Synthesis of the large subunit of ribulose-1,5-bisphosphate carboxylase in anin vitro partially definedE. coli system

Abstract

Thein vitro DNA- or RNA-directed synthesis of the large subunit (LS) of spinach chloroplast ribulose-1,5-biphosphate carboxylase (RuP₂C) has been examined in a highly definedE. coli transcription-translation system. Spinach chloroplast DNA, RNA and recombinant plasmids containing the spinach chloroplast LS gene (rbcL) have been used as templates in thein vitro system and a quantitative assay has been developed to measure LS formation. Thein vitro formed product contains formylmethionine at the N-terminal position and sediments primarily as a monomer. There is no detectable enzymatic activity associated with thein vitro product. To determine where theE. coli RNA polymerase used in these systems initiates, we have examined the transcripts produced by this enzymein vitro. Measurements of run-off transcripts indicate thatE. coli RNA polymerase initiates at the same position on the gene as is seenin vivo. In addition, the complete nucleotide sequence of therbcL gene including previously unsequenced 3′ and 5′ flanking regions has been determined. The sequence agrees, except at two nucleotide positions, with previously published sequencing data for this gene (Zurawski, G, Perrot, B, Bottomley, W, Whitfeld, PR, 1981. Nucleic Acids Res. 9:3251–3270).