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).

An 11.2-kilobase pair (kbp) BamHI restriction nuclease fragment from spinach chloroplast DNA has been found to contain the gene for the large subunit (LS) The resulting recombinant plasmid, pSoe3101, was used to direct the synthesis of a protein, which was immunoprecipitable with antibody to RuP2 carboxylase, in a partially defined in vitro transcription-translation system derived from Escherichia coli. The product synthesized in vitro has a molecular weight identical to that ofauthentic spinach LS. By using pSoe3101 DNA cleaved at various positions with restriction nucleases, and the in vitro transcription-translation system, the LS gene has been mapped to a 1.5-kbp region located at one end of the 11.2-kbp BamHI fragment. The direction of transcription of the LS gene on the plasmid as well as on the chloroplast chromosome has also been determined. The position of the LS gene on circular spinach chloroplast DNA is approximately 27 kbp from the start of one of the inverted repeat regions and 180°from one ofthe rRNA-coding regions.

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