A study of T7 RNA polymerase‐Template interaction utilizing CRISPR/dCas9 protein

Abstract

In vitro transcription is a simple and broadly applicable procedure which synthesizes RNA from DNA template by using bacteriophage RNA polymerase. Beside its importance for gene regulations and cell functions, transcriptional mechanism is not fully understood yet. In previous transcriptional pausing studies, it has been found that selected phage RNA polymerases exhibit different behaviors when they encounter a blockade during transcription. This research is investigating structural and chemical interactions between the DNA template, RNA polymerase and CRISPR-dCas9 protein at the point of an engineered transcription blockade. The CRISPR system is an adaptive immune mechanism present in many bacteria and the majority of characterized Archaea. This system binds to nucleic acids, which can be RNA or DNA and modifies specific sequences at desired locations. The dCas9 (nuclease deficient) protein retains an ability to bind template DNA at a specific location when guided by a complementary RNA molecule. This complementary RNA is called guide RNA (gRNA). T7 RNA polymerase in vitro transcription of a purified U5 DNA template was optimized with respect to time, template and rNTP concentration. This established conditions under which to pause transcription, utilizing dCas9 protein, and analyze protein, polymerase, and nucleic acid interactions. We also hypothesized that the structural interactions between RNA polymerase and dCas9 may affect the binding between the DNA template and RNAP. Simple molecular modeling data were generated by using 3D visualization software. This research is important for creating an efficient and reversible dCas9 mediated transcription control mechanism.