Purification and Characterization of an RNA Dodecamer Sequence Binding Protein from Mitochondria of Saccharomyces cerevisiae

Abstract

Saccharomyces cerevisiae mitochondrial mRNAs terminate at their 3′ ends with a conserved dodecamer sequence, 5′-AAUAA(U/C)AUUCUU-3′. We have identified a nuclear-encoded protein (DBP) which specifically binds to the dodecamer sequence and have purified it to apparent homogeneity by RNA affinity chromatography. DBP consists of a single polypeptide of 55 kDa and binds to its RNA substrate with a 1:1 stoichiometry. Scatchard analysis determines that Kd is 0.93 nM for the canonical dodecamer sequence (5′-AAUAAUAUUCUU-3′) and 0.46 nM for the only naturally occurring variant (5′-AAUAACAUUCUU-3′) unique to oli1 gene. Based on the studies using mutant oligonucleotides, DBP appears to recognize primarily the nucleotide sequence of an RNA rather than its potential secondary structure.