Site-specific photo crosslinking has been used
to investigate the RNA neighborhood of 16S rRNA positions
U788/U789 in Escherichia coli 30S subunits. For
these studies, site-specific psoralen (SSP) which contains
a sulfhydryl group on a 17 Å side chain was first
added to nucleotides U788/U789 using a complementary guide
DNA by annealing and phototransfer. Modified RNA was purified
from the DNA and unmodified RNA. For some experiments,
the SSP, which normally crosslinks at an 8 Å distance,
was derivitized with azidophenacylbromide (APAB) resulting
in the photoreactive azido moiety at a maximum of 25 Å
from the 4′ position on psoralen (SSP25APA). 16S
rRNA containing SSP, SSP25APA or control 16S rRNA were
reconstituted and 30S particles were isolated. The reconstituted
subunits containing SSP or SSP25APA had normal protein
composition, were active in tRNA binding and had the usual
pattern of chemical reactivity except for increased kethoxal
reactivity at G791 and modest changes in four other regions.
Irradiation of the derivatized 30S subunits in activation
buffer produced several intramolecular RNA crosslinks that
were visualized and separated by gel electrophoresis and
characterized by primer extension. Four major crosslink
sites made by the SSP reagent were identified at positions
U561/U562, U920/U921, C866 and U723; a fifth major crosslink
at G693 was identified when the SSP25APA reagent was used.
A number of additional crosslinks of lower frequency were
seen, particularly with the APA reagent. These data indicate
a central location close to the decoding region and central
pseudoknot for nucleotides U788/U789 in the activated 30S
subunit.