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Published online 3 March 2004

Nucleic Acids Research, 2004, Vol. 32, No. 4 1518-1526
© 2004 Oxford University Press

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

Tatjana Shapkina, Simon Lappi1, Stefan Franzen1 and Paul Wollenzien*

Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695-7622, USA and 1 Department of Chemistry, North Carolina State University, Raleigh, NC 27695-8204, USA

*To whom correspondence should be addressed. Tel: +1 919 515 5703; Fax: +1 919 515 2047; Email: paul_wollenzien{at}ncsu.edu

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 109 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 109 W m–2, an ~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.


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