RNA-protein interactions in the bacteriophage MS2

Abstract: The bacteriophage MS2 coat protein interacts specifically with a 19 nucleotide long hairpin formed within the viral RNA genome. In the current work the structural details of this interaction have been examined using X-ray crystallography of complexes where the RNA hairpin sequence has been varied.The requirements for binding of this hairpin to the coat protein have been examined by studying the structures of longer and shorter hairpin variants. The results show that a hairpin as short as eight nucleotides in length can bind and form a stable complex with the protein. The important -5 position, which is a uracil in the wild type complex, interacts with the protein both by stacking onto a tyrosine side chain and by forming a hydrogen bond to an asparagine side chain. The structure of an RNA hairpin containing a pyridin-4-one base at the -5 position in complex with the coat protein shows a different binding than in all other studied complexes so far. The -6 base in this complex is stacked onto the tyrosine side chain instead of the -5 base. The explanation for this different behaviour has been proposed to be the changed electrostatic properties of the pyridin-4-one base. This result shows the importance of combining biochemical studies with structural studies. Other variant bases, both natural and non-natural have also been incorporated at the -5 position and they all bind to the coat protein in a way similar to the wild-type uracil. This is true also for the larger purine bases which have been previously found by biochemical methods to bind about 100 times weaker than wild type hairpin.Two of the other positions in the RNA hairpin which have been shown to be important for binding have also been varied and the complexes have been structurally studied. The -7 base is not involved in any interaction with the protein, still the identity of this base is important for binding. A pyrimidine at this position does, however, bind to the coat protein and the structure of this complex has been determined. The bulged out base at position -10 has also been varied. The results show that when the -10 base is changed to a guanine, the sequence of the hairpin stem also has to be changed in order to keep a unique conformation needed to obtain an interpretable electron density map when using X-ray crystallography.