Everything you always wanted to know about Sxy but were afraid to ask

(Sorry about the title - I'm going to post only a fraction of what we know, only what will be in our Sxy paper, but I couldn't resist. And no, I'm not a big Woody Allen fan.)

You can find some Sxy background in this post.

I'm basically going to describe what's in our figures and what they tell us. But no, I'm not going to post the figures themselves; I fear this would be perilously close to the kind of "prior publication" that journal editors forbid.

1. We have 4 more sxy mutations that cause hypercompetence (5 mutations in all). They all increase competence at least 100-fold in non-inducing conditions, and about 20-50-fold in partially inducing conditions (Fig. 1).

2. Each of these mutations changes 1 base pair of sxy DNA, but 4 of them don't change the Sxy protein at all (Fig. 2). So we suspected that they cause hypercompetence by increasing the amount of Sxy protein in the cell, not by changing what Sxy protein can do.

3. Each mutant strain does have more Sxy protein (between 3-fold and 50-fold more, depending on the mutation and the growth conditions) (Fig. 3A). The competence of each strain (measured as transformation frequency) increases with the amount of Sxy protein in the cells (Fig. 3B). How could the mutations cause this? We propose that they cause this by interfering with how part of the sxy mRNA folds back on itself. The folding lets the two different parts of the mRNA where the mutations occur form base pairs with each other (Fig. 4).

4. We show that sxy mRNA does fold by examining the digestion of pure sxy mRNA with RNase enzymes (Fig. 5). This confirms that, in wildtype sxy mRNA, the nucleotides where the mutations occur are involved in base pairing, as are many other nucleotides in the 'internal' part of the mRNA between the mutation locations. Almost all of the base pairing is consistent with a complicated folded structure predicted by a computer program called Mfold (part of Fig. 5).

Problem with the data for Figure 5: The gel images are not reproducing well - either everything is washed out or it's too dark. This isn't just a problem with the printer resolution, as the screen image has it too. I hope this can be corrected using the imaging software that created the gel image (I hope we don't need to run a new gel).

5. RNase analysis of mRNA with the sxy-1 mutation shows almost the same structure. We predicted that the sxy-1 mutation weakens the folded structure because it decreases by 1 the number of base pairs holding it together. We see this only as a modest increase in the RNase digestion of all the nucleotides in the paired region affected by the mutations (another part of Fig. 5).

6. The best test of our hypothesis that the mutations cause their effects by preventing base pairing is to restore base pairing while keeping the mutations. We can do that easily because two of our mutations, when combined in the same chromosome, can base pair with each other, giving a 'double mutant' that has both mutations but the normal number of base pairs. If we're right, this double mutant will not be hypercompetent. And it's not (Fig. 6A).

To be continued....