The Genetic Code

The Genetic Code

Some of the most important features of the code can be specified by saying that the genetic message is contained in a triplet, nonoverlapping, commaless, degenerate,universal code. Each of these terms has a definite meaning that describes the wayin which the code is translated.

A triplet code means that a sequence of three bases (called a codon) is needed to specify one amino acid. The genetic code must translate the lan-guage of DNA, which contains four bases, into the language of the 20 common amino acids that are found in proteins. If there were a one-to-one relationship between bases and amino acids, then the four bases could encode only four amino acids, and all proteins would have to be combinations of these four. If it took two bases to make a codon, then there would be 4² possible combina-tions of two bases for 16 possible amino acids, which is still not enough. Thus, one could have guessed that a codon would have to be at least three bases long. With three bases, there are 4³ possibilities, or 64 possible codons, which is more than enough to encode the 20 amino acids. The term nonoverlapping indicates that no bases are shared between consecutive codons; the ribosome moves along the mRNA three bases at a time rather than one or two at a time (Figure 12.2). If the ribosome moved along the mRNA more than three bases at a time, this situation would be referred to as “a punctuated code.” Because no intervening bases exist between codons, the code is commaless. In a degener-ate code, more than one triplet can encode the same amino acid. Sixty-four(4 X 4 X  4) possible triplets of the four bases occur in RNA, and all are used to encode the 20 amino acids or one of the three stop signals. Note that there is a big difference between a degenerate code and an ambiguous one. Each amino acid may have more than one codon, so the genetic code is a little redundant, but no codon can encode more than one amino acid. If it did, the code would be ambiguous, and the protein-synthesizing machinery would not know which amino acid should be inserted in the sequence. All 64 codons have been assigned meanings, with 61 of them coding for amino acids and the remaining - > serving as the termination signals (Table 12.1).

Two amino acids, tryptophan and methionine, have only one codon each, but the rest have more than one. A single amino acid can have as many as six codons, as is the case with leucine and arginine. Originally, the genetic code was thought to be a random selection of bases encoding amino acids. More recently, it is becoming clear why the code has withstood billions of years of natural selection. Multiple codons for a single amino acid are not randomly distributed in Table 12.1 but have one or two bases in common. The bases that are common to several codons are usually the first and second bases, with more room for variation in the third base, which is called the “wobble” base. The degeneracy of the code acts as a buffer against deleterious mutations. For example, for eight of the amino acids (L, V, S, P, T, A, G, and R), the third base is completely irrelevant. Thus, any mutation in the third base of these codons would not change the amino acid at that location. A mutation in the DNA that does not lead to a change in the amino acid translated is called a silent mutation.

In addition, the second base of the codon also appears to be very important for determining the type of amino acid. For example, when the second base is U, all the amino acids generated from the codon possibilities are hydrophobic. Thus, if the first or third base were mutated, the mutation would not be silent, but the damage would not be as great because one hydrophobic amino acid would be replaced with another. Codons sharing the same first letter often code for amino acids that are products of one another or precursors of one another. A recent paper in Scientific American looked at the error rate for other hypothetical genetic codes and calculated that, of 1 million possible genetic codes that could be conceived of, only 100 would have the effect of reducing errors in protein function when compared with the real code. Indeed, it seems that the genetic code has withstood the test of time because it is one of the best ways to protect an organism from DNA mutations.

However, to make matters more interesting, scientists have recently discov-ered that some silent mutations are not as silent as they once thought.

How did scientists determine the genetic code?

The assignment of triplets in the genetic code was based on several types of experiments. One of the most significant experiments involved the use of synthetic polyribonucleotides as messengers. When homopolynucleotides (polyribonucleotides that contain only one type of base) are used as a syntheticmRNA for polypeptide synthesis in laboratory systems, homopolypeptides(polypeptides that contain only one kind of amino acid) are produced. When poly U is the messenger, the product is polyphenylalanine. With poly A as the messenger, polylysine is formed. The product for poly C is polyproline, and the product for poly G is polyglycine. This procedure was used to establish the code for the four possible homopolymers quickly. When an alternating copolymer (a polymer with an alternating sequence of two bases) is the messenger, the product is an alternating polypeptide (a polypeptide with an alternating sequence of two amino acids). For example, when the sequence of the polynucleotide is –ACACACACACACACACACACAC–, the polypeptide produced has alternating threonines and histidines. There are two types of coding triplets in this polynucleotide, ACA and CAC, but this experiment cannot establish which one codes for threonine and which one codes for histidine. More information is needed for an unambiguous assignment, but it is interesting that this result proves that the code is a triplet code. If it were a doublet code, the product would be a mixture of two homopolymers, one specified by the codon AC and the other by the codon CA. (The terminology for the different ways of reading this message as a doublet is to say that they have different reading frames, /AC/ AC/ and /CA/CA/. In a triplet code, only one reading frame is possible, namely, /ACA/CAC/ACA/CAC/, which gives rise to an alternating polypeptide.) Use of other synthetic polynucleotides can yield other coding assignments, but, as in our example here, many questions remain.

Other methods are needed to answer the remaining questions about codon assignment. One of the most useful methods is the filter-binding assay (Figure 12.3). In this technique, various tRNA molecules, one of which is radioactively labeled with carbon-14 (¹⁴C), are mixed with ribosomes and synthetic trinu-cleotides that are bound to a filter. The mixture of tRNAs is passed through the filter, and some bind and others pass through. If the radioactive label is detected on the filter, then it is known that the particular tRNA did bind. If the radioactive label is found in a solution that flowed through the filter, then the tRNA did not bind. This technique depends on the fact that aminoacyl-tRNAs bind strongly to ribosomes in the presence of the correct trinucleotide. In this situation, the trinucleotide plays the role of an mRNA codon. The possible trinucleotides are synthesized by chemical methods, and binding assays are repeated with each type of trinucleotide. For example, if the aminoacyl-tRNA for histidine binds to the ribosome in the presence of the trinucleotide CAU, the sequence CAU is established as a codon for histidine. About 50 of the 64 codons were identified by this method.

Codon–Anticodon Pairing and Wobble

A codon forms base pairs with a complementary anticodon of a tRNA when an amino acid is incorporated during protein synthesis. Because there are 64 possible codons, one might expect to find 64 types of tRNA but, in fact, the number is less than 64 in all cells.

If there are 64 codons, how can there be less than 64 tRNA molecules?

Some tRNAs bond to one codon exclusively, but many of them can recognize more than one codon because of variations in the allowed pattern of hydrogen bonding. This variation is called “wobble” (Figure 12.4), and it applies to the first base of an anticodon, the one at the 5' end, but not to the second or the third base. Recall that mRNA is read from the 5' to the 3' end. The first (wobble) base of the anticodon hydrogen-bonds to the third base of the codon, the one at the 3' end. The base in the wobble position of the anticodon can base-pair with several different bases in the codon, not just the base specified by Watson–Crick base pairing (Table 12.2).

When the wobble base of the anticodon is uracil, it can base-pair not only with adenine, as expected, but also with guanine, the other purine base. When the wobble base is guanine, it can base-pair with cytosine, as expected, and also with uracil, the other pyrimidine base. The purine base hypoxanthine fre-quently occurs in the wobble position in many tRNAs, and it can base-pair with adenine, cytosine, and uracil in the codon (Figure 12.5). Adenine and cyto-sine do not form any base pairs other than the expected ones with uracil and guanine, respectively (Table 12.2). To summarize, when the wobble position is occupied by I (from inosine, the nucleoside made up of ribose and hypoxan-thine), G, or U, variations in hydrogen bonding are allowed; when the wobble position is occupied by A or C, these variations do not occur.

The wobble model provides insight into some aspects of the degeneracy of the code. In many cases, the degenerate codons for a given amino acid differ in the third base, the one that pairs with the wobble base of the anticodon. Fewer different tRNAs are needed because a given tRNA can base-pair with several codons. As a result, a cell would have to invest less energy in the synthesis of needed tRNAs. The existence of wobble also minimizes the damage that can be caused by misreading of the code. If, for example, a leucine codon, CUU, were to be misread as CUC, CUA, or CUG during transcription of mRNA, this codon would still be translated as leucine during protein synthesis; no damage to the organism would occur. We saw in earlier that drastic consequences can result from misreading the genetic code in other codon positions, but here we see that such effects are not inevitable.

A universal code is one that is the same in all organisms. The universality of the code has been observed in viruses, prokaryotes, and eukaryotes. However, there are some exceptions. Some codons seen in mitochondria are different from those seen in the nucleus. 

There are also at least 16 organisms that have code variations. For example, the marine alga Acetabularia translates the stan-dard stop codons, UAG and UAA, as a glycine rather than as a stop. Fungi of the genus Candida translate the codon CUG as a serine, where that codon would specify leucine in most organisms. The evolutionary origin of these differences is not known at this writing, but many researchers believe that understanding these code variations is important to understanding evolution.

Summary

The genetic code is based on a series of three bases coding for an amino acid.

The code is nearly universal in all organisms from viruses through humans. The code has no punctuation, meaning the mRNA is read three bases at a time with no spaces in between. The code is nonoverlapping as well, meaning that each base is part of only one codon.

The genetic code was determined by a variety of techniques, such as using synthetic mRNA with known sequences to see what proteins would be translated from them.

Although there are 64 combinations of three bases leading to 64 codons, there are fewer types of tRNA anticodons. This means that standard Watson–Crick base pairing must be broken on occasion. The wobble model of codon–anticodon base pairing shows that some bases at the 5' end of the anticodon of the tRNA can base-pair with multiple bases on the codon.