Chemical Methods for the Structural Study of the Ribosome: Judgment Day

Abstract

The data on RNA–RNA interactions between the components of the E. colitranslation machinery obtained by X-ray crystallography and chemical methods are compared. The approaches to the study of RNA secondary and tertiary structure are assessed. The following conclusions are made: comparative sequence analysis and compensatory mutations approach both give reliable data on the RNA secondary structure. The chemical modification technique provides good results. Local cleavage of internucleotide bonds by hydroxyl radicals is reliable in the frame of its 40 A resolution, in contrast to the application of copper–phenanthroline complex as a cleavage reagent, which is unreliable. Direct UV irradiation and nitrogen mustard treatment are the best methods of crosslink generation. In vitrotranscription is the only good method for the incorporation of nucleotide analogs in RNA. RNase H hydrolysis and/or nucleotide-specific RNases fingerprints must be applied for the crosslink site determination in parallel with reverse transcription.