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.
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.
We live in a time when cloning a gene is a trivial matter done by technicians in the lab using tools that have been around for decades. However, this does not mean that it can be accomplished without knowing precisely how the biology works.
It is often said, even in molecular biology texts, that the DNA of the target gene and a plasmid vector simply need to be cut with the same restriction enzyme so that their ‘sticky ends’ can come together and be ligated into a new construct. This is often diagrammed as a cut and paste procedure (below), suggesting that no further attention to detail is required.
There are two issues (the bulleted items) I have with such a simplified cartoon:
This cartoon does not indicate any preference for the direction that the insert takes once it is incorporated into the plasmid.
Inserts have directionality. A gene has a start codon on one end and a stop codon on the other. These must be in line with the promoter sequence and polyA signals that are typically not a part of the insert, but are contributed by the plasmid.
The Promoter sequence is required for binding of the DNA-Dependent RNA-Polymerase that will make the mRNA. This must be upstream of the start codon, ATG. The Poly Signal is responsible for attaching untemplated ‘A’ residues to the 3′ end of the mRNA. It must be downstream of the stop codon, [TAA/TAG/TGA].
The easiest way to prevent this problem is to ensure directional cloning by cutting each end of the vector and insert with different enzymes that don’t possess compatible ends.
The second easiest way to control for cloning your insert in the wrong way is to have a digest with another enzyme to screen for the proper orientation after miniprepping multiple clones. In the figure below, we can see that there is one XhoI site within the insert and one within the plasmid. Because the one in the insert is offset to one side, the size of the fragments generated by this digestion will depend on the orientation of the insert. Knowing which fragment size is associated with proper orientation of the insert will help us to quickly screen our minipreps by XhoI digestion.
This cartoon suggests that the recombinant plasmid is the only product of this ligation reaction.
The other problem with this cartoon is that it assumes that the desired ligation of one insert with one plasmid is the most abundant product, when in fact, it probably occurs in a tiny minority of cases. More likely, the plasmid curls up on itself and religates without the insert being incorporated.
The reason that the plasmid religates is because the two ends of the plasmid can religate and they’re close enough together that they probably run into one another more often than an insert does. The way to prevent this is to recall the chemistry involved in this ligation reaction.
This reaction requires a 3′ hydroxyl group and a 5′ phosphate group to come together to form the phosphodiester bond. This happens on each strand of the DNA and is mediated by a ligase enzyme.
However, only one strand being ligated is sufficient to hold the recombinant plasmid together until it can be fixed inside the cell by DNA repair mechanisms. If you remove the phosphate from the plasmid molecule, it cannot come together and be ligated to itself. Instead, only when an insert is incorporated are there the phosphates required to ligate one strand at either end.
The phosphates can be removed from the plasmid DNA using the enzyme, Calf Intestinal Phosphatase (CIP). The only thing you need to remember is that the CIP must be destroyed at the end of its reaction with the plasmid. Otherwise, it can go on to also dephosphorylate the insert DNA when that is added for a ligation reaction. Luckily, CIP is easily inactivated by heat (NEB suggests 80 C for 2 minutes).
The ultimate goal of the new and emerging SARS-CoV-2 antibody tests is to determine when and how we can safely get back to work and restart our stalled economies. While knowing how many people are sick is valuable in tracking the rate of disease spread and identifying people who need to isolate or seek treatment, it misses out on telling us who has had the virus and has recovered (or may have not been symptomatic at all). The idea is that people with antibodies may be protected from (re-)infection and are therefore safe to return to “normal” life. (caveats are that these antibodies are actually protective and will last, conferring immunity).
A good article discussing what antibodies are and how these tests operate was recently published in USA Today. The form of many of these tests is very similar to home pregnancy tests (lateral flow ELISAs), enabling quick, rapid results with no special training to perform.
Eventually, once enough people have been exposed to the virus and recovered or been vaccinated against the virus (probably early next year), then we will have herd immunity sufficient to protect the vulnerable people around us from being exposed to a life-threatening illness.
In the meantime, we’ll probably have to be content with a ‘new normal’ of living in a way that doesn’t prevent the spread of infection but limits it to a level that our hospitals can bear.
This is an excellent graphic from the laboratory of Carl June, who is a pioneer in CAR T Cell therapy at UPenn (Moore and June. Science 17 Apr 2020). One of the problems associated with CAR T Cell therapy is the resulting cytokine story often accompanying such a large infusion of immune cells. A similar problem is seen in COVID-19 patients (as well as in those infected with SARS-CoV and MERS-CoV), where it is thought to be the actual cause of death in infected patients.
Preliminary trials using drugs interfering with IL-6 signaling are showing signs of hope for severely sick COVID-19 patients.
Thank you to Michael McHeyzer-Williams (@mmw_lmw) of the Scripps Institute for his tweet pointing me toward this paper.
I have to admit, I’m getting a little bit tired of COVID-19. Or more specifically, I am getting tired of all the social distancing and not being able to work from my campus office. More than anything, this comes out in my frustration at having to use online meetings to conduct classes and student one-on-ones. Perhaps I’m not as effective a communicator as I would like to be, but I miss being able to sketch out ideas on paper to show students how systems work or using a whiteboard to supplement PowerPoint slides.
I am very much looking forward to exposure testing (an antibody test – most likely laminar flow, e.g. pregnancy tests) for SARS-cov-2 to demonstrate that we’ve either had the virus or are otherwise immune to it so we can get back to work!
I suggest that the Whitehouse coronavirus taskforce spend some time watching ‘Contagion’ for a roadmap of how we can return to a normally functioning society after an outbreak.
Another front in the fight against this virus that I am eagerly awaiting is a vaccine such as that made by the company Moderna, which uses RNA-like molecules to deliver protein-encoded messages to our cells. I’m eager to see the results of this vaccine’s trials (now at Emory University) both for the immediate effect in preventing COVID-19 as well as to see how this new vaccine platform performs.
I have my fingers crossed that I can get back to work as usual and, equally important, get back to my climbing gym, RoKC.
Complement is an ancient component of our innate immune system that was initially discovered in the 19th century and named for its ability to complement antibody in the lysis of cells.
In the simplest of terms, complement is triggered by one of three mechanisms (Antibody Triggers the Classical Pathway; Carbohydrates Trigger the Lectin Pathway; The Alternative Pathway is triggered spontaneously.) Once triggered, a cascade of events leads to the assembly of a C3 Convertase, which breaks C3 into the soluble anaphylatoxin C3a and the insoluble C3b, which precipitates onto the surface of the cell and forms a component of additional C3 convertase, thus amplifying the reaction, and also a C5 convertase.
C5 is then digested into an additional, more powerful soluble anaphylatoxin, C5a, and the insoluble C5b, which cooperates with other components to lead to the formation of the Membrane Attack Complex (MAC) comprised of C9 molecules inserted into the plasma membrane.
The anaphylatoxins, C3a, C5a (and, to a lesser extent, C4a), function to induce contraction of the smooth muscle and then an increase in vascular permeability of the capillaries. They further increase the expression of adhesion molecules on these same vascular epithelial cells so as to recruit immune cells to the location. Finally, they promote receptor-mediated chemoattraction of leukocytes.
In summary, complement can be activated through three somewhat distinct pathways, each one converging at a C3 Convertase. Complement will lead to direct cell death via pore formation (MAC complex formation), it will recruit leukocytes to the area of infection via chemotaxis, and will facilitate phagocytosis of pathogens via complement receptor-mediated endocytosis.
As a last note, it is relevant to bring up the presence of ‘natural antibody.’ These (IgM) antibodies are made by a special group of innate (B-1a) B Cells found most prevalently in young organisms and at lower concentrations later in life. Interestingly, these antibodies are produced prior to antigen exposure and have a pre-defined array of specificities including targets such as phosphorylcholine and certain carbohydrates common on bacteria. As such, they are distinctly innate mediators of immunity and are specifically capable of fixing complement on their bacterial targets.
Comparison of chemotaxis of eosinophils mediated by C3a and C5a.Discipio 1999
There are several simple problems that are often at the root of communication failures. Sometimes, this may be because the speaker is not thinking clearly. Sometimes this might be because the speaker really does not understand what they are talking about at all. Sometimes, it might even be that the speaker knows what they are talking about too well. In his recent book, The Sense of Style: The Thinking Person’s Guide to Writing in the 21st Century, Harvard Psychologist, Steven Pinker, argues that the curse of knowledge is often to blame. Check out this Inc. article by Glenn Leibowitz on the topic.