Nucleic Acids Research, Vol 27, Issue 11 2283-2290, Copyright © 1999 by Oxford University Press
M Osswald and R Brimacombe
Three contiguous fragments of Escherichia coli 5S rRNA were prepared by T7
transcription from synthetic DNA templates. The central fragment,
comprising residues 33-71 of the molecule, was transcribed in the presence
of 4-thiouridine triphosphate together with [32P]UTP. The three transcripts
were ligated together, yielding a 5S rRNA analogue carrying 4-thiouridine
residues at positions 40, 48, 55 and 65 in helices II and III. After
ligation, the 4-thiouridine residues were derivatised with p -azidophenacyl
bromide. The modified 5S rRNA was reconstituted into 50S subunits and these
subunits were used to prepare 70S ribosomes in the presence or absence of
tRNA and mRNA. The azidophenyl groups were then photoactivated by mild
irradiation at 300 nm and the products of cross-linking analysed by our
standard procedures. Multiple cross-links from 5S rRNA to two distinct
regions of the 23S rRNA were observed. The first region was located in
helix 38 in Domain II of the 23S molecule, with cross-links at sites
between nucleotides 885 and 922. The second region covered helices 81-85 in
Domain V, with sites between nucleotides 2272 and 2345. Taken together with
previous data, these results serve to define the arrangement of the 5S rRNA
molecule relative to the 23S rRNA within the 50S subunit.
ARTICLES
The environment of 5S rRNA in the ribosome: cross-links to 23S rRNA from sites within helices II and III of the 5S molecule
Max-Planck-Institut fur Molekulare Genetik, Ihnestrasse 73, 14195 Berlin, Germany.
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