Today I want to discuss an experiment that led to the discovery of messenger RNA (mRNA). Why did I choose to focus on one specific experiment? First, because of its importance in the history of molecular biology. Second, the experiment highlights the use of radioisotopes like those Russ Hobbie and I describe in Chapter 17 of Intermediate Physics for Medicine and Biology. Third, the recent development and of mRNA vaccines for Covid and other diseases makes this a good time to review how our knowledge of mRNA was established.
A crucial experiment was performed by Arthur Pardee and Monica Riley at the University of California, Berkeley, and published in 1960. Let me provide some context and set the stage. The structure of DNA had been discovered by Watson and Crick in 1953. By 1960, scientists knew that individual genes in DNA coded for individual proteins. The question was how the genetic information got from DNA to the protein. RNA was suspected to be involved, in part because ribosomes—the stable cellular macromolecules where DNA was produced—are made from RNA. Were the ribosomes the messenger, or was there something else? Many key experiments in biology, like the one by Pardee and Riley, are performed using a simple model system: E coli bacteria. Another important tool of early modern biology was radioisotopes, a product of modern physics from the first half of the twentieth century that was essential for biology during the second half of the century.
The experiment Pardee and Riley had done in Berkeley was new, technically amusing, and persuasive. It amounted to removal of the gene from the cell after it had begun to function. They had grown… bacteria… carrying [a specific gene to produce the protein enzyme beta-galactosidase]… in a broth where the available phosphorus [an important element in DNA] was the radioactive isotope 32P. The bacteria, with their DNA heavily labeled, were then centrifuged out... [and] resuspended in a nonradioactive broth… [Next] they added glycerol [a type of antifreeze]. Then they took one sample to test for enzyme activity [to check if beta-galactosidase was produced]. They put other samples into small glass ampules, sealed the ampules by fusing the glass at the neck, and lowered them into a vacuum-insulated flask of liquid nitrogen. The bacteria were frozen almost instantly at 196 degrees below zero centigrade. Protected from bursting by the glycerol, the bacteria were not killed, but their vital processes were arrested while the radiophosphorus in the DNA… continued to decay… From day to day, Riley raised ampules of the frozen bacterial suspension from the liquid nitrogen and thawed them… For comparison, they ran the whole [experiment] in parallel without the radioactivity [this was their control].
Before telling you the result, let me digress a bit about phosphorus-32. It’s an unstable isotope that undergoes beta decay to stable sulfur-32. This means the 32P ejects an electron (and an antineutrino) and transforms to 32S. In many cases (such as in sodium-24 examined in Fig. 17.9 of IPMB), beta decay occurs to an excited state that then emits gamma rays. But 32P is “pure” meaning there are no gamma rays, or even different competing beta decay paths. The book MIRD: Radionuclide Data and Decay Schemes by Eckerman and Endo, often cited in IPMB, shows this simple process with this figure and table.
Note the half-life of 32P is two weeks, and the average energy of the ejected electron is 695 keV.
What happens when 32P decays? First, the electron can damage the cells. An electron of this energy has a range of about a millimeter, so that damage would not be localized to an individual bacterium (with a size on the order of 0.001 mm). However, when the 32P isotope decays, it will recoil, which could eject it from the DNA molecule, causing a strand break. Even if the recoil is not strong enough remove the atom from DNA, there would now be a sulfur atom where a phosphorus atom should be, and these two atoms, being in different columns of the periodic table, will have different chemical properties which surely would disrupt the DNA structure and function. As Judson says
An atom of 32P decays by emitting a beta particle, which is a high-speed electron, whereupon it is transformed into an atom of sulphur. The transformation, and the recoil of the atom as the electron leaves, breaks the bonds of the backbone of the DNA at that point… Half of those decayed in fourteen days. The [beta-galactosidase] genes were being killed.So, what was the result? Judson summarizes,
The nonradioactive bacteria sampled before freezing were synthesizing enzyme copiously. So were the radioactive ones before freezing… Thawed after ten days, samples of nonradioactive bacteria synthesized beta-galactosidase just as vigorously as those never frozen. But the bacteria whose [beta-galactosidase] genes had suffered ten days of radioactive decay made the enzyme at less than half the rate they had before. Inactivation of the gene… abolished protein synthesis without delay. Stable intermediates between the gene and its protein—in other words, ribosomes whose RNA carried information to specify the sequence of amino acids—were ruled out. Continual action of the gene was necessary, either directly or by way of an intermediate that was unstable and so had to be steadily renewed.When Francis Crick and Sydney Breener learned of Pardee and Riley’s results, they combined their knowledge of this experiment with a previous one by Elliot Volkin and Lazarus Astrachan using bacteriophages [a virus that infects bacteria] to hypothesize that a new type of RNA, called messenger RNA, was the unstable intermediary connecting DNA and protein. And the rest is history.
The Pardee and Riley experiment (which made up Monica Riley’s PhD dissertation… wow, what a dissertation topic!) is beautiful and important. It is also relevant today. Why do mRNA vaccines (like the Pfizer and Moderna Covid vaccines) have to be kept so cold when being transported and stored before use? As Pardee and Riley showed, the mRNA is unstable. It will decay quickly if not kept ultra-cold. Can mRNA change the DNA in your cells? No, the mRNA is simply a messenger that transfers the stored genetic information in DNA to the proteins formed on ribosomes. Moreover, one difference between E coli bacteria and human cells is that in humans the DNA is located inside the cell nucleus (bacteria don’t have nuclei) and the ribosomes are in the cytoplasm outside the nucleus. DNA can’t leave the nucleus, and mRNA can only go out of, not into, the nucleus. So an mRNA vaccine will cause human cells to make virus proteins (for the covid vaccine, it will produce the spike protein) that will be detected by your immune system, but the mRNA will only be present a short time before it decays and will not affect your DNA. Finally, the vaccine contains mRNA for only the spike protein, not for the entire virus. So, no actual intact viruses are produced by the vaccine. The spike protein simply activates your immune system, without exposing you to an infection.
Isn’t science great?
