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Nucleic Acids Research, 1983, Vol. 11, No. 6 1687-1706
© 1983

MOLECULAR BIOLOGY

Localisation of a series of intra–RNA cross-links in the secondary and tertiary structure of 23S RNA, induced by ultraviolet irradiation of Escherichia 50S ribosomal subunits

Wolfgang Stiege, Carola Gkotz and Richard Brimacombe

Max-Planck-Institut fur Molekulare Genetik, Abteilung Wittmann D-1000 Berlin-Dahlem, FRG

Received January 20, 1983. Accepted February 23, 1983.

Intra–RNA cross-links were introduced into E. coli 50S ribosomal subunits by mild ultraviolet irradiation. The sub-units were partially digested with cobra venom nuclease, and the cross-linked RNA complexes were isolated by two-dimensional electrophoresis. Many of the complexes were submitted to a second partial digestion procedure. Oligonucleotide analysis of the RNA fragments obtained in this manner enabled cross-links between the following ribonuclease T1 oligonucleotides in the 23S RNA to be established: positions 292-296 and 339-350; 601-604 and 652-656; 1018-1022 and 1140-1149; 1433-1435 and 1556-1560; 1836-1839 and 1898-1903; 2832-2834 (tentative) and 2878-2885; 2849-2852 and 2865-2867 (tentative); 739-748 and 2609-2618; 571-577 and 2030-2032; 1777-1792 (tentative) and 2584-2588. The first seven of these cross-links lie within the secondary structure of the 23S RNA, whereas the last three are tertiary structural cross--links. The degree of precision of the individual determinations was variable, depending on the nucleotide sequence in the vicinity of the cross-link site concerned.


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