Efficiency and pattern of UV pulse laser-induced RNA-RNA cross-linking in the ribosome

Escherichia coli ribosomes were irradiated with a KrF excimer laser (248 nm, 22 ns pulse) with incident pulse energies in the range of 10-40 mJ for a 1 cm2 area, corresponding to fluences of 4.5 to 18 x 10(9) W m(-2), to determine strand breakage yields and the frequency and pattern of RNA-RNA cross- linking in the 16S rRNA. Samples were irradiated in a cuvette with one laser pulse or in a flow cell with an average of 4.6 pulses per sample. The yield of strand breaks per photon was intensity dependent, with values of 0.7 to 1.3 x 10(-3) over the incident intensity range studied. The yield for RNA-RNA cross-linking was 3 x 10(-4) cross-links/photon at the intensity of 4.5 x 10(9) W m(-2), an approximately 4-fold higher yield per photon than obtained with a transilluminator. The cross-link yield/photon decreased at higher light intensities, probably due to intensity-dependent photoreversal. The pattern of cross-linking was similar to that observed with low intensity irradiation but with four additional long-range cross-links not previously seen in E.coli ribosomes. Cross- linking frequencies obtained with one laser pulse are more correlated to internucleotide distances than are frequencies obtained with transilluminator irradiation.