Chapter 14 - A Rapid Genetic Method for the Study of RNA Binding Proteins

Abstract

This chapter introduces a rapid genetic method for the study of ribonucleic acid (RNA) binding proteins (RNA-BP). This method is useful for: (1) identifying mutations in the RNA or the RNA-BP that affect binding affinity and/or specificity, (2) obtaining quantitative estimates of relative binding affinities from repression ratios easily measured in E. coli, and (3) developing models for RNA–protein interaction on the basis of the altered specificity or enhanced affinity of RNA-BP suppressor mutants thereby, identified and characterized. Using this approach, translational repression of modified lacZ transcripts is achieved not only with Rev, but also with other heterologous RNA-BPs including: U1A, nucleolin, and alfalfa mosaic virus coat protein. A future goal is to adapt this method for cloning complementary deoxyribonucleic acids (cDNAs) encoding RNA-BPs that bind to specific RNA sequences. This chapter discusses about the quantitative model for translation repression by heterologous RNA-BPS. The chapter also explains concepts related to methods such as strains, plasmids, and RNA-BP plasmid. Preparation and use of competent cells is also discussed. In addition, the chapter highlights the conditions for expression of the RNA-BP in E.coli and elaborates upon estimating dissociation constants.