19] Intra-RNA and RNA—protein cross-linking techniques in Escherichia coli ribosomes

Abstract

This chapter describes the methods currently in use in the laboratory for the analysis of RNA-protein and intra-RNA cross-link sites in E. coli ribosomal RNA. The methods are still by no means ideal, and are continually being modified and improved. In particular, the resolution of the RNA-protein cross-linked complexes on two-dimensional gels is not yet as sharp as that of the corresponding intra-RNA cross-linked complexes. Nonetheless the system has enabled us to isolate a considerable number of individual complexes, and from the example shown that eleven specific cross-link sites could be identified, involving nine of the 30S ribosomal proteins linked to RNA sequences comprising 30–150 nucleotides. The total data accumulated so far include nearly 40 intra-RNA cross-link sites identified to within two or three nucleotides in the E. coli 30S and 50S subunits, as well as a similar number of RNA-protein cross-link sites involving 19 different ribosomal proteins.