Essentials of the Living World 5th Edition by George Johnson

Exercise 1

Building Proteins in a Test Tube
The complex mechanisms used by cells to build proteins were not discovered all at once. Our understanding came slowly, accumulating through a long series of experiments, each telling us a little bit more. To gain some sense of the incremental nature of this experimental journey and to appreciate the excitement that each step gave, it is useful to step into the shoes of an investigator back when little was known and the way forward was not clear.
The shoes we will step into are those of Paul Zamecnik, an early pioneer in protein synthesis research. Working with colleagues at Massachusetts General Hospital in the early 1950s, Zamecnik first asked the most direct of questions: Where in the cell are proteins synthesized? To find out, they injected radioactive amino acids into rats. After a few hours, the labeled amino acids could be found as part of newly made proteins in the livers of the rats. And, if the livers were removed and checked only minutes after injection, radioactive-labeled proteins were found only associated with small particles in the cytoplasm. Composed of protein and RNA, these particles, later named ribosomes, had been discovered years earlier by electron microscope studies of cell components. This experiment identified them as the sites of protein synthesis in the cell.
After several years of trial-and-error tinkering, Zamecnik and his colleagues had worked out a "cell-free" protein-synthesis system that would lead to the synthesis of proteins in a test tube. It included ribosomes, mRNA, and ATP to provide energy. It also included a collection of required soluble "factors" isolated from homogenized rat cells that somehow worked with the ribosome to get the job done. When Zamecnik's team characterized these required factors, they found most of them to be proteins, as expected, but also present in the mix was a small RNA, very unexpected.
To see what this small RNA was doing, they performed the following experiment. In a test tube, they added various amounts of 14 C-leucine (that is, the radioactively labeled amino acid leucine) to the cell-free system containing the soluble factors, ribosomes, and ATP. After waiting a bit, they then isolated the small RNA from the mixture and checked it for radioactivity. You can see the results in graph (a) above.
In a follow-up experiment, they mixed the radioactive leucinesmall RNA complex that this experiment had generated with cell extracts containing intact endoplasmic reticulum (that is, a cell system of ribosomes on membranes quite capable of making protein). Looking to see where the radioactive label now went, they then isolated the newly made protein [red in graph (b)] as well as the small RNA [blue in graph (b)].
Interpreting Data Does the amount of leucine added to the test tube have an effect on the amount of leucine found bound to the small RNA?