The large subunit gene (rbcL) of ribulose 1,5-bisphosphate carboxylase was transcribed in vitro by using maize and pea chloroplast extracts and a cloned plastid DNA template containing 172 base pairs (bp) of the maize rbcL protein-coding region and 791 bp of upstream sequences. Three major in vitro RNA species were synthesized which correspond to in vivo maize rbcL RNAs with 5' termini positioned 300, 100 to 105, and 63 nucleotides upstream of the protein-coding region. A deletion of 109 bp, including the "-300" 5' end (the 5' end at position -300), depressed all rbcL transcription in vitro. A plasmid DNA containing this 109-bp fragment was sufficient to direct correct transcription initiation in vitro. A cloned template, containing 191 bp of plastid DNA which includes the -105 and -63 rbcL termini, did not support transcription in vitro. Exogenously added -300 RNA could be converted to the -63 transcript by maize chloroplast extract. These results established that the -300 RNA is the primary maize rbcL transcript, the -63 RNA is a processed form of the -300 transcript, and synthesis of the -105 RNA is dependent on the -300 region. The promoter for the maize rbcL gene is located within the 109 bp flanking the -300 site. Mutagenesis of the 109-bp chloroplast sequence 11 bp upstream of the -300 transcription initiation site reduced rbcL promoter activity in vitro.Ribulose 1,5-bisphosphate carboxylase (RUBISCO) catalyzes CO2 fixation, the first step of the Calvin cycle (40). In higher plants, the chloroplast enzyme is composed of eight identical, catalytic polypeptides of 50,000 to 60,000 kilodaltons and eight smaller polypeptides of 12,000 to 20,000 kilodaltons (2). The small subunit, whose function is unknown, is encoded in the nucleus by a small multigene family (7,14,19). The large subunit gene (rbcL) is present as a single copy on the multicopy chloroplast genome (5). Under most conditions, the synthesis of the two RUBISCO subunits is coordinated (55) and regulated by light (15, 50) and cytokinins (23). In maize and other C4 plants, RUBISCO expression is leaf cell-type specific (30). The maize mRNAs encoding the two subunits are present in bundle sheath cells but not in mesophyll cells, which assimilate CO2 via ribulose 1,5-bisphosphate and C4 dicarboxylic acids, respectively (11,35).In chloroplast genomes of higher plants, the genes for the large subunit of RUBISCO and the beta subunit of ATP synthetase (atpB) are adjacent and transcribed divergently (31, 60). The 5' ends of the two genes are separated by approximately 150 base pairs (bp) (52). Northern hybridization analyses of transcripts from a broad spectrum of angiosperms indicate that the rbcL gene is generally transcribed as a 1.6-kilobase mRNA, although larger transcripts are also observed (46). Si nuclease protection experiments have revealed the presence of two 5' termini for the rbcL RNAs of maize, barley, spinach, and peas (16,21,41,49,62.) Tobacco has a single rbcL RNA with its 5' end at position -180 relative to the coding region (53). In maize ...