DNA Helicase Reduces Production of Aberrant Run-off Transcripts during in vitro RNA Synthesis with T7 RNA Polymerase

Abstract

In vitro transcription using the bacteriophage RNA polymerase is an efficient way to synthesize RNA molecules in a large scale. There are several RNA polymerases and each enzyme needs a different DNA promoter sequence for initiation of transcription.1-3 Among the RNA polymerases used to synthesize RNAs in vitro, bacteriophage T7 RNA polymerase (T7 RNAP) is the most widely used due to its convenience and ample synthesis of RNA using DNA template harboring conserved sequences of upstream promoter.T7 RNAP is a single subunit enzyme with a molecular weight of 98 kDa, which can catalyze RNA synthesis without any accessory proteins.4-6 T7 RNAP binds to a variety of promoter sequences present in bacteriophage genome, all of which contain a 17-base-pair of consensus sequence as promoter.6,7 In this study, forked DNA construct was designed, which contains the 5' and 3' overhangs comprised of thymine nucleotides and the 35-base-pair duplex harboring the T7 promoter sequence ( Fig. 1(a)).The fidelity of the in vitro transcription process is usually sufficient for low scale synthesis of RNAs. However, nontemplated 3'-extension incorporating extra nucleotides at the 3'-terminal of nascent RNA strands has been often recognized as a propensity of T7 RNAP, producing aberrant run-off transcripts in some cases.8,9 Especially, these incorrect run-off transcripts are often generated in a relatively short RNA synthesis less than 20 bases.9 Thus, oligoribonucleotide synthesis with defined sequences entails verification of exact length as well as 3'-terminal sequences. Since translocation of the T7 RNAP along the DNA template is elemental for the nascent RNA synthesis, we hypothesized that T7 RNAP could be stalled at the end of DNA template without being released from the DNA template in the case of run-off products generation. This circumstance would make the T7 RNAP add a few more extra nucleotides to the transcript and generate aberrant run-off RNA products.T7 bacteriophage gene gp4A encodes another class of DNA translocating proteins, T7 DNA helicase, which is a 63-kDa replicative primase-helicase protein 10 that assembles into a ring-shaped hexamer in the presence of dTTP.11-13 T7 helicase can form both hexamer and heptamer, but hexamer Figure 1. (a) Forked DNA template for both T7 RNAP and T7 helicase is shown, which contains T7 promoter sequence (indicated in a box) and two tails at the end of the template. In vitro transcription with the template generates aberrant RNA products as well as 14-mer RNA. +1 indicates initiation point of RNA synthesis. (b) Unwinding of the forked DNA substrate by T7 DNA helicase.3 2 P-labeled forked DNA was used as a substrate, and an excess amount of unlabeled DNA was added as a trap DNA. Reaction aliquots of the unwinding reaction at increasing time points were analyzed on the native-PAGE (10%).