Nucleic Acids Research, 1981, Vol. 9, No. 13 3187-3198
© 1981
MOLECULAR BIOLOGY |
Fluorescence modification of Escherichia coli 5S RNA
,Max-Planck-Institut für Molekulare Genetik Abt. Wittmann, Ihnestrasse 63-73, 1000 Berlin 33, GFR Qayton Foundation Biochemical Institute, Department of Chemistry, The University of Texas Austin, TX 78712, USA
For offprints contact : V. A. Erdmann
Received March 23, 1981.
Reaction of 5S RNA with cloracetaldehyde leads to the conversion of unpaired adenines to the fluorescent 1,N6 -etheno-adenine derivatives. Up to 16 of the 23 adenines in free 5S RNA can be modifed, the fastest reacting are A29' A34' A39 and A57–59. Partial modification of adenines in this area resultsin a 20% reduction in the efficiency of 5S RNA incorporation into 50S subunits during reconstitution and a 15% reduction in the activity of these subunits in peptide synthesis. Fluorescence from 1,N6 -etheno-adenine is quenched in free 5S RNA and is not detectably further influenced by the binding of proteins E-L5, E-L18 and E-L25, nor by the first stage of the two step E. coli 50S subunit reconstitution procedure. However, the fluorescence is further reduced to near zero after the second step of the reconstitution. Thus, 5S RNA free in solution contains 16 unpaired adenines, those in the region between A29 and A59 being particularly accessible to modification bychloracetaldehyde. This portion of the 5S RNA molecule appears to undergoeither a conformational change or interacts with other ribosomal componentsin the last stage of subunit reassembly.
*Current address: Institut für Biochemie, FB 21, Thielallee 69–73, Freie Universität Berlin, D-1000 Berlin 33 (Dahlem)