Photoaffinity Modification of Human 80S Ribosomes
with an mRNA Analog, a Hexaribonucleotide
pUUUGUU Derivative Carrying an Arylazido Group
at the Guanine Residue

D. M. Graifer, A. V. Lyakhovich, N. A. Demeshkina, A. V. Ivanov, M. N. Repkova,
A. G. Veniyaminova, and G. G. Karpova

Novosibirsk Institute of Bioorganic Chemistry, Siberian Division, Russian Academy of Sciences, Novosibirsk, 630090 Russia

Received April 1, 1998

Abstract—For photoaffinity modification of 80S ribosomes from human placenta, an mRNA analog was used,
namely, a derivative of hexaribonucleotide pUUUGUU (comprising Phe and Val codons) with an arylazido
group at the guanine residue. This derivative was prepared in two stages: UUUGUU was alkylated with
4[(N2-chloroethyl-N-methylamino)]benzylamine, ClRCH₂NH₂, at N7 of guanine to give an N-RCH₂NH₂
derivative. Then an arylazido group was selectively introduced at the aliphatic amino group by treatment with
p-azidotetrafluorobenzoic acid N-hydroxysuccinimide ester. The mRNA analog was also 5'-³²P-labeled before
cross-linking experiments. Irradiation of complexes of the pUUUGUU derivative with 80S ribosomes and cog-
nate tRNAs with mild UV light (270–380 nm) caused covalent attachment of the mRNA analog preferentially
to 40S subunits. In all cases, both 18S rRNA and proteins were modified; the ratio of the modification extents
depended on the type of the complex. The modification site in 18S rRNA was located using RNase H hydrolysis
of the modified rRNA in the presence of 20-mer oligodeoxyribonucleotides complementary to various
sequences in the rRNA. In complexes of the pUUUGUU derivative with 80S ribosomes and Phe-tRNA^Phe in
the P site (or with two tRNAs simultaneously), the degree of 18S rRNA modification was considerable, and the
attachment site of the mRNA analog was located in the 3'-terminal fragment (1831–1869). In a complex with
Val-tRNA^Val in the P site, modification of rRNA was substantially lower; in this case, two attachment sites were
identified: in the 3'-terminal fragment (1831–1869) and in the central part of 18S rRNA (674–1196). Protein
analysis by 1D and 2D gel electrophoresis showed that in all cases protein S15 was preferentially modified. We
also observed minor labeling of proteins S2 and S30 in complex with Phe-tRNA^Phe in the P site, and of proteins
S2 and S6 in complex with Val-tRNA^Val in the P site.

Key words: 80S ribosomes, photoaffinity modification, mRNA analog, decoding region, ribosomal proteins,
ribosomal RNA

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