Most mutations in the sequence of the RNA hairpin that specifically binds MS2 coat protein either reduce the binding affinity or have no effect. However, one RNA mutation, a uracil to cytosine change in the loop, has the unusual property of increasing the binding affinity to the protein by nearly 100-fold. Guided by the structure of the protein-RNA complex, we used a series of protein mutations and RNA modifications to evaluate the thermodynamic basis for the improved affinity: The tight binding of the cytosine mutation is due to (i) the amino group of the cytosine residue making an intra-RNA hydrogen bond that increases the propensity of the free RNA to adopt the structure seen in the complex and (ii) the increased affinity of hydrogen bonds between the protein and a phosphate two bases away from the cytosine residue. The data are in good agreement with a recent comparison of the cocrystal structures of the two complexes, where small differences in the two structures are seen at the thermodynamically important sites.

An RNA binding assay measuring cooperative protein binding has been used to evaluate the effects of mutations in the MS2 phage coat protein expected to disrupt capsid assembly. By using the crystal structure of the virus as a guide, six different mutations in the FG loop structure were selected in which hydrophobic residues were replaced with charged residues. Most of these proteins form capsids in Escherichia coli, but not in an in vitro assembly assay, suggesting that interdimer interactions are weaker than wild type. These mutant proteins reduce the free energy of cooperative protein binding to a double-hairpin RNA from its wild-type value of -1.9 kcal/mol. Several of the variants that have large effects on cooperativity have no effect on RNA affinity, suggesting that protein-RNA interactions can be affected independently of dimer-dimer interactions. The V75E;A81G protein, which shows no measurable cooperativity, binds operator RNA equally well as the wild-type protein under a variety of buffer conditions. Because this protein also exhibits similar specificity for variant RNA sequences, it will be useful for studying RNA binding properties independent of capsid assembly.

We have used a combination of physical and molecular biological techniques to examine the structure of Leptomonas collosoma spliced leader RNA. We confirm the general features of the previously proposed structure for the 3' half of the RNA, in which a single-stranded region is flanked by two stem loops. However, we find that the 5' half of the RNA, which contains the splice site, has two competing secondary structures which differ only slightly in stability and which can interconvert on a fast (<1 s) time scale. In the favored conformation, a stable hairpin helix is augmented by conserved complementarity between the splice site and the 5' end of the SL RNA. This putative helix has anomalous nuclease sensitivity and thermal stability features, suggesting that it is probably coupled by unknown tertiary interactions to other nucleotides in the 5' half-molecule. The structure offers intriguing parallels with RNA-RNA interactions in the mammalian splicing system.

The spliced leader RNA from Leptomonas coUosoma has two competing secondary structures of nearly equal free energy. Short, complementar oligonucleotides can drive the structure from one form to the other. We report stopped-flow rapid-mixing and temperature-jump measurements of the kinetics of the strtural switch. At high concentrations of oligonucleotide, the rate of binding becomes limited by the rate ofthe structural switch, which occurs on a time scale of a fraction of a second. The low activation energy observed for the process implies a branch migration type of anism in which portions of the two competing helices transiently coexist.RNA conformational switching is thought to be fundamental to a number of biological processes, including translational regulation, protein synthesis, and mRNA splicing. For example, in translational attenuation, alternative RNA hairpin conformers have been shown to regulate translation in Escherichia coli and Bacillus subtifis by forming either terminator or antiterminator structures (1-3). Also, during protein synthesis, the elongation factors EF-Tu and EF-G have been proposed to bind to alternative RNA conformations in 28S rRNA during each elongation cycle (4). Finally, in yeast intron splicing, U6 small nuclear (snRNA) has been shown to bind to U4 snRNA in an inactive conformation and with U2 snRNA in the active splicing complex (5). Since the two pairings are mutually exclusive, it can be inferred that a conformational rearrangement is required.The dissociation of nucleic acid helices occurs with a large activation energy, approximately equal to the heat of dissociation (6, 7), and as a consequence the process becomes extremely slow at temperatures substantially below the melting temperature (tm) for the helix that is disrupted. A process that may take hours to accomplish is not well suited as a biological regulatory switch. Therefore it is reasonable to suppose that functional RNA conformational switches must occur by an alternative kinetic pathway that is both faster and less temperature sensitive than predicted for a reaction pathway that involves a fully melted intermediate.The spliced leader (SL) RNA of trypanosomes and nematodes is a short RNA whose 5' exon sequence is donated to mRNAs by the process of trans splicing (8)(9)(10)(11)(12). Techniques such as nuclease mapping, RNA base modification/ interference, mutagenesis, and equilibrium melting curves have been used to show that the 5' half of the SL RNA from Leptomonas collosoma has two competing secondary structures that differ in tm by only 60C (13). Form 1 (Fig. 1) is the more stable of the two structures in the wild-type (WT) sequence, but point mutations can switch the structural preference. For example, mutation M3 (Fig. 1), in which a G&U base pair in form 2 is replaced by G-C, raises the tm of the form 2 helix by 15'C, making it more stable than form 1 (13). The 60C tm difference between the two forms for the wild-type sequence, along with the enthalpy of melting estimated from the equilibrium meltin...

The thermodynamic contribution of a stacking interaction between Tyr85 in MS2 coat protein and a single‐stranded pyrimidine in its RNA binding site has been examined. Mutation of Tyr85 to Phe, His, Cys, Ser and Ala decreased the RNA affinity by 1–3 kcal/mol under standard binding conditions. Since the Phe, His and Cys 85 proteins formed UV photocrosslinks with iodouracil‐containing RNA at the same rate as the wild‐type protein, the mutant proteins interact with RNA in a similar manner. The pH dependence of KD for the Phe and His proteins differs substantially from the wild‐type protein, suggesting that the titration of position 85 contributes substantially to the binding properties. Experiments with specifically substituted phosphorothioate RNAs confirm a hydrogen bond between the hydroxyl group of tyrosine and a phosphate predicted by the crystal structure.