By now, you have likely become familiar with the central dogma of molecular biology, as proposed by Francis Crick:
DNA → RNA → Protein
British physician Archibald Garrod was among the first researchers to suggest a connection between DNA and protein, but it was the work of Sydney Brenner, Francois Jacob, and Matthew Meselson that explained the middle step, specifically identifying the nature of the RNA intermediate, which we now call messenger RNA (mRNA). Moreover, through the use of radioactive labeling, these scientists also discovered the precise role that ribosomes play in protein synthesis.
Earlier Hypotheses About RNA and Protein Synthesis
![Schematic of the bacteriophage infection protocol.](javascript:dispOrigImg("20192/ribosome_heavy_light-f1_FULL.gif", "Schematic of the bacteriophage infection protocol.", "Y", "Figure 1", "1219785409054-3799432876_1", 'N', "Copyright 2008 Nature Education", '570', '218', "http://www.natureeducation.com/"); "Schematic of the bacteriophage infection protocol.")
Before the publication of Brenner, Jacob, and Meselson's results in 1961, the prevailing hypothesis regarding the relationship between DNA and protein was that the RNA associated with ribosomes (called rRNA) must be the intermediate gene copy. This view, as proposed by Francis Crick, required a complex mechanism of protein regulation in which each individual ribosome and its associated rRNA produced a unique protein. Crick's hypothesis came to be known as the "one gene, one ribosome, one protein" concept.
According to this concept, one would expect to observe that the sequence of each rRNA would vary widely, because each one would correspond to a different gene. In addition, one would expect that the rRNA template would allow for protein translation in the absence of the gene encoding the rRNA. However, these expectations did not hold true. For one, rRNA sequences did not exhibit the diversity one would expect; rather, they were homogenous in size and nucleotide composition. This observation led Brenner, Jacob, and Meselson to hypothesize that another type of RNA must mediate gene-to-protein translation and that ribosomes (and ribosomal RNA) were simply general machines that could translate any or all protein templates.
Testing the New Hypothesis
![Three potential models of information transfer in phage-infected cells.](javascript:dispOrigImg("19580/10.1038_190576a0-f1_full.jpg", "Three potential models of information transfer in phage-infected cells.", "Y", "Figure 2", "1221674870694-491385474_2", 'Y', "Copyright 1961 Nature Publishing Group, Brenner, S., et al., An unstable intermediate carrying information from genes to ribosomes for protein synthesis, Nature 190, 576–581", '755', '640', "http://www.nature.com/nrc/index.html"); "Three potential models of information transfer in phage-infected cells.")
Brenner, Jacob, and Meselson decided to conduct their experiments using the T2 bacteriophage, a virus that infects bacteria and causes almost immediate disruption of host protein production; when infected with this virus, cells quickly and efficiently switch to producing phage proteins. The timing of such disruption was already well established, so Brenner et al. knew that they could use a "pulse" of radioactivity followed by a "chase" of unlabeled similar molecules (i.e., a pulse-chase experiment) to follow the fate of several different biomolecules within a bacterium both before and after infection with the T2 bacteriophage. Furthermore, based on their preexisting knowledge of T2 infection (Figure 1), they described three different hypotheses that could be tested (Figure 2).
![Schematic of RNA labeling protocol.](javascript:dispOrigImg("20780/ribosome_heavy_light-f3_FULL.jpg", "Schematic of RNA labeling protocol.", "Y", "Figure 3", "1221675143257-8896798694_3", 'Y', "Copyright 2008 Nature Education", '600', '154', "http://www.natureeducation.com/"); "Schematic of RNA labeling protocol.")
A key step in the protocol these investigators used to distinguish between the three models was the differential ultracentrifugation of so-called "heavy" and "light" ribosomes. Here, the heavy ribosomes were those that were labeled with two radioactive isotopes, 13C and 15N, that were present in the food source of the bacteria, while the light ribosomes were those that remained unlabeled. After switching the bacteria away from 13C- and 15N-containing media, 32P was added to label the nascent RNA that arose following bacteriophage infection. The bacterial samples were then subject to ultracentrifugation. The extremely high speeds used in the ultracentrifugation, along with the salt gradient that was used to buoy up the molecules, resulted in separation of the two types of ribosomes (Figure 3). After centrifugation, the heavier ribosomes were found closer to the bottom of the centrifuge tube; thus, to collect these ribosomes, Brenner et al. simply poked a hole in the bottom of the tube and collected the fractions that dripped out. Naturally, the earliest fractions corresponded to those at the bottom of the tube (i.e., the heaviest particles). Each fraction was then tested to determine where the radioactivity was. This process produced the result depicted in Figure 4.
Figure 4: RNA after isotope transfer in phage-infected cells.
Brenner and colleagues were able to indirectly observe the presence of ribosomes in the samples because it was known that the fractions with high concentrations of ribosomes could be detected by measuring the optical density at a wavelength (λ) of 254 nm. Using this wavelength, the researchers measured the two peaks for the ribosomes, one for the heavy ribosomes (here, at approximately fraction number 43) and one for the light ribosomes (here, at approximately fraction number 53). When the heavy versus light ribosomes (fractions 43 versus 53) were examined, it was noted that the peak of radioactivity from 32P, which had been incorporated into newly synthesized RNA, was under both peaks, but it reached its maximum in the heavy fraction. This suggested that model 1 could not be true, because the heavy ribosomes represented those ribosomes that were in the bacteria before infection with T2 bacteriophages.
Next, to differentiate between models 2 and 3, Brenner et al. had to determine whether preexisting ribosomes were responsible for the protein synthesis observed after bacteriophage expression. Model 2 suggested that host ribosomes were not necessary for this process, while model 3 required that host ribosomes be nonspecialized so that they could facilitate translation of RNA from either the bacterium itself or the invading bacteriophage.
For this experiment, the investigators grew cells in the presence of 15N to label the host ribosomes "heavy." Then, the cells were infected and transferred to 14N ("light") media that also contained radioactive sulfur (35S). Radioactive sulfur effectively labeled new proteins because it labeled methionine and cysteine residues in growing peptides. Upon centrifugation and analysis, the 35S label was found to correspond completely to the fraction in which the preexisting ribosomes were found, suggesting that these native ribosomes facilitated synthesis of bacteriophage proteins. These results were therefore consistent with model 3, which required bacteriophage proteins to be synthesized using the ribosomes of the cells they infect. Together, these experiments revealed that ribosomes serve as nonspecialized sites for protein synthesis and that these organelles work to perform this function no matter the source of the RNA template.
However, much remained to be discovered about the nature of this RNA template, which investigators called messenger RNA or mRNA. For example, was it a direct copy of a gene? How did mRNA stability affect protein expression? If ribosomal RNA was not involved in the process of protein synthesis as originally suspected, what role did it have? In addition, what exactly was the language of translation that connected mRNA to protein? Those questions were left for later investigators to explore.
References and Recommended Reading
Brenner, S., Jacob, F., & Meselson, M. An unstable intermediate carrying information from genes to ribosomes for protein synthesis. Nature 190, 576–581 (1961) doi:10.1038/190576a0 (link to article)
