Molecular cloning and characterization of the yeast ribosomal RNA enhancer binding protein (REB1) gene

Abstract

REB1 is a DNA binding protein that recognizes sites within both the enhancer and the promoter of the rRNA transcription unit as well as upstream of many genes transcribed by RNA polymerase II in Saccharomyces cerevisiae. Thus, REB1 is a general DNA-binding protein with currently unknown function. The gene encoding REB1 has been cloned by screening a yeast genomic {dollar}\lambda{dollar}gt11 library using specific double stranded oligonucleotide containing the REB1 binding site consensus sequence and confirmed by gel retardation and footprint assays. The REB1 gene was sequenced, revealing an intronless open reading frame of 809 amino acids. The predicted protein is highly hydrophilic, with numerous hydroxy amino acids and glutamine residues, features common to the DNA binding proteins such as: ABF1, RAP1, GCN4 and HSF1. REB1 is a single copy gene and has been mapped on chromosome II. Gene disruption experiments have shown that REB1 is an essential gene. Interestingly, there is some homology between a portion of REB1 and the conserved DNA binding domain of the oncogene myb, especially for a set of regularly spaced tryptophan residues. Two potential REB1 binding sites have been found upstream of the REB1 gene and confirmed by gel retardation assays. Analysis of the upstream sequence also revealed one potential heat shock factor-binding site. Using an antibody raised against the LacZ-REB1 fusion protein, it was found that REB1 is phosphorylated in vivo. The cloning and characterization of REB1 gene has provided useful information for studying the biological function of REB1.