Technical Requirements

Background Information

Genetic information is stored in cells as deoxyribonucleic acid (DNA). In addition to functioning as the hereditary material for living organisms, the information stored in DNA as genes is the basis for cell metabolism because all proteins are synthesized from genes. However DNA is not directly copied into protein. Protein synthesis requires a deciphering of the genetic information stored within DNA whereby the sequence of deoxyribonucleotides in DNA are copied into strands of ribonucleic acid (RNA) during a process called transcription. During translation, different RNA molecules-specifically messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA) - are used to specify the amino acids that are incorporated into a protein. Because it has been well established that this flow of genetic information is universally followed by living cells-including bacteria, yeast, and human cells-this concept is often referred to as the central dogma of the genetic code.

The structure of DNA and its role as a genetic material has not always been so clearly understood. In the 1900s, many scientists suggested that proteins were a key component of cell metabolism; however, the association between genes and proteins was not known. During this time, Sir Archibald Garrod and William Bateson observed human patients who demonstrated rare diseases caused by deficiencies in metabolic pathways involving amino acids. Garrod and Bateson followed the patterns of genetic inheritance of these disorders within families and concluded that inherited information controls metabolism in a cell. The terms gene and enzyme were not even used at this time.

In the 1930s, George Beadle and Edward Tatum performed experiments with a bread mold, Neurospora crassa, that provided substantial evidence for the relationship between genes and proteins. Beadle and Tatum developed and observed several mutant groups of Neurospora that were identified by comparing the ability of these mutants to grow on plates with minimal nutritional medias with that of wild-type Neurospora. Beadle and Tatum discovered different mutants that were defective in their ability to synthesize the amino acid arginine. Supplementing these mutants with other amino acids allowed some of these mutants to synthesize arginine. Beadle and Tatum hypothesized that the amino acid supplements that allowed the mutants to synthesize arginine were amino acids that the mutants could not synthesize on their own due to a mutation that caused a loss of enzyme activity. These results led Beadle and Tatum to suggest a one gene - one enzyme hypothesis, in which they reasoned that a single gene is important for determining the synthesis of a single enzyme. This hypothesis, however, did not account for how DNA was deciphered by a cell to produce a protein. In the 1940s and early 1950s, several other scientists performed experiments with bacteria and viruses to present strong evidence that genes were made of DNA, but the process by which a protein could be produced from a gene was still unknown.

The discovery of the double-helical structure of DNA by James Watson and Francis Crick in 1953 clearly established that hereditary information in cells is encoded in the nucleotide sequences contained within DNA. Although this landmark discovery represented a significant advance in the history of biological research, a basic question still existed: How was the nucleotide sequence of genes interpreted or decoded by a cell to provide that cell with the instructions for the synthesis of a protein?

A number of different investigators carried out studies to demonstrate that RNA was synthesized from DNA and that RNA performed a central role in protein synthesis. In 1961, Marshall Nirenberg and Heinrich Matthaei used an in vitro cell-free protein- synthesizing system to provide the first evidence for protein-coding sequences of RNA nucleotides. In a cell-free system, organelles such as ribosomes and other factors including amino acids, tRNAs, a mRNA template, and a number of cofactors required for translation­can be added together to synthesize proteins in a test tube. Extracts of organelles and the molecules required for translation are often isolated by the lysis of bacteria or animal cells that are highly active in protein synthesis. Nirenberg and Matthaei used extracts from bacteria called Escherichia coli. The addition of radioactive amino acids to a cell-free extract allows biologists to follow the rate and specific sequences of proteins that are translated in the assay. Because mRNA had only recently been discovered and was not yet easily isolated from cells, Nirenberg and Matthaei synthesized RNA homopolymers, single strands of RNA containing only one ribonucleotide in each strand (for example, UUUUUUU, AAAAAAA), by using a bacterial enzyme called polynucleotide phosphorylase. This enzyme does not require a DNA template to synthesize strands of RNA. The homopolymers synthesized by polynucleotide phosphorylase were then added to the cell-free system, and the incorporation of radioactive isotopes into protein was measured. In a cell-free system, translation begins at multiple and random sites along a nucleotide sequence. Keep this in mind when completing the assignments for this laboratory.

Although these early experiments did not determine the number of nucleotides required for a codon, Nirenberg and Matthaei concluded that certain RNA sequences coded for specific amino acids. For example, poly A codes for lysine. Subsequent experiments by Nirenberg and others using RNA heteropolymers, combinations of different ribonucleotides, served to further delineate the assignment of specific nucleotide sequences to individual amino acids. In 1964, Marshall Nirenberg and Philip Leder used an increased understanding of the function of tRNA and the function of ribosomes as RNA-binding organelles to establish that the genetic code is interpreted as three-ribonucleotide sequences, called codons, that specify only one amino acid. Nirenberg and Leder's experiments also provided a better understanding of how the anticodon portion of a tRNA molecule interacts with a codon by base pairing during translation.

Gobind Khorana performed similar experiments with a cell-free system to which long sequences of RNA molecules consisting of repeating dinucleotides (for example, ACACAC), trinucleotides, and tetranucleotides were added. The results of Khorana's experiments, and the work of many others, served to identify new codons as well as confirm the specificity of many codons that were previously identified. In particular, Khorana concluded that certain sequences, such as a triplet contained in polymers of GAUA, function as termination signals because they do not code for the incorporation of an amino acid into a peptide.

It was apparent from many of these studies that the genetic code is degenerate or redundant, because although each codon codes for only one amino acid, most amino acids are specified by more than one codon. The wobble hypothesis was proposed by Francis Crick to explain how the first two nucleotides of a codon are more important for tRNA binding to an anticodon than the third nucleotide. Modified base-pair rules that occur with U at the third position (for example, U may pair with A or G at the third position of a codon) suggested a rationale for why the number of different tRNAs inside a cell does not need to equal the number of codons that code for amino acids.

In this laboratory you will have the opportunity to simulate many of the early experiments involving cell-free extracts that were essential for deciphering and determining the genetic code. You will investigate how polyribonucleotide sequences that you create can be translated in a cell-free system to produce sequences of amino acids, and you will interpret the results of your experiments to help you learn how the genetic code is deciphered.

References

  1. Alberts, B., et al. Essential Cell Biology, 1st ed. New York: Garland Publishing, 1998.

  2. Beadle, G. W., and Tatum, E. L. "Genetic Control of Biochemical Reactions in Neurospora." Proceedings of the National Academy of Science, USA 27 (1941): 499-506.

  3. Nirenberg, M. W. "The Genetic Code: II." Scientific American, March 1963.