RNA Polymerase Activity Assay on Biochips: Correlation between Template DNA Density and RNA Synthesis

摘要

In vitro synthesis of RNAs by bacteriophage T7 RNA polymerase (T7 RNAP) has been applied extensively to the investigation of RNA serving as a biologically active molecule. T7 RNAP is a single subunit enzyme with a molecular weight of 98 kDa that is capable of catalyzing transcription without any accessory proteins. Other RNA polymerases derived from bacteriophage (SP6 and T3) were also useful for in vitro synthesis of RNA. Each RNA polymerase has different DNA promoter sequences for initiation of transcription. The behavior of T7 RNAP is critically dependent on its interaction with the promoter element in template DNA. The T7 bacteriophage genome contains a variety of promoters that are recognized by T7 RNAP, all of which are related to a 23-base pair consensus sequence (see Figure 1). The promoter can be divided into a recognition domain, encompassing positions 17 through 5, and an initiation domain, encompassing positions 4 through +6 with a transcription start site initiates at the +1 position.