12] Isolation and characterization of colicin fragments of bacterial 16S ribosomal RNA

Abstract

This chapter describes the large-scale purification of the colicin fragment from Escherichia coli ribosomes and illustrates its usefulness for physical studies. Essentially the same procedure can be followed to isolate colicin fragments from other bacteria. Ribosomal RNAs are complex molecules that elude detailed analysis of their structures by physical techniques. Nevertheless, by the method of comparative sequence analysis highly sophisticated models of their secondary structures have been proposed. Analysis of the final product is done by SDS-gel electrophoresis on cylindrical gels followed by scanning of the gel with UV light. Although the purity of the colicin fragment may also be checked by gel electrophoresis and autoradiography of 5'- 32 p-labeled RNA, such an analysis is a hazardous criterion for purity since especially high-molecular weight RNA contaminants may escape detection. This chapter also describes the properties of the colicin fragments such as melting characteristics and high-resolution NMR.