What's UP, Sxy?

One of the grad students has been doing experiments to clarify how the transcriptional activator protein Sxy turns on genes that have its recognition sequence, the "CRP-S" site. He's given me a draft of a paper he's writing about this work.

Sxy works by interacting with another transcriptional activator protein called CRP. CRP's job is to bind to DNA at CRP sites and, by bending the DNA, help RNA polymerase start making RNA. CRP-S sites are much harder to bend than normal "CRP-N" sites, and we have been thinking that Sxy acts by helping CRP to bind (if it can't bend the DNA it lets go).

But several pieces of his data tell us that binding isn't enough. A protein from E. coli (we work mostly in another bacterium, Haemophilus influenzae) binds CRP-S sites fairly strongly in the test tube, but when it's in H. influenzae cells it can't turn the CRP-S genes on without Sxy's help. And changing the CRP-S sequence so H. influenzae's CRP can bind it without Sxy in the test tube doesn't enable CRP to turn the gene on in cells without Sxy's help.

So the grad student is suggesting that Sxy may also affect how RNA polymerase interacts with the DNA. RNA polymerase binds to DNA more effectively if its 'tail' makes contact with an AT-rich sequence called the "UP" sequence. Many genes have an UP sequence, and at normal CRP-N sites CRP is thought to help RNA polymerase's tail bind to it.

He's found that the genes with CRP-S promoters have what look like three precisely-spaced UP sequences beside their CRP-S sites, and in his draft paper he proposes that Sxy acts partly by helping RNA polymerase make contact with these sites. We could test this by changing these sequences to not fit the UP consensus, and seeing if this makes the promoter unable to be activated by Sxy.

This work doesn't address the question of whether Sxy does help CRP bend the DNA. But we won't be able to address this until we have conditions where purified CRP and Sxy both interact. Right now we have the CRP but not the Sxy - it's a very difficult protein to work with.

Rachael's boyfriend's plasmid, and rifampicin

I ran into a colleague at the coffee pot today, and asked his expert advice about ways to investigate whether RNA polymerase pauses or stalls when transcribing the sxy gene.

He said that pausing is quite easy to show using a commercially-available E. coli in vitro system (as I had hoped it would be), but that showing that a H. influenzae sequence causes pausing in this system would only be significant if we first showed that the H. influenzae sxy gene was regulated in vivo in E. coli as it is in H. influenzae. The alternative is to use a H. influenzae in vitro system, but we would have to purify the components ourselves, which is well beyond both our abilities and our real interests.

We might be able to show the regulation in E. coli, if the sxy gene wasn't so toxic to E. coli (see Making lemonade). Well, we could perhaps work with a truncated gene, subject to the same transcriptional controls but not producing Sxy protein... Hey! In fact, one of the very first sxy plasmids I made would be just the thing! The plasmid is named pDBJ90 (the name I think is the initials and year of the boyfriend of the student who made it); it contains only the 5' half of the sxy coding region but all of the upstream sequences that affect its transcription. And it's in a high-copy vector. And as far as I know the insert is stable, though we've learned that we should always check the sequence. I don't know whether our Sxy antibody will recognize the truncated protein it should produce.

What would the experiment be? Grow the E. coli cells with the plasmid in minimal medium with added purines and pyrimidines. Add cAMP to induce the sxy promoter, and transfer half the culture to the same medium with no purines or pyrimidines. Measure the amounts of sxy mRNA and protein at several time points for each half of the culture. If the ratio of protein to RNA is higher when the purines and pyrimidines are absent, then we can do the in vitro experiment.

The colleague also suggested an entirely new way to look at the relationship between sxy transcription and sxy translation. He reminded me that mutations in the genes for RNA polymerase can make the polymerase resistant to the antibiotic rifampicin, and told me that these mutations can affect the efficiency of transcription in ways that might change sxy expression. So here's the experiment plan:

Starting with wildtype (rifS) H. influenzae cells, select cells that are resistant to rifampicin. I think these are quite easy to select; we could try several different rifampicin concentrations. Pool all the colonies that grow up on rifampicin plates, and select hypercompetent ones by transforming the pooled cells with novR DNA while they are in log-phase growth in sBHI. If we get any hypercompetent cells, check whether the hypercompetence is caused by the rifR mutation, by using their DNA to transform fresh cells to rifR. If yes, we've shown that mutations in RNA polymerase can cause hypercompetence.

We do already have one RNA polymerase mutation that affects competence induction. It's an insertion that doesn't change the sequence of RNA polymerase but probably reduces the amount of polymerase in the cell, and it decreases competence rather than increasing it. I can make up a just-so-story that fits this mutant into our current model, but it's just handwaving.