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Nucleic Acids Research, 1985, Vol. 13, No. 19 6919-6936
© 1985

Articles

Investigation of the tertiary folding of Escherichia coli 16s RNA by in situ intra-RNA crosslinking within 30S ribosomal subunits

Johannes Atmadja, Richard Brimacombe, Helmut Blöcker and Ronald Frank

Max-Planck-Institut für Molekulare Genetik, Abteilung Wittmann D-1000 Berlin-Dahlem, FRG Gesellschaft für Biotechnologische Forschung Abteilung DNA-Synthese, D-3000 Braunschweig-Stöckheim, FRG

Received July 16, 1985. Accepted September 6, 1985.

Intra-RNA cross-links were introduced into E. coli 30S ribosomal subunits by mild ultraviolet irradiation. The subunits were partially digested with cobra venom nuclease, followed in some cases by a second partial digestion with ribonuclease H in the presence of the hexanucleotide d-(CTTCCC). The cross-linked RNA complexes were separated by two-dimensional gel electrophoresis and the sites of cross-linking analysed by our published procedures. Tertiary structural cross-links in the 16S RNA were identified between positions 31 and 48, between oligonucleotides 1090–1094 and 1161–1164, and between oligonucleotides 1125–1127 and 1280–1281. The first of these imposes a rigid constraint on the relative orientations of helices 3 and 4 of the 16S secondary structure. A further tertiary cross-link (which could not be precisely localised) was found between regions 1–72 and 1020–1095, and secondary structural cross-links were identified between positions 497 and 545–548, and positions 1238–1240 and 1298.


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