In vitro promoter recognition by the catalytic subunit of plant phage-type RNA polymerases

We identified sequence motifs, which enhance or reduce the ability of the Arabidopsis phage-type RNA polymerases RPOTm (mitochondrial RNAP), RPOTp (plastidial RNAP), and RPOTmp (active in both organelles) to recognize their promoters in vitro with help of a 'specificity loop'. The importance of this data for the evolution and function of the organellar RNA polymerases is discussed. The single-subunit RNA polymerase (RNAP) of bacteriophage T7 is able to perform all steps of transcription without additional transcription factors. Dicotyledonous plants possess three phage-type RNAPs, RPOTm-the mitochondrial RNAP, RPOTp-the plastidial RNAP, and RPOTmp-an RNAP active in both organelles. RPOTm and RPOTp, like the T7 polymerase, are able to recognize promoters, while RPOTmp displays no significant promoter specificity in vitro. To find out which promoter motifs are crucial for recognition by the polymerases we performed in vitro transcription assays with recombinant Arabidopsis RPOTm and RPOTp enzymes. By comparing different truncated and mutagenized promoter constructs, we observed the same minimal promoter sequence supposed to be needed in vivo for transcription initiation. Moreover, we identified elements of core and flanking sequences, which are of critical importance for promoter recognition and activity in vitro. We further intended to reveal why RPOTmp does not efficiently recognize promoters in vitro and if promoter recognition is based on a structurally defined specificity loop of the plant enzymes as described for the yeast and T7 RNAPs. Interestingly, the exchange of only three amino acids within the putative specificity loop of RPOTmp enabled the enzyme for specific promoter transcription in vitro. Thus, also in plant phage-type RNAPs the specificity loop is engaged in promoter recognition. The results are discussed with respect to their relevance for transcription in organello and to the evolution of RPOT enzymes including the divergence of their functions.