Quantitative Analysis Another important feature of RNA (or DNA, for that matter) is its base composition, that is, the percentage of each of the nucleotides that make it up. The four bases that, with ribose and phosphoric acid, comprise the RNA molecule are guanine, adenine, cytosine, and uracil. It will be noted that three of the four—guanine, adenine, and cytosine—are the same as those in DNA, but thymidine in DNA has been replaced by another base, uracil. To determine the percentage of each base in a given RNA molecule, we must digest RNA with alkali to produce mononucleotides, which are smaller molecules, each consisting of a base, ribose, and phosphoric acid. We can now separate the four nucleotides by using paper chromatography (see [Figure 20]).
Figure 21 A paper chromatography showing separation of amino acids in two directions. Radioactivity in samples then produced this record by radioautography.
In this technique a mixture of compounds is deposited on the edge of a special type of paper. This edge is then immersed in a solvent that slowly permeates the paper (at a constant speed) by capillary action. As the solvent moves from the immersed edge toward the other edge, which is hanging freely, it carries the mixture of nucleotides with it. Each of the compounds in the mixture travels at a different speed, however; thus, as the solvent front moves along the paper, the dissolved compounds are separated from each other and appear as distinct spots on the paper. To locate the nucleotides on the paper and to determine the percentage composition, we can use a chromatogram scanner, a device that scans the paper chromatograms, measures the radiation from them, and thus locates the labeled substances (see [Figure 22]).
Figure 22 Recording of radioactivity in a sample by radioautography and paper chromatography. The peaks of the trace prepared by a chromatogram scanner coincide with the areas of separated components on the same chromatogram, as revealed by radioautography. The radioautograph is superimposed on the chromatogram recording.
Another technique used to separate the nucleotides of RNA is column chromatography. In this method mixtures of nucleotides are separated as they pass down a column of chemicals (see [Figure 23]).
Figure 23 Students visiting Argonne National Laboratory listen to a scientist explain the column chromatography process, in which mixtures of nucleotides are separated as they pass down a column of chemicals.
We have now learned how to use radioisotopes to investigate the synthesis of RNA, the molecule that translates the DNA message into the language of proteins. Let us now see what we can learn about the synthesis and function of proteins.