Fig. 4

Oligoribonucleotide synthesis by chimeric primases of A. thaliana mitochondria and bacteriophage T7. A) Representative diagram of the regions used to construct the chimeras between the ZFDs of T7 Primase and AtPrimase. The green blocks represent the ZFDs and the red blocks the RNAP domains. The numbers indicate the amino acids of the wild-type enzymes that were used to construct the chimeras by swapping the ZFD and RNAP domains. B) SDS-PAGE gel showing purified chimeric primases. C) Denaturing sequencing gel showing oligoribonucleotide synthesis by wild-type AtPrimase and T7 Primase on their canonical templates (lane 1 and 2, respectively), chT7At chimera (lanes 3 to 5 and 9 to 11) and chAtT7 chimera (lanes 6 to 8 and 12 to 14). The identity of the canonical substrates for AtPrimase and T7 Primase is 5’-T6GGGAT7-3’ substrate and 5’-T6GGTCT7-3’ substrate, respectively. The cryptic elements are underlined. The chT7At chimera efficiently synthesizes primers and harbors an increased substrate-independent oligoribonucleotide synthesis activity. This chimera was only able to synthesize primers on the substrate containing the bacteriophage T7-primase recognition trinucleotide. chAtT7 was extremely inefficient, synthesizing a product of maximum size of 4 nucleotides. Primase chimeras were tested at 100, 200 and 400 nM. The ssDNA substrate was used at 5 µM. D) Wild-type and chimeric AtPrimases prime organellar DNA polymerase (AtPolIB). Coupled AtPrimase-DNA polymerase reactions on a canonical AtPrimase ssDNA recognition sequence labeled with [α-³²P]-dATP. AtPolIB, generates DNA:RNA products that correspond to products longer than 36 nts in the presence of increasing concentrations of AtPrimase and ChAtT7 chimera (lanes 2 to 5 and 6 to 9, respectively). The AtPrimase recognition sequence is underlined. In this experiment AtPrimase was present at concentration of 150, 300, and 600 nM whereas AtPolIB was maintained at a fixed concentration of 150 nM