2.2.1: The Genetic Code

The Central Dogma: RNA Encodes Protein

The flow of genetic information in cells from DNA to mRNA to protein is described by the central dogma, as previously discussed. Genes specify the sequence of mRNAs, which in turn specify the sequence of amino acids making up all proteins. The decoding of one molecule to another is performed by specific proteins and RNAs. Because the information stored in DNA is so central to cellular function, it makes sense that the cell would make mRNA copies of this information for protein synthesis, while keeping the DNA itself intact and protected. The copying of DNA to RNA is relatively straightforward, as we saw in the previous chapter, with one nucleotide being added to the mRNA strand for every nucleotide read in the DNA strand. The translation to protein is a bit more complex because three mRNA nucleotides correspond to one amino acid in the polypeptide sequence. However, the translation to protein is still systematic and colinear, such that nucleotides 1 to 3 correspond to amino acid 1, nucleotides 4 to 6 correspond to amino acid 2, and so on.

Figure 4.2 Instructions on DNA are transcribed onto messenger RNA. Ribosomes are able to read the genetic information inscribed on a strand of messenger RNA and use this information to string amino acids together into a protein.

The Genetic Code Is Degenerate and Universal

Because the mRNA 'alphabet' consists of only four nucleotide bases while the protein 'alphabet' consists of 20 amino acids, scientists realized that single letters could not specify individual amino acids. Combinations of two nucleotides (doublets) are also insufficient, yielding only 16 possible arrangements (\(4^{2}\)). In contrast, combinations of three nucleotides (triplets) yield 64 possible arrangements (\(4^{3}\)), providing far more than enough combinations to encode the 20 universal amino acids. This mathematical surplus led researchers to theorize that the genetic code must be degenerate, meaning a single amino acid can be specified by more than one distinct nucleotide triplet, now known as a codon.

This triplet hypothesis was famously confirmed by Francis Crick and Sydney Brenner using the chemical mutagen proflavin to insert nucleotides into a viral gene. Translation relies on a continuous reading frame composed of these triplets. When Crick and Brenner inserted one or two nucleotides, it shifted the entire downstream sequence, completely altering the message for every subsequent amino acid and preventing functional proteins from forming. However, when they inserted exactly three nucleotides, the reading frame was restored downstream; the resulting protein simply gained a single extra amino acid but otherwise maintained its structural integrity. This experiment definitively proved that the genetic code is read as a continuous series of non-overlapping nucleotide triplets. Scientists painstakingly solved the genetic code by translating synthetic mRNAs in vitro and sequencing the proteins they specified (Figure 4.4).

Figure 4.3 The deletion of two nucleotides shifts the reading frame of an mRNA and changes the entire protein message, creating a nonfunctional protein or terminating protein synthesis altogether.

Figure 4.4 This figure shows the genetic code for translating each nucleotide triplet in mRNA into an amino acid or a termination signal in a protein. (credit: modification of work by NIH)

In addition to codons that instruct the addition of a specific amino acid to a polypeptide chain, three of the 64 codons terminate protein synthesis and release the polypeptide from the translation machinery. These triplets are called nonsense codons, or stop codons. Another codon, AUG, also has a special function. In addition to specifying the amino acid methionine, it also serves as the start codon to initiate translation. The reading frame for translation is set by the AUG start codon near the 5' end of the mRNA. Following the start codon, the mRNA is read in groups of three until a stop codon is encountered.

The arrangement of the coding table reveals the structure of the code. There are sixteen "blocks" of codons, each specified by the first and second nucleotides of the codons within the block, e.g., the "AC*" block that corresponds to the amino acid threonine (Thr). Some blocks are divided into a pyrimidine half, in which the codon ends with U or C, and a purine half, in which the codon ends with A or G. Some amino acids get a whole block of four codons, like alanine (Ala), threonine (Thr) and proline (Pro). Some get the pyrimidine half of their block, like histidine (His) and asparagine (Asn). Others get the purine half of their block, like glutamate (Glu) and lysine (Lys). Note that some amino acids get a block and a half-block for a total of six codons.

Degeneracy is believed to be a cellular mechanism to reduce the negative impact of random mutations. Codons that specify the same amino acid typically only differ by one nucleotide. In addition, amino acids with chemically similar side chains are encoded by similar codons. For example, aspartate (Asp) and glutamate (Glu), which occupy the GA* block, are both negatively charged. This nuance of the genetic code ensures that a single-nucleotide substitution mutation might specify the same amino acid but have no effect or specify a similar amino acid, preventing the protein from being rendered completely nonfunctional.

The genetic code is nearly universal. With a few minor exceptions, virtually all species use the same genetic code for protein synthesis. Conservation of codons means that a purified mRNA encoding the globin protein in horses could be transferred to a tulip cell, and the tulip would synthesize horse globin. That there is only one genetic code is powerful evidence that all of life on Earth shares a common origin, especially considering that there are about 10⁸⁴ possible combinations of 20 amino acids and 64 triplet codons.

Scientific Method Connection

Which Has More DNA: A Kiwi or a Strawberry?

Question: Would a kiwi and strawberry that are approximately the same size also have approximately the same amount of DNA?

Background: Genes are carried on chromosomes and are made of DNA. All mammals are diploid, meaning they have two copies of each chromosome. However, not all plants are diploid. The common strawberry is octoploid (8n) and the cultivated kiwi is hexaploid (6n). Research the total number of chromosomes in the cells of each of these fruits and think about how this might correspond to the amount of DNA in these fruits’ cell nuclei. What other factors might contribute to the total amount of DNA in a single fruit? Read about the technique of DNA isolation to understand how each step in the isolation protocol helps liberate and precipitate DNA.

Hypothesis: Hypothesize whether you would be able to detect a difference in DNA quantity from similarly sized strawberries and kiwis. Which fruit do you think would yield more DNA?

Test your hypothesis: Isolate the DNA from a strawberry and a kiwi that are similarly sized. Perform the experiment in at least triplicate for each fruit

1. Prepare a bottle of DNA extraction buffer from 900 mL water, 50 mL dish detergent, and two teaspoons of table salt. Mix by inversion (cap it and turn it upside down a few times).
2. Grind a strawberry and a kiwi by hand in a plastic bag, or using a mortar and pestle, or with a metal bowl and the end of a blunt instrument. Grind for at least two minutes per fruit.
3. Add 10 mL of the DNA extraction buffer to each fruit, and mix well for at least one minute.
4. Remove cellular debris by filtering each fruit mixture through cheesecloth or porous cloth and into a funnel placed in a test tube or an appropriate container.
5. Pour ice-cold ethanol or isopropanol (rubbing alcohol) into the test tube. You should observe white, precipitated DNA.
6. Gather the DNA from each fruit by winding it around separate glass rods.

Analyze your data: Did you notice an obvious difference in the amount of DNA produced by each fruit? Were your results reproducible?

Draw a conclusion: Given what you know about the number of chromosomes in each fruit, can you conclude that chromosome number necessarily correlates to DNA amount? Can you identify any drawbacks to this procedure? If you had access to a laboratory, how could you standardize your comparison and make it more quantitative?