACCCA3', is critical for recognition by RAP1. The less conserved flanking regions are not as important. Interactions between RAP1 and these regions probably stabilise the interaction between RAP1 and the core binding site. Each of the sequences isolated in the SAAB analysis was used to search release 78 of the EMBL+GenBank DNA data base. The searches identified 102 potential binding sites for RAP1 within promoters of yeast genes.