Nucleic Acids Research, 1985, Vol. 13, No. 11 3953-3968
© 1985
Articles |
Analysis of a sequence region of 5S RNA from E. coli cross-linked in situ to the ribosomal protein L25
Max-Planck-Institut für Molekulare Genetik, Abt. Wittmann Ihnestr. 63–73, D-1000 Berlin 33, GDR
Received March 8, 1985. Revised May 13, 1985. Accepted May 13, 1985.
70S ribosomes from E. coli were chemically cross-linked under conditions of in vitro protein biosynthesis. The ribosomal RNAs were extracted from reacted ribosomes and separated on sucrose gradients. The 5S RNA was shown to contain the ribosomal protein L25 covalently bound. After total RNase T1 hydrolysis of the covalent RNA-protein complex several high molecular weight RNA fragments were obtained and identified by sequencing. One fragment, sequence region U103 to U12O, was shown to be directly linked to the protein first by protein specific staining of the particular fragment and second by phosphor cellulose chromato-graphy of the covalent RNA-protein complex. The other two fragments, U89 to G106 and A34 to G51, could not be shown to be directly linked to L25 but were only formed under cross-linking conditions. While the fragment U89 to G106 may be protected from RNase T1 digestion because of a strong interaction with the covalent RNA-protein complex, the formation of the fragment A34 to G51 is very likely the -result of a double monovalent modification of two neighbouring guanosines in the 5S RNA
The RNA sequences U103 to U12O established to be in direct contact to the protein L25 within the ribosome falls into the sequence region previously proposed as L25 binding site from studies with isolated 5S RNA-protein complexes