We have determined the X-ray structures of six MS2 RNA hairpin–coat-protein complexes having five different substitutions at the hairpin loop base −5. This is a uracil in the wild-type hairpin and contacts the coat protein both by stacking on to a tyrosine side chain and by hydrogen bonding to an asparagine side chain. The RNA consensus sequence derived from coat protein binding studies with natural sequence variants suggested that the −5 base needs to be a pyrimidine for strong binding. The five −5 substituents used in this study were 5-bromouracil, pyrimidin-2-one, 2-thiouracil, adenine, and guanine. The structure of the 5-bromouracil complex was determined to 2.2 Å resolution, which is the highest to date for any MS2 RNA–protein complex. All the complexes presented here show very similar conformations, despite variation in affinity in solution. The results suggest that the stacking of the −5 base on to the tyrosine side chain is the most important driving force for complex formation. A number of hydrogen bonds that are present in the wild-type complex are not crucial for binding, as- they are missing in one or more of the complexes. The results also reveal the flexibility of this RNA-protein interface, with respect to functional group variation, and may be generally applicable to other RNA–protein complexes.

The well-studied interaction between the MS2 coat protein and its cognate hairpin was used to test the utility of the methylphosphonate linkage as a phosphate analog. A nitrocellulose filter binding assay was used to measure the change in binding affinity upon introduction of a single methylphosphonate stereoisomer at 13 different positions in the RNA hairpin. Comparing these data to the available crystal structure of the complex shows that all phosphates that are in proximity to the protein show a weaker binding affinity when substituted with a phosphorothioate and control positions show no change. However, in two cases, a methylphosphonate isomer either increased or decreased the binding affinity where no interaction can be detected in the crystal structure. It is possible that methylphosphonate substitutions at these positions affect the structure or flexibility of the hairpin. The utility of the methylphosphonate substitution is compared to phosphate ethylation and phosphorothioate substitution experiments previously performed on the same system.