ISOLATION AND FUNCTIONAL-ANALYSIS OF A KLUYVEROMYCES-LACTIS RAP1 HOMOLOG

Saccharomyces cerevisiae RAP1 (Sc RAP1) is an essential protein which interacts with diverse genetic loci within the cell. RAP1 binds site-s pecifically to the consensus sequence, 5'-AYCYRTRCAYYW (UAS(RPG), wher e R=A or G, W=A or T, Y=C or T). In Kluyveromyces (Kl) ribosomal prote in-encoding genes (rp) retain functional RAP1-binding elements, sugges ting the presence of a RAP1-like factor. Kl extracts display an activi ty capable of specifically binding to rp fragments bearing UAS(RPG). W e subsequently isolated the Kl RAP1-encoding gene by homology to a sub fragment which encodes the N terminus of the DNA-binding domain of Sc RAP1. The predicted amino acid (aa) sequence of KI RAP1 indicates it i s smaller than Sc RAP1 (666 vs. 827 aa) with the N terminus being trun cated. The DNA-binding domain is virtually identical between the two R AP1 proteins, while the RIF1 domain is moderately conserved. The regio n between these two domains and the N-termini are highly divergent. Tw o potential UAS(RPG) were identified in the 5' flanking region, sugges ting an autoregulatory role for RAP1. Despite the similarities between the two proteins, Kl RAP1 is unable to complement Sc rap1(ts) mutants , suggesting that domains essential for function in Sc are absent from the Kl protein.