Nucleic Acids Research, Vol 26, Issue 11 2519-2525, Copyright © 1998 by Oxford University Press
P Sergiev, S Dokudovskaya, E Romanova, A Topin, A Bogdanov, R Brimacombe and O Dontsova
Two photoreactive diazirine derivatives of uridine were used to study
contacts between 5S rRNA and 23 rRNA in situ in Escherichia coli ribosomes.
2'-Amino-2'-deoxy-uridine or 5-methyleneaminouridine were introduced into
5S rRNA by T7 transcription. After incorporation of these uridine analogues
into the transcript their amino groups were modified with
4-[3-(trifluoromethyl)-3 H -diazirin-3-yl]benzyl isothiocyanate or the N
-hydroxysuccinimide ester of 4-[3- (trifluoromethyl)-3 H
-diazirin-3-yl]benzoic acid respectively. 5S rRNA carrying the
photoreactive diazirine groups (referred to as the 2'- aminoribose
derivative and the 5-methyleneamino derivative respectively) was
reconstituted into 50S subunits or 70S ribosomes. After mild UV irradiation
cross-links formed to 23S rRNA were analysed by standard procedures. All of
the observed cross-links involved residue U89 of the 5S rRNA. Three
nucleotides of 23S rRNA were cross- linked to this residue with the
5-methyleneamino derivative, namely U958, G1022 and G1138. With the
2'-aminoribose derivative a single cross-link was found, to U958. The
significance of these cross-links for our understanding of the structure
and function of 5S rRNA and its environment in the ribosome are discussed.
ARTICLES
The environment of 5S rRNA in the ribosome: cross-links to the GTPase- associated area of 23S rRNA
A. N. Belozersky Institute of Physico-Chemical Biology and Department of Chemistry, Moscow State University, Moscow 119899, Russia.
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