Aurintricarboxylic acid (ATA) is a general inhibitor of nucleases. ATA has been shown to inhibit the following enzymes in vitro: DNAse I, RNAse A, S1 nuclease, exonuclease III, and restriction endonucleases Sal I, Bam HI, Pst I and Sma I. The observed inhibition is consistent with the proposal by Blumenthal and Landers (BBRC 55, 680, 1973) that most nucleic acid binding proteins will be sensitive to ATA. The action of ATA as a nuclease inhibitor can be used to advantage in the isolation of cellular nucleic acids.

We have compared therbcL andatpB transcription units from spinach, maize, and pea. In most cases multiple transcripts were found for a given chloroplast gene. The 5' termini of these transcripts were determined by S1 nuclease protection and primer extension analyses. TherbcL transcripts have 5' termini 178-179 and 64 nucleotides (spinach), 300 and 59-63 nucleotides (maize), and 178 and 65 nucleotides (pea) upstream from their respective protein coding regions. TheatpB transcripts have 5' termini (453-454, 272-273, 179, and 99 nucleotides (spinach), 298-302 nucleotides (maize), and 351-355 nucleotides (pea) upstream from their respective protein coding regions. The intergenic distance between therbcL andatpB genes is relatively constant (152 to 157 base pairs) among the three chloroplast genomes. In spinach, maize, and pea, the 80 base pairs surrounding the 5' end of therbcL gene (±40 base pairs) have 85% sequence homology. Similarly, the 60 base pairs preceding theatpB gene have 48% sequence homology. Both genes have '-10' and '-35' regions that resemble the prokaryotic consensus promoter sequence. The larger, but not smaller,rbcL transcripts from spinach and pea can be labeled with alpha-(32)P-GTP by guanylyltransferase. These data suggest that DNA sequences 178-179 (spinach), 300 (maize), and 178 (pea) base pairs before therbcL protein coding regions represent sites of transcription initiation. The sequences 59-65 base pairs before therbcL protein coding regions may correspond to sites of RNA cleavage.

We have developed an homologous in vitro system from spinach chloroplasts that correctly initiates transcription of the plastid genes for the large subunit of ribulose-1,5-bisphosphate carboxylase (rbcL) and the beta subunit of the plastid ATPase (atpB). The transcriptionally active extracts from spinach chloroplasts require circular DNA templates for specific initiation. The RNA polymerase activity is insensitive to rifampicin. The extent of transcription in vitro is a function of the extract:template ratio. The efficiency of the rbcL transcription in vitro is more than one transcript per one hundred templates per hour.

To determine whether chloroplast RNA polymerase will accurately terminate transcription in vitro, we have fused the spinach chloroplast rbcL promoter to the 3' end of the rbcL gene as well as to various factor independent transcription terminators from E. coli. Transcription of the rbcL minigene did not result in production of the expected 265 nucleotide RNA. However, the spinach chloroplast RNA polymerase did terminate transcription with varying efficiency at the thra, rrnB, rrnC and gene 32 terminators. The most efficient transcription termination was observed for the threonine attenuator. For each of the prokaryotic terminators, the chloroplast enzyme ceased transcription at essentially the same position as the E. coli RNA polymerase. These data indicate that the transcription termination process in chloroplasts has some features in common with the mechanism used in prokaryotes.