Use of synthetic oligoribonucleotides to probe RNA-protein interactions in the MS2 translational operator complex.
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. These results are discussed in the light of our data in support of Michael adduct formation.
Department of Genetics, School of Chemistry, University of Leeds, UK.
S J
SJ
Talbot
S
S
Goodman
S R
SR
Bates
C W
CW
Fishwick
P G
PG
Stockley
D001482:Base Composition; D001483:Base Sequence; D002213:Capsid / Q000378:metabolism*; D036022:Capsid Proteins; D002851:Chromatography, High Pressure Liquid; D007700:Kinetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D015345:Oligonucleotide Probes / Q000032:analysis; D009875:Operator Regions, Genetic; D012313:RNA / Q000032:analysis / Q000378:metabolism*; D016601:RNA-Binding Proteins; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism; D013816:Thermodynamics
D036022:Capsid Proteins; D015345:Oligonucleotide Probes; D016601:RNA-Binding Proteins; D012313:RNA; D012324:RNA-Dependent RNA Polymerase