Much of today was spent on a still-very-rough paper about how the regulatory protein Sxy is itself regulated. The regulation of Sxy is critical to understanding how bacteria decide to take up DNA, because the amount of Sxy protein in the cell determines how strongly the DNA uptake genes are themselves turned on. Hey, I just realized that the statement I just made didn't have much direct experimental support until now. The first solid evidence for this statement will be in this paper: we have mutants that increase the amount of Sxy in the cell, and we have measured both how much Sxy protein is present and how much DNA they take up, so we can show that increasing Sxy causes increased uptake of DNA.
This morning, when I and the two grad student authors started working through it, the rough draft we had was pretty bad - the experiments and ideas weren't in a logical order, so the draft didn't tell an interesting story. But after we talked it through we found a much better order for the experiments, and now the importance of the results is clearer to me.
And what is this importance?
1. We show that mutations that cause more Sxy to be made act by disrupting the base pairing in a key part of the mRNA that codes for Sxy. Mutations that weaken base-pairing = more sxy mRNA and Sxy, mutations that strengthen base-pairing = less Sxy.
2. We can be quite sure that this base-paired mRNA helps the cell decide how much Sxy should be made.
3. We show that cells make less sxy mRNA and Sxy protein when they are provided with purine nucleotides. This is important because we think cells take up DNA because they have run short on nucleotides. This result tells us that Sxy is the 'transducer' that connects the nucleotide-sensing component to the genes for taking up DNA. We would very much like to know whether the base-paired part of sxy mRNA is the sensor. To find this out we would need to show that nucleotide sensing is altered in the base-pairing mutants, which we haven't done yet.
4. We show that the base pairing mutations change both the amount of sxy mRNA in the cell and the amount of Sxy protein that's made from each sxy mRNA. The effect on the amount of mRNA could arise because the mutations relieve an obstacle to making the mRNA (they help the elongation step of transcription) or because they make the mRNA molecules more stable (they hinder the degradation step). The effect on the amount of Sxy protein per mRNA must arise because the mutations relieve an obstacle to protein synthesis. Our best hypothesis is that they make it harder for the base pairing to block the ribosome's access to its binding site on the mRNA.
The paper still needs LOTS of work, but I'm much more pleased with it than I was this morning.
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3 weeks ago in Genomics, Medicine, and Pseudoscience
I am not at all from this field but what I find interesting is how shortage of nucleotides might increase the production of Sxy. Could it be that a shortage of nucleotides leads to an increase of some RNA binding proteins that helps regulate the secondary structure to increase the production of the protein ?
ReplyDeleteIf the loop is so important then the loop structure itself should be very conserved in other species.
It sounds a bit strong to say that DNA import is only due to nucleotide shortage. It makes it sound like recombination and horizontal transfer are a byproduct of nucleotide import. Very interesting thought.
Pedro, you're exactly on track; we do think thatrecombination and horizontal transfer are byproducts. But most people think we're wrong.
ReplyDeleteThe sequences of sxy genes from related species don't look like their mRNAs could form loops, but we're hoping to use a genetic test on one of the species to see if base pairing is important.
Blogging and research. Nothing better. Please have a look at my similar research at http://mammals-shed.blogspot.com your comments would be appreciated.
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