Assay of Intrinsic Transcript Termination by E. Coli RNA Polymerase on Single-Stranded and Double-Stranded DNA Templates

Abstract

This chapter describes the principal techniques used for biochemical characterization of transcript termination. The chapter focuses on intrinsic termination by Escherichia coli (E. coli) RNA polymerase on double-stranded (dsDNA) and single-stranded (ssDNA) DNA templates. These methods can be easily adapted to study transcript termination of other RNA polymerases and factor-dependent termination, or extended to study aspects of transcript elongation. To study intrinsic termination of E. coli RNA polymerase, 5ʹ biotinylated linear dsDNA templates generated by polymerase chain reaction (PCR) using one 5ʹ biotinylated oligonucleotide and one unmodified oligonucleotide. The template contains a promoter and an intrinsic termination site. Two solid-phase transcription methods are developed to prepare transcript elongation complexes halted at a distinct position along the DNA template. Use of biotinylated templates provides an attractive alternative to using T4 polynucleotide kinase and [γ 32 P] ATP to radiolabel one of two oligonucleotides required to amplify a linear DNA template by PCR.