Large subunit ribulosebisphosphate carboxylase messenger RNA from Euglena chloroplasts

Abstract

RNA from wild-type Euglena, aplastidic mutant cells, and purified chloroplasts was chromatographed on oligo(dT)-cellulose at 4 degrees. The poly(A)-containing and poly(A)-lacking fractions from each were then tested in a cell-free protein-synthesizing system from wheat germ for their abilities to specifically stimulate synthesis of large subunit ribulosebisphosphate carboxylase [EC 4.1.1.39; 3-phospho-D-glycerate carboxy-lyase (dimerizing)]. The large subunit polypeptide (59,000 molecular weight) was identified in the in vitro reaction by two-dimensional electrophoresis involving isoelectric focusing and size filtration on polyacrylamide gels. Template activity for the large subunit was detected in wild-type cells but not in aplastidic mutant strains; it was highly enriched in the isolated chloroplasts. This messenger was present only in the poly(A)-lacking RNA fraction, where it constituted the most prominent template species of chloroplast RNA. The large subunit message was freed of considerable non-messenger RNA contamination and localized to the 10-20S fraction by sucrose gradient centrifugation.