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Nucleic Acids Research, 1990, Vol. 18, No. 12 3521-3528
© 1990

MOLECULAR BIOLOGY

Use of synthetic oligoribonucleotides to probe RNA-protein interactions in the MS2 translational operator complex

S.J. Talbot, S. Goodman1, S.R.E. Bates2, C.W.G. Fishwick1 and P.G. Stockley

Department of Genetics, University of Leeds Leeds LS2 9JT 1School of Chemistry, University of Leeds Leeds LS2 9JT 2Applied Biosystems (UK) Ltd, Kelvin Close, Birchwood Science Park Warrington WA3 7PB, UK

Received March 22, 1990. Revised May 3, 1990. Accepted May 3, 1990.

Synthetic oligoribonucleotides have been used to probe the interaction of MS2 coat protein with the translational operator of the MS2 replicase gene. We have investigated the possible formation of a transient covalent bond between the single-stranded uridine residue, at position –5, and a cysteine side-chain on the coat protein, by the incorporation of a chemically modified residue (5-BrU) at this position. This chemically synthesised operator variant has a binding constant of between 10 and 50 times greater than that of the wild type and is therefore comparable with the tight binding variant having a cytidine substituted at the –5 position. Dissociation kinetics show that the complex with the 5-BrU operator is more stable than the –5C variant; a result which is consistent with the formation of a Michael adduct at the –5 position. In addition, a number of other chemical variants of the operator have been analysed. These include operators incorporating deoxyadenine residues at each of the important single-stranded adenine sites.

Recently the Michael adduct proposal has been challenged on the basis of mutagenesis of the coat protein cysteine residues (1). These results are discussed in the light of our data in support of Michael adduct formation.


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