Quite a while ago, I wrote a blog entry here discussing the regulation of sigma factors in E. coli.
Briefly, sigma factors are the subunit of prokaryotic DNA-Dependent RNA Polymerases (RNAP) that mediates the attachment of the polymerase to the promoter region. See the illustration below (taken from The Cell, ASM Press), where the various subunits of the RNAP are illustrated along with a DNA strand. The s subunit is required for the precise positioning of the RNAP on the promoter of the amplified gene. Without this subunit, the polymerase does not know where to bind or initiate transcription.
In that earlier post, I referenced an article by De Las Peñas et al., which predicted that a regulator of s24, RseA, was a membrane protein. This week, in another class I teach, we are looking at ways to determine if a specific protein contains membrane-spanning regions, so I thought I would use this as an example.
The first thing we need to do is to get the amino acid sequence for RseA. The easiest way to do this is to query the NCBI’s protein database.
It looks like a fair number of RseA proteins are known, but I’m selecting the fourth one down, from E. coli strain K-12.
This gives us the AA sequence:
With this in hand, we can go to a hydrophobicity plotter that will scan the AA sequence and provide a moving average score (similar to those sometimes used in the stock market) of the hydrophobicity of each amino acid. The moving average includes scores from the neighboring AAs in a way that we can get a sense for the hydrophobicity of a region of a protein taken together. I prefer the Kyte-Doolittle plot which I use through the Swiss protein group’s ProtScale tool found here.
Pasting the AA sequence into the box and checking “Hphob. / Kyte & Doolittle” will return the following plot:
Note that a score of ‘0’ is midway along the y axis. ‘0’ corresponds to neither hydrophobic nor hydrophilic. As this is a hydrophobicity plot, positive scores identify hydrophobic regions. The largest hydrophobic region found on this plot occurs just after AA 100 and goes on for about 20 amino acids after that. This tells us that this bit of the protein may be hydrophobic enough to be a transmembrane region.
Although this is suggestive, it would take some experimental work to demonstrate that it is true. However, because this protein is well characterized, we can check this using another web tool known as UniProt.
On UniProt’s website, we can find an entry for this exact protein which includes a subcellular location and topology section with references to back up our conclusion.
I hope this exercise is useful in describing how these tools can be used to learn more about your favorite protein before you even step into the lab.
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