Engineering of Elongation Complexes of Bacterial and Yeast RNA Polymerases

Abstract

This chapter discusses the elongation of RNA, accompanied by stepwise forward translocation of RNA polymerase (RNAP) along the template. To understand the mechanisms by which DNA sequences and protein factors regulate elongation, the need to identify the components of the elongation complex (EC) that are targeted by regulatory signals is required. This chapter describes posttranscriptional modifications of the RNA in the EC, utilized to introduce changes in the RNA without altering the sequence of the DNA template and to address the effects of transcript composition on EC stability and catalytic activity. Another set of experimental techniques is based on the reconstitution (assembly) of the EC from RNAP and synthetic RNA and DNA oligonucleotides. This approach is useful for the introduction of mismatches in the RNA, and for obtaining ECs with RNAs shorter than 8 nt. Experiments with assembled ECs allows to address the specific roles of the RNA and the DNA within the regions protected by the enzyme, which could not be accomplished using promoter-initiated ECs.