Genetic and Molecular Analysis of RNA Splicing in Escherichia coli

This chapter discusses the genetic and molecular analysis of RNA splicing in Escherichia coli. Non-targeted mutagenesis approaches based on phenotypic selections are useful alternatives to site-directed mutagenesis. It is also important to consider the type of mutational event desired, as in splicing the effective change is at the RNA level. As will be considered in more detail, the molecular consequences of transition versus transversion events for an RNA molecule, as opposed to a protein, can be great. The NH2-terminal polypeptide assay for td RNA splicing (the NH2-TS assay) is based on the ratio of NH2-TS, the translation product of the premRNA, to TS, the product of the mature mRNA. This ratio is elevated when splicing is defective. The suitability of E. coli as the host for this rapid assay rests on its meeting three criteria. Junction-specific oligodeoxynucleotide hybridization probes have been valuable tools for screening and confirming splicing defects. These probes have been used successfully with RNA isolated from phage-infected cells and after transcriptional induction of transformed E. coli.