Nucleic Acids Research, 1983, Vol. 11, No. 7 2035-2052
© 1983
MOLECULAR BIOLOGY |
Nuclease mapping of the secondary structure of the 49-nucleotide 3' terminal cloacin fragment of Escherichia coli 16S RNA and its interactions with initiation factor 3
Department of Chemistry, University of South Florida Tampa, FL 33620, USA
Received January 7, 1983. Revised March 11, 1983. Accepted March 11, 1983.
Escherichia coli translational initiation factor 3 (IF3) may be crosslinked to the 3' end of 16S RNA in 30S ribosomal subunits. In order to determine the sequence to which IF3 may bind in vivo, samples of 5'-32p labelled 3' terminal 49-nucleotide fragment of 16S RNA were incubated 5 min. at 37° in 40 mM Tris-HOAc, pH 7.4, 100 mM NaCl, 1 mM Mg(0Ac)2, 1 mM ZnSO4, with or without IF3, then reacted a further 5 min with nuclease S1, RNase Tl, or RNase A. Base pairing between the 5' and 3' legs of the fragment occurs in the absence of IF3, but is disrupted by IF3 binding. IF3 appears to protect some residues near the 5' end of the fragment (U1495, A1499, A1500, A1502, and A1503) from nuclease Sl, and potentiates S1 attack on others (G1494, G1497, C1501, G1504, G1505, U1506, G1517, G1529, G1530, and C1533). A series of equimolar reactions at increasing dilution imply an association constant range of 1.4–7.0x107 M–1.
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