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I think the first thing we should do is knock out the H. influenzae hfq gene. The authors of the SELEX study say that "Hfq is not essential for growth, but the adaptation to changing environmental conditions is hampered in the absence of Hfq." That would fit with Sxy's function. I could imagine the mutation either increasing or decreasing sxy expression and thus the transformation frequency, depending on how Hfq interacts with the sxy mRNA. Provided it doesn't have dramatic effects on viability or cell growth, we should be able to detect specific effects on transformation frequency. We could then mutate one or more of the positions Hfq is predicted to interact with, but that would be more difficult, and first I'll need to read a lot more about Hfq. Of course, long strings of As and Us are very common in H. influenzae mRNAs because of its AT-rich base composition, so I won't get my hopes too far up until we see a mutant phenotype.
The students' supervisor gave me the url for a web site that will, I think, search a genome for sRNAs complementary to a given mRNA. I've lost the url, and all the websites I can find do the complementary search (start with small RNA, identify possible targets), so I can't check this until he sends me the url again by email.
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Hi Dr Redfield,
ReplyDeleteI was one of Dr. Haniford students you were talking to at lunch (the Australian one if that helps). One thing you might want to do, which is quite easy, is to first test if Hfq is able to bind Sxy mRNA in vitro. Making hot RNA and doing some quick EMSA's is a fairly simple way to find out if Hfq might play a role in post-transcriptional modification of Sxy mRNA. While I'm sure you don't have any purified H. influenzae Hfq, one thing you might consider trying is using E coli Hfq. Hfq is highly conserved, so if E coli Hfq binds Sxy mRNA tightly (with a Kd < 100 nM) it is probably likely that H. influenzae Hfq will bind the mRNA as well.
As for introducing mutations to the putative Hfq binding site in the 5'UTR of Sxy, this can be a little more challenging than you might expect. I have spent some time in my project attempting a very similar thing with IS10 mRNA, and have had some success, but nothing greater than a 5-fold decrease in binding affinity. I have found a recent structural paper to be helpful for designing mutations (Link et al, 2009). And while most published information about Hfq concerns the E coli protein, it seems to be conserved enough (particularly with respect to RNA-Binding properties) that you could assume the H. influenzae Hfq behaves the same.
It was great meeting you last week, and good luck with your possible Hfq-Sxy work. If you are interested in any of our RNA protocols I would be happy to pass them on.
Mike
Link, T. M., P. Valentin-Hansen, et al. (2009). "Structure of Escherichia coli Hfq bound to polyriboadenylate RNA." Proceedings of the National Academy of Sciences.
An Hfq knock out may alter the expression of a lot of genes, untangling direct from indirect influences may be quite a challenge.
ReplyDeleteRfam should give you annotation of a number of H. influenza sRNAs (http://rfam.sanger.ac.uk/genome/browse#H). Pick your favourite genome sequence and pull them out (e.g. http://rfam.sanger.ac.uk/genome/281310#tabview=tab1). Interaction prediction tools such as RNAcofold (http://www.tbi.univie.ac.at/RNA/) or IntaRNA (http://www.bioinf.uni-freiburg.de//Software/index.html?de) will assess whether interactions between sRNAs and sxy are likely (predictions model both intra- and inter- molecular interactions).
However, this relies on the Rfam annotations which only include the known RNAs. In a perfect world you would know all the H. influenza RNAs first.
Another possible route is to look for unusual conservation in the UTRs and terminal coding regions - this may provide a signal of RNA:RNA interaction. You can use these conserved blocks and the above tools to hunt for possible interacting sRNAs in intergenic regions (with appropriate negative controls). However, I recently read an (unpublished) manuscript that showed that targets of sRNAs were often poorly conserved.
PS. Is sxy expression temperature (see PrfA and the other thermoregulators) and/or pH sensitive? Both conditions could influence that structure you show.
ReplyDeleteHI Mike and Paul.
ReplyDeleteYour comments inspired me to do more work, so see the next post. Competence regulation doesn't appear to be temperature sensitive.
I look forward to hearing more. I think gene regulation is very sexy!
ReplyDelete