Assay of Transcription Termination by Ribosomal Protein L4

Abstract

Publisher Summary This chapter discusses assaying of transcription termination by ribosomal protein L4. The chapter discusses the two approaches, reporter genes and in vitro transcription that characterize attenuation control of the S10 ribosomal protein operon in Escherichia coli (E. coli) and other bacteria. The use of reporter genes usually assumes that transcription rates can be deduced from the rate of accumulation of a protein product. However, the outcome of the measurement depends not only on accumulation of transcript, but also on the rate of its translation. The mechanism of L4-mediated transcription control of the S10 operon by using a cell-free transcription system is discussed. The principal approaches used in the experimentation have included kinetic analysis of regulation, mutational analysis of the mRNA target, disruption of nascent RNA structures with oligonucleotides, and competition for L4 by addition of other RNAs. Furthermore, purification and restart of the transcription complex in the presence or absence of its ligands allow ordering of the NusA- and L4-dependent steps in the regulation. In vitro transcription studies shows that L4-mediated transcription termination is preceded by a NusA-stimulated pause of the RNA polymerase at the site of termination.