I've just returned from a visit to the University of Western Ontario, where I gave two talks, one about #arseniclife and one about competence. In the competence talk I briefly described how sxy translation is limited by the secondary structure of its mRNA, and said that we don't have a good understanding of the mechanism.
Later I had lunch with some grad students, and a couple of them told me that they'd noticed a potential binding site for the protein Hfq in the sxy mRNA secondary structure. Hfq contributes to gene regulation by helping small regulatory RNAs (sRNAs) find and bind to their target mRNAs. My earlier discussion with their supervisor had reminded me that we've never investigated whether Hfq plays a role in sxy regulation. What the students noticed is that a loop in the structure ('A' in the figure) exposes the sequence AAUAAU, which is most of the Hfq motif identified by a recent SELEX study (citation below).
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.
et al. Genomic SELEX for Hfq-binding RNAs identifies genomic aptamers predominantly in antisense transcripts Nucl. Acids Res. (2010) 38(11): 3794-3808 first published online March 26, 2010 doi:10.1093/nar/gkq032
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Later I had lunch with some grad students, and a couple of them told me that they'd noticed a potential binding site for the protein Hfq in the sxy mRNA secondary structure. Hfq contributes to gene regulation by helping small regulatory RNAs (sRNAs) find and bind to their target mRNAs. My earlier discussion with their supervisor had reminded me that we've never investigated whether Hfq plays a role in sxy regulation. What the students noticed is that a loop in the structure ('A' in the figure) exposes the sequence AAUAAU, which is most of the Hfq motif identified by a recent SELEX study (citation below).
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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