RNA's involvement in polypeptide synthesis, which is taken for granted today, was not firmly established until the 1950s. The earliest clues that RNA plays some role in information flow between genes and polypeptides came from the independent work of Torbjörn Caspersson and Jean Brachet in the late 1930s and early 1940s. Caspersson used cytochemical techniques to show that: (1) most of a eukaryotic cell's DNA and RNA are in its nucleus and cytoplasm, respectively; (2) cells that are actively engaged in protein synthesis have high levels of cytoplasmic RNA; and (3) cytoplasmic RNA tends to be concentrated in small spherical particles. Brachet, using cell fractionation techniques to separate nuclear and cytoplasmic fractions, also observed a correlation between active protein synthesis and high cytoplasmic RNA levels. In addition, Brachet centrifuged the cytoplasmic fraction at high speed to obtain a pellet containing the RNA-rich spherical particles that Caspersson had observed in the cell. Because these RNA-rich particles contained tightly bound proteins, they are more properly described as ribonucleoproteins. By the early 1950s, electron microscopy techniques had improved to the point where they could be used to visualize the spherical ribonucleoprotein particles, which appeared to be about 250 Å in diameter. These particles were known by a variety of different names until 1957, when Richard B. Roberts coined the now universally accepted term ribosome.
    Some eukaryotic ribosomes appear to be free in the cytoplasm, whereas others are attached to the outer surface of a continuous intracellular tubular membrane network that Keith Porter named the endoplasmic reticulum. Regions of the ER that are studded with ribosomes appear rough or grainy in electron micrographs and are therefore known as the rough endoplasmic; reticulum (RER). Regions that lack ribosomes have a smooth appearance and are therefore called the smooth endoplasmic reticulum (SER). The ER cannot be isolated as a continuous membrane network because methods that disrupt the cell membrane also rupture the endoplasmic reticulum. However, pieces of endoplasmic reticulum can be isolated by differential centrifugation of a crude cell homogenate. The crude cell extract is centrifuged at about 600 X g for five minutes to remove intact cells and nuclei, and then the postnuclear supernatant is centrifuged at about 15,000 X g for one hour to remove mitochondria, lysosomes, and peroxisomes. Further centrifugation at 100,000 X g for two hours produces a pellet called the microsomal fraction that contains pieces of the endoplasmic reticulum and free ribosomes.
    In 1953, Paul Zamecnik and coworkers provided the first convincing evidence that the microsomal fraction makes an important contribution to protein synthesis. Their approach was to inject rats with radioactive amino acids, kill groups of rats at intervals, remove the livers, disrupt the fresh livers, fractionate the disrupted liver cells, and determine the amount of radioactive protein that was present in the different cellular fractions. They observed that radioactive proteins first appeared in ribosomes associated with the microsomal fraction, suggesting that ribosomes are protein synthetic factories.
    Building on these in vivo studies, Zamecnik and coworkers worked to develop a cell-free system that would permit them to characterize components of the protein synthetic system and learn how the components work. Their efforts were rewarded by the discovery that a suspension containing rat liver ribosomes, the 100,000 X g supernatant, adenosine triphosphate (ATP), and guanosine triphosphate (GTP) converts acid- soluble [C] amino acids into acid-insoluble radioactive proteins.