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.
Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695-7622, USA.
Tatjana
T
Shapkina
Simon
S
Lappi
Stefan
S
Franzen
Paul
P
Wollenzien
D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D007834:Lasers; D008969:Molecular Sequence Data; D017785:Photons; D011684:Purine Nucleosides / Q000528:radiation effects; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000528:radiation effects*; D012270:Ribosomes / Q000528:radiation effects*; D014466:Ultraviolet Rays
D011684:Purine Nucleosides; D012336:RNA, Ribosomal, 16S