IDENTIFICATION OF A PROTEIN COMPLEX THAT BINDS TO A DODECAMER SEQUENCE FOUND AT THE 3' ENDS OF YEAST MITOCHONDRIAL MESSENGER-RNAS

An activity from Saccharomyces cerevisiae mitochondria was identified that specifically bound to a 12-nucleotide sequence, AAUAA(U/C)AUUCUU, that is a site for processing of pre-mRNAs so as to generate the matu re 3' ends of mRNAs. Because processing occurs 3' to the end of the do decamer site, all mRNAs in yeast mitochondria terminate with that sequ ence. RNase T1 digestion fragments which terminated precisely at their 3' ends with the dodecamer sequence bound the activity, indicating th at mRNAs in vivo would be capable of binding. Gel mobility shift analy ses using RNA oligonucleotides showed that binding was reduced by a U- to-A substitution at position 3 of the dodecamer sequence; a C-to-A su bstitution at position 10 eliminated binding. UV cross-linking identif ied three polypeptides with approximate molecular masses of 19, 60, an d 70 kDa as constituents of the binding activity. These estimates incl uded the contribution of the P-32-labeled RNA oligonucleotide used to tag these polypeptides. An oligonucleotide with a UA-->AU substitution at positions 3 and 4 of the dodecamer site formed complexes deficient in the 19-kDa species, suggesting that binding specificity was inhere nt to the higher-molecular-weight polypeptides. Assembly of the comple x at a dodecamer site on an RNA protected sequences located 5' to the dodecamer site from digestion by a nucleoside triphosphate-dependent 3 ' exoribonuclease found in yeast mitochondria. Since mitochondrial mRN As terminate with an intact dodecamer sequence, the binding activity m ay function in the stabilization of mRNAs in addition to 3'-end format ion of mRNAs.