Characterization of Protein–Nucleic Acid Interactions that are Required for Transcription Processivity

Abstract

This chapter provides a detailed protocol for developing a minimal nucleic acid scaffold that defines each of three nucleic acid binding sites and integrates them in the fully stable elongation complex (EC). To determine the minimal requirements for the DNA to sustain the fully stable and functional EC, two in vitro approaches were combined in manipulating with the EC: “walking” and “template switching.” After the switch, transcription continues on the secondary template, which can be a single-stranded, double stranded or partially double-stranded DNA oligonucleotides. The template-switching phenomenon provides a powerful method to study protein–DNA interaction in the EC as the portion of DNA normally protected by RNAP from the chemical and enzymatic attack can be changed at will in the secondary template. Template switching has been observed with other bacterial RNAP and also mammalian RNAP II in vitro and in vivo. To prepare the EC with the shortest possible RNA without disrupting the complex, the reaction of pyrophosphorolysis is utilized. Processive pyrophosphorolysis drives a “normal” EC carrying transcript of 11 nt or longer in a reverse reaction, resulting in progressive shortening of RNA. The methods of DNA and RNA minimization described can be used to measure the parameters of the nucleic acid scaffold for other bacterial and also eukaryotic RNAPs. This information is essential for understanding the mechanisms of transcription elongation and termination, and their regulation by various intrinsic signals and protein factors.