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

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Abstract
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.