Promoter Length Affects the Initiation of T7 RNA Polymerase In Vitro: New Insights into Promoter/Polymerase Co-evolution.

Polymerases are integral factors of gene expression and are essential for the maintenance and transmission of genetic information. RNA polymerases (RNAPs) differ from other polymerases in that they can bind promoter sequences and initiate transcription de novo and this promoter recognition requires the presence of specific DNA binding domains in the polymerase. Bacteriophage T7 RNA polymerase (T7RNAP) is the prototype for single subunit RNA polymerases which include bacteriophage and mitochondrial RNAPs, and the structure and mechanistic aspects of transcription by T7 RNAP are well characterized. Here, we describe experiments to determine whether the prototype T7 RNAP is able to recognize and initiate at truncated promoters similar to mitochondrial promoters. Using an in vitro oligonucleotide transcriptional system, we have assayed transcription initiation activity by T7 RNAP. These assays have not only defined the limits of conventional de novo initiation on truncated promoters, but have identified novel activities of initiation of RNA synthesis. We propose that these novel activities may be vestigial activities surviving from the transition of single subunit polymerase initiation using primers to de novo initiation using promoters.

A specific, promoter-independent activity of T7 RNA polymerase suggests a general model for DNA/RNA editing in single subunit RNA Polymerases.

Insertional RNA editing has been observed and characterized in mitochondria of myxomycetes. The single subunit mitochondrial RNA polymerase adds nontemplated nucleotides co-transcriptionally to produce functional tRNA, rRNA and mRNAs with full genetic information. Addition of nontemplated nucleotides to the 3' ends of RNAs have been observed in polymerases related to the mitochondrial RNA polymerase. This activity has been observed with T7 RNA polymerase (T7 RNAP), the well characterized prototype of the single subunit polymerases, as a nonspecific addition of nucleotides to the 3' end of T7 RNAP transcripts in vitro. Here we show that this novel activity is an editing activity that can add specific ribonucleotides to 3' ends of RNA or DNA when oligonucleotides, able to form intramolecular or intermolecular hairpin loops with recessed 3' ends, are added to T7 RNA polymerase in the presence of at least one ribonucleotide triphosphate. Specific ribonucleotides are added to the recessed 3' ends through Watson-Crick base pairing with the non-base paired nucleotide adjacent to the 3' end. Optimization of this activity is obtained through alteration of the lengths of the 5'-extension, hairpin loop, and hairpin duplex. These properties define a T7 RNAP activity different from either transcriptional elongation or initiation.