Monthly Archives: November 2020

Often simply called a gel shift assay, these are performed to identify the binding of proteins (or other molecules, conceivably) to DNA. The basic idea is that you have a section of DNA in excess supply that contains a sequence that you suspect may be bound by known or unknown proteins. You first label your DNA by incorporation of labelled bases via PCR or end-labeling. This used to be done using radioactive bases, but there are other alternatives available now. For our purposes, I’m going to assume a radioactive label as it is simple to imagine.

Once you have your radiolabelled DNA, you now need to add some proteins that you suspect might bind. This could be isolated protein from a recombinant source or it could be a cell lysate from cells that have been treated in some way. Again, I’ll imagine that we have stimulated cells with something for a progressively longer time (say, 0 minutes stimulation – 30 minutes of stimulation at 5 minute intervals). Finally, after the get runs, you can blot it onto a membrane and expose it to film where the radioactive DNA will light up. Assuming this, I guess we also have a radioactive DNA ladder too

Here’s an example… (remember, this is entirely fictitious)

We stimulate cultured B cells given the survival cytokine, BLyS. At the timepoints indicated above (0-30 minutes), we harvest cells and lyse them in the cold to obtain nuclear fractions. Our DNA is derived from the promoter region of a protein we are interested in and we are wondering if we will see transcription initiation factors assemble.

What we are seeing is a 100bp DNA ladder run with our samples. Keep in mind that the ONLY thing we can see here is labeled DNA. In the first several lanes, none of our DNA is being bound and it is running according to its size (~50bp). At 15 minutes, we start to see the shadow of a band that has shifted our DNA up to an apparent ~700bp. IMPORTANT: DNA ladders are not protein ladders! Also, this is a native protein with unknown charge that is bound to our DNA. All we know is that we are getting binding, we can’t assume knowledge of the exact size of the protein (however, in general, we do see shifts going up as more proteins accumulate)

By 30 minutes, we see an appreciable amount of protein binding our DNA. But how can we know what protein(s) are binding?

One way, if we know what we are expecting, is to use an antibody against that suspected protein. Assuming we have extra lysate + DNA mixture to run a second gel, we could spike our antibody into one tube with DNA and lysate and not into the other (or spike an irrelevant antibody).

If we see that the antibody results in a ‘supershift’ where the DNA/protein band has moved up to a larger apparent size, this means that our antibody is binding its target and that target is binding the labeled DNA.

Let’s assume we suspect that Nf-kB is what is binding our DNA, so we use and Nf-kB antibody to do a supershift assay.

These data support our model and we can now go on to ask new questions.

Francis Crick is well known, not only for his work in elucidating the structure of DNA along with James Watson, but also for his hypothesizing about the nature of biology that he thought would be discovered in the wake of the publication of the structure.

Among these hypotheses was the notion of a ‘central dogma’ that described the flow of information from DNA, through RNA, to protein. It was clear how information could flow from DNA to RNA as these were written in the same language of nucleic acids. What was less clear was how this information could then be translated into a protein.

 In 1955 Crick proposed the idea of adaptor molecules that would be required to read the information on the nucleic acid and translate this into the language of proteins, comprised of Amino Acids (AA). “In its simplest form there would be 20 different kinds of adaptor molecules, one for each amino acid”

We now understand these adaptor molecules to be transfer RNAs (tRNAs), which bind both messenger RNA (mRNA) and AAs in a way that the AAs can engage in protein synthesis. These tRNAs bind the AAs on one end of the molecule and have three bases, called anticodons, that interact with the codons of the mRNA. This alone would be enough to prove Crick as prescient.

However, that only covers the first part of his statement. He goes on to say, “…and twenty different enzymes to join the amino acid to their adaptor.”

Twenty enzymes to build the adaptor molecules (tRNA with AA).

Yet again, Crick’s mental model was correct, we have a number of aminoacyl tRNA synthetase (aaRS) enzymes that specifically bind the tRNAs and AAs individually and then promote their conjugation. One might expect these synthetase enzymes to be, themselves, at least partially comprised of nucleic acids that can identify the tRNAs by their anticodons and then charge them with their appropriate, cognate, AAs. However, this is not always the case. Each tRNA is, instead, recognized by an identity element that is often discrete from the anticodon, but nevertheless serves to distinguish the correct tRNA that should be bound. These identity elements (aka identity determinants) are often located along the anticodon stem or the acceptor arm of the tRNAs, however they are not restricted to such (see Fig 2 from Giegé et al.)

Figure 2 from Giege et al: Cloverleaf folding of tRNA with location of known identity determinants and its three-dimensional L-shaped organisation.

Although it is conceivable that every individual tRNA would have a specific aaRS to charge it with its AA, “[w]ith some exceptions there is only one aaRS per isoacceptor tRNAfamily (the cognate set of tRNAs).”¹ However, I will not go into the specifics of this flexibility here.

Once a tRNA is recognized and bound by its associated aaRS, the appropriate adenylated AA is brought in to the synthetic site where it will become charged to the tRNA. Once charged, the AA will be shifted to a second binding pocket, the editing site, where it is again checked. Together, this is known as the double sieve model, where the first, coarse, sieve (occurring in the synthesis site) allows placement of correctly sized (or smaller) AAs, while the second sieve (occurring in the editing site), allows only AAs of the correct hydrophilicity, mischarged AAs are cleaved by deacetylase enzymes.²

References

1. Giegé, Richard & Eriani, Gilbert. (2014). Transfer RNA Recognition and Aminoacylation by Synthetases. 10.1002/9780470015902.a0000531.pub3.
2. Dino Moras, Proofreading in translation: Dynamics of the double-sieve model PNAS December 21, 2010 107 (51) 21949-21950; https://doi.org/10.1073/pnas.1016083107
3. Shuya Fukai et al. Structural Basis for Double-Sieve Discrimination of L-Valine from L-Isoleucine and L-Threonine by the Complex of tRNA^Val and Valyl-tRNA Synthetase. Open ArchiveDOI:https://doi.org/10.1016/S0092-8674(00)00182-3