Nucleic Acids Research, Vol 24, Issue 12 2228-2235, Copyright © 1996 by Oxford University Press
EV Alexeeva, OV Shpanchenko, OA Dontsova, AA Bogdanov and KH Nierhaus
The contacts of phosphate groups in mRNAs with ribosomes were studied. Two
mRNAs were used: one mRNA contained in the middle two defined codons to
construct the pre- and the post-translocational states, the other was a
sequence around the initiation site of the natural cro- mRNA.
Phosphorothioate nucleotides were randomly incorporated at a few A, G, U or
C positions during in vitro transcription. Iodine can cleave the thioated
positions if they are not shielded by ribosomal components. Only a few
minor differences in iodine cleavage of ribosome bound and non-bound mRNA
were observed: the nucleotide two positions upstream of the decoding codons
(i.e. those codons involved in codon- anticodon interactions) showed a
reduced accessibility for iodine and the nucleotide immediately following
the decoding codons an enhanced accessibility in both elongating states. In
initiating ribosomes where the mRNA contained a strong Shine-Dalgarno
sequence, at least five phosphates were additionally slightly protected
covering the Shine- Dalgarno sequence and nucleotides downstream including
the initiator AUG in the P site (Al, G3, G-2, G-5 and A-7). The low contact
levels of the phosphates in the mRNA with the elongating ribosome
strikingly contrast with the pronounced contact patterns previously
described for tRNAs. The data obtained in this study, as well as results of
previous studies, suggest that mRNA regions downstream and upstream of
decoding codons form only weak contacts with ribosomal components and that
the mRNA thus is mainly fixed by codon-anticodon interaction on the
elongating ribosome.
ARTICLES
Interaction of mRNA with the Escherichia coli ribosome: accessibility of phosphorothioate-containing mRNA bound to ribosomes for iodine cleavage
Max-Planck-Institut fur Molekulare Genetik, Berlin, Germany.
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