Nucleic Acids Research, 1983, Vol. 11, No. 2 515-524
© 1983
MOLECULAR BIOLOGY |
Crosslinking of Escherichia coli 50S ribosomal subunits with chlorambucilyl oligoprolyl phenylalanyltRNA molecular rulers
Department of Chemistry, University of Denver Denver, CO 80208, USA
+To whom reprint requests should be sent, at: Department of chemistry, University of south Florida, Tampa, Florida 33620, USA.
Received October 4, 1982. Revised December 14, 1982. Accepted December 14, 1982.
A series of P-site probes, chlorambucilyl-(Pro)n-phe-tRNAPhe, were prepared and reacted with poly(U)-directed Escherichia coli MRE 600 ribosomes. Upon binding of the probes to ribosomes, 90% of the cpm bound were not released following subsequent interaction with puromycin. In the absence of poly(U) or in the presence of poly(C), binding was limited to the amount of cpm bound if ribosomes were incubated in the presence of puromycin before adding modified tRNA and poly(U). AcPhe-tRNAPhe was a competitive inhibitor of chlorambucilyl Phe-tRNAPhe, Binding to 50S subunits was strongly stimulated by poly (U), while binding to 30S subunits was not. Crosslinked 50S proteins were analyzed by two-dimensional gel electrophoresis. Cross1inking with molecular rulers containing zero prolines led to poly(U)-dependent labeling of L1 and L27. With rulers containing five prolines, L6, L25, L28, and the group L18,23,24 were labeled. Analysis of crosslinked ribosomal RNA on sucrose density gradients revealed almost no cpm in the 16S or 23S peaks, but only in the 5S peaks. This was observed with molecular rulers containing either zero or five proline residues.
*Present address: Department of science, Western Montana college, Dillon, Montana 59725, USA.