Deconstructing the structure?

A lab meeting presentation yesterday got me worrying about how having an explicit technical drawing of a structure makes it difficult to think about alternative structures.

We started 10 or 12 years ago with a hand-drawn sketch of how parts of sxy mRNA might pair with each other. But now we've been relying on a computer program called Mfold, which takes any RNA sequence and uses thermodynamic principles to deduce the most stable configurations it could fold into. It gives nice professional-looking drawings, and I'm afraid we're being seduced by them into not fully considering the other possibilities.

It's not Mfold's fault. Mfold offers several alternative structures, but it's all too easy to focus on the one with the highest thermodynamic stability score and ignore the others. This would be the right thing to do if we knew that the score correctly predicted the real structure of the RNA in the cell, but there are lots of reasons to doubt this.

First, the thermodynamics is only an approximation of reality. Second, the foldings are too simple - each position interacts with at most one other position - whereas far more complex interactions are likely to occur in real molecules. Third, interactions are dynamic and stochastic; a population of RNA molecules will include many different conformations, and each molecule's conformation is likely to fluctuate over time. Fourth, many biologically significant effects of secondary structures depend on competition between two or more different possible foldings, as in the following made-up example. Part B of an RNA strand can either pair with part A or with part C. If it pairs with A, the ribosome-binding site in part C is available and the protein can be made, but if it pairs with C the site is blocked and no protein is made.

We have genetic evidence for one short section of pairing in sxy mRNA, between sequences separated by about 90 bases. This is very solid; we know which bases pair with which in this section. But what about the rest of the molecule, especially the intervening 90 bases? We now have biochemical (RNase digestion) evidence over the whole region. This tells us that some parts of the RNA are in a base-paired state, but it doesn't tell us which other bases they are paired with. It also tells us that some bases are not base-paired, and it gives us no information about the states of still other bases, The 'paired' and 'not paired' states match quite well to those predicted by the most stable Mfold structure, but I think we should spend a bit more time considering whether they also fit alternative foldings. Alternatives include other Mfold predictions, predictions from other programs, or even structures we've come up with ourselves, folding 'by eye' rather than by computer.