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-specifically to the consensus sequence, 5'-AYCYRTRCAYYW (UAS(RPG), where R=A or G, W=A or T, Y=C or T). In Kluyveromyces (Kl) ribosomal protein-encoding genes (rp) retain functional RAP1-binding elements, suggesting the presence of a RAP1-like factor. Kl extracts display an activity capable of specifically binding to rp fragments bearing UAS(RPG). We subsequently isolated the Kl RAP1-encoding gene by homology to a subfragment which encodes the N terminus of the DNA-binding domain of Sc RAP1. The predicted amino acid (aa) sequence of KI RAP1 indicates it is smaller than Sc RAP1 (666 vs. 827 aa) with the N terminus being truncated. The DNA-binding domain is virtually identical between the two RAP1 proteins, while the RIF1 domain is moderately conserved. The region between these two domains and the N-termini are highly divergent. Two potential UAS(RPG) were identified in the 5' flanking region, suggesting 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.