27.1 The Genetic Code in Chapter 27 Protein Metabolism

27.1 The Genetic Code

By the 1960s, it was apparent that at least three nucleotide residues of DNA are necessary to encode each amino acid. The four code letters of DNA (A, T, G, and C) in groups of two can yield only $4^{2} = 16$ $4 squared equals 16$ different combinations, insufficient to encode 20 amino acids. Groups of three, however, yield $4^{3} = 64$ $4 cubed equals 64$ different combinations. Deciphering the genetic code quickly became a major goal.

The Genetic Code Was Cracked Using Artificial mRNA Templates

Several key properties of the genetic code were established in early genetic studies. A codon is a triplet of nucleotides that codes for a specific amino acid. In all living systems, translation occurs in such a way that these nucleotide triplets are read in a successive, nonoverlapping fashion (Figs. 27-3, 27-4). A specific first codon in the sequence establishes the reading frame, in which a new codon begins every three nucleotide residues. There is no punctuation between codons for successive amino acid residues. The amino acid sequence of a protein is defined by a linear sequence of contiguous triplets. In principle, any given single-stranded DNA or mRNA sequence has three possible reading frames. Each reading frame gives a different sequence of codons (Fig. 27-5), but only one is likely to encode a given protein. A key question remained: what were the three-letter code words for each amino acid?

A figure shows the difference between a nonoverlapping code and an overlapping code. — FIGURE 27-3 Overlapping versus nonoverlapping genetic codes. In a nonoverlapping code, codons (numbered consecutively) do not share nucleotides. In an overlapping code, some nucleotides in the mRNA are shared by different codons. In a triplet code with maximum overlap, many nucleotides, such as the third nucleotide from the left (A), are shared by three codons. A nonoverlapping code provides much more flexibility in the triplet sequence of neighboring codons and therefore in the possible amino acid sequences designated by the code.

The top horizontal strand is labeled nonoverlapping code. It contains sets of three nucleotides enclosed in numbered brackets. From left to right, the sequence is A U A enclosed in bracket 1, C G A enclosed in bracket 2, G U C enclosed in bracket 3, and the left half of a bracket with three vacant dashes. The horizontal strand below is labeled overlapping code. From left to right, it contains A U A enclosed in bracket 1, then a space, then C G A, then a space then G U C. Beneath, bracket 2 begins to the left of U in bracket 1 and ends to the right of C to the right, in the central set of three nucleotides. This produces a sequence of U A C enclosed in bracket 2. Beneath, bracket 3 begins to the left of A in bracket 1 and ends to the right of G in the central set of three nucleotides. This produces a sequence of A C G enclosed in bracket 3. Each bracket in the row showing overlapping code includes nucleotides also included in the other brackets whereas each bracket in the row showing nonoverlapping code contains a unique set of nucleotides.

A figure shows evidence for the triplet, nonoverlapping code by showing the effects of insertion mutations, deletion mutations, and mutations that include insertion and deletion. — FIGURE 27-4 The triplet, nonoverlapping code. Evidence for the general nature of the genetic code came from many types of experiments, including genetic experiments on the effects of deletion and insertion mutations. Inserting or deleting one base pair (shown here in the mRNA transcript) alters the sequence of all amino acids coded by the mRNA following the change. Combining insertion and deletion mutations affects some amino acids but can eventually restore the correct amino acid sequence. Adding or subtracting three nucleotides (not shown) leaves the remaining triplets intact, providing evidence that a codon has three, rather than four or five, nucleotides. The triplet codons shaded in gray are those transcribed from the original gene; codons shaded in blue are new codons resulting from the insertion or deletion mutations.

Four horizontal rows are shown. The top row is labeled m R N A. It begins at 5 prime followed by three dashes, then has five gray boxes containing bases follows: G U A, then G C C, then U A C, then G G A, then U with three dashes to 3 prime. The second row is labeled insertion. It begins with three dashes, then has a gray box containing G U A, a gray box containing G C C, a blue box containing U C A indicated by a red arrow from a red (+) symbol, a blue box containing C G G, and a blue box containing A U followed by three dashes. The third row is labeled deletion and begins with three dashes, then has a gray box containing G U A, then has a red arrow pointing down from a red (-) symbol to the space between the gray box and the blue box to its right, then has a blue box containing C C U, then a blue box containing A C G, then a blue box containing G A U followed by three dashes. The fourth row is labeled insertion and deletion. It begins with three dashes, then has a gray box containing G U A, then a blue box labeled A G C with a red arrow pointing down from a red (+) symbol to the left-hand A, then a blue box containing C A C with a red arrow pointing down from a red (-) symbol to the space between the left-hand C and A, then a gray box containing G G A, then a gray box containing U followed by three dashes. A vertical line from the space between the blue box containing C A C and the gray box containing G G A connects with a horizontal arrow pointing right above text reading, reading frame restored. This G G A followed by U in the next box matches the G G A followed by U in the original m R N A strand shown above with no mutations.

A figure shows three potential reading frames m R N A that has a triplet, nonoverlapping code. — FIGURE 27-5 Reading frames in the genetic code. In a triplet, nonoverlapping code, all mRNAs have three potential reading frames, shaded here in different colors. The triplets, and hence the amino acids specified, are different in each reading frame.

Three horizontal rows show three different reading frames. From top to bottom, these are labeled reading from 1, reading frame 2, and reading frame 3. All of the reading frames show the same sequence of bases, but the bases are divided up into different codons. Reading frame 1 is gray and begins with 5 prime followed by three dashes and then the following eight gray boxes with three nucleotides each as follows: U U C, U C G, G A C, C U G, G A G, A U U, C A C, and A G U, then three dashes to 3 prime. Reading frame 2 is blue and begins with three dashes followed by a single U, then seven blue boxes each with three nucleotides as follows: U C U, C G G, A C C, U G G, A G A, U U C, and A C A. It then has a blue box with G U followed by three dashes. Reading frame 3 is yellow and begins with three dashes followed by a box with U U, then seven yellow boxes each with three nucleotides as follows: C U C, G G A, C C U, G G A, G A U, U C A, and C A G. It then has a yellow box with U followed by three dashes.

In 1961, Marshall Nirenberg and J. Heinrich Matthaei reported the first breakthrough. They incubated synthetic polyuridylate, poly(U), with an Escherichia coli extract, GTP, ATP, and a mixture of the 20 amino acids in 20 different tubes, each tube containing a different radioactively labeled amino acid. Because poly(U) mRNA is made up of many successive UUU triplets, it should promote the synthesis of a polypeptide containing only the amino acid encoded by UUU. A radioactive polypeptide was formed only in the tube containing radioactive phenylalanine. Nirenberg and Matthaei therefore concluded that the triplet codon UUU encodes phenylalanine. The same approach soon revealed that polycytidylate, poly(C), encodes a polypeptide containing only proline (polyproline), and that polyadenylate, poly(A), encodes polylysine. Polyguanylate did not generate any polypeptide in this experiment because it spontaneously forms tetraplexes (see Fig. 8-20d) that cannot be bound by ribosomes.

The synthetic polynucleotides used in such experiments were prepared by using polynucleotide phosphorylase (p. 987), which catalyzes the formation of RNA polymers starting from ADP, UDP, CDP, and GDP. This enzyme, discovered by physician/biochemist Severo Ochoa, requires no template and makes polymers with a base composition that directly reflects the relative concentrations of the nucleoside $5'$ $5 prime$ -diphosphate precursors in the medium. If polynucleotide phosphorylase is presented with only UDP, it makes only poly(U). If it is presented with a mixture of five parts ADP and one part CDP, it makes a polymer in which about five-sixths of the residues are adenylate and one-sixth is cytidylate. This random polymer is likely to have many triplets of the sequence AAA; smaller numbers of AAC, ACA, and CAA triplets; relatively few ACC, CCA, and CAC triplets; and very few CCC triplets (Table 27-1). Using a variety of artificial mRNAs made by polynucleotide phosphorylase from different starting mixtures of ADP, GDP, UDP, and CDP, the Nirenberg and Ochoa groups soon identified the base compositions of the triplets coding for almost all the amino acids. Although these experiments revealed the base composition of the coding triplets, they usually could not reveal the sequence of the bases.

TABLE 27-1 Incorporation of Amino Acids into Polypeptides in Response to Random Polymers of RNA
Amino acid	Observed frequency of incorporation $(Lys = 100)$ $left-parenthesis bold upper L y s equals bold 100 right-parenthesis$	Tentative assignment for nucleotide composition of corresponding codon^a	Expected frequency of incorporation based on assignment $(Lys = 100)$ $left-parenthesis bold upper L y s equals bold 100 right-parenthesis$
Asparagine	24	$A_{2} C$ $upper A Subscript 2 Baseline upper C$	20
Glutamine	24	$A_{2} C$ $upper A Subscript 2 Baseline upper C$	20
Histidine	6	${AC}_{2}$ $AC Subscript 2$	4
Lysine	100	AAA	100
Proline	7	${AC}_{2}, CCC$ $AC Subscript 2 Baseline comma CCC$	4.8
Threonine	26	$A_{2} C, {AC}_{2}$ $upper A Subscript 2 Baseline upper C comma AC Subscript 2 Baseline$	24
Note: Presented here is a summary of data from one of the early experiments designed to elucidate the genetic code. A synthetic RNA containing only A and C residues in 5:1 ratio directed polypeptide synthesis, and both the identity and the quantity of incorporated amino acids were determined. Based on the relative abundance of A and C residues in the synthetic RNA, and assigning the codon AAA (the most likely codon) a frequency of 100, there should be three different codons of composition $A_{2} C$ $upper A Subscript 2 Baseline upper C$ , each at a relative frequency of 20; three of composition ${AC}_{2}$ $AC Subscript 2$ , each at a relative frequency of 4.0; and CCC at a relative frequency of 0.8. The CCC assignment was based on information derived from prior studies with poly(C). Where two tentative codon assignments are made, both are proposed to code for the same amino acid. ^a These designations of nucleotide composition contain no information on nucleotide sequence (except, of course, AAA and CCC).

Key convention

Much of the following discussion deals with tRNAs. The amino acid specified by a tRNA is indicated by a superscript, such as ${tRNA}^{Ala}$ $tRNA Superscript Ala$ , and the aminoacylated tRNA is designated by a hyphenated name, such as ${alanyl-tRNA}^{Ala}$ $alanyl hyphen tRNA Superscript Ala$ or ${Ala-tRNA}^{Ala}$ $Ala hyphen tRNA Superscript Ala$ .

In 1964, Nirenberg and Philip Leder achieved another experimental breakthrough. Isolated E. coli ribosomes would bind a specific aminoacyl-tRNA in the presence of the corresponding synthetic polynucleotide messenger. For example, ribosomes incubated with poly(U) and ${phenylalanyl-tRNA}^{Phe} ({Phe-tRNA}^{Phe})$ $phenylalanyl hyphen tRNA Superscript Phe Baseline left-parenthesis Phe hyphen tRNA Superscript Phe Baseline right-parenthesis$ bind both RNAs, but if the ribosomes are incubated with poly(U) and some other aminoacyl-tRNA, the aminoacyl-tRNA is not bound, because it does not recognize the UUU triplets in poly(U) (Table 27-2). Even trinucleotides could promote specific binding of appropriate tRNAs, so these experiments could be carried out with chemically synthesized small oligonucleotides. With this technique, researchers determined which aminoacyl-tRNA bound to 54 of the 64 possible triplet codons. For some codons, either no aminoacyl-tRNA or more than one would bind. Another method was needed to complete and confirm the entire genetic code.

TABLE 27-2 Trinucleotides That Induce Specific Binding of Aminoacyl-tRNAs to Ribosomes
	Relative increase in ${}^{14}C$ $Superscript bold 14 Baseline bold upper C$ -labeled aminoacyl-tRNA bound to ribosome^a
Trinucleotide	${Phe-tRNA}^{Phe}$ $bold Phe hyphen tRNA Superscript bold Phe$	${Lys-tRNA}^{Lys}$ $bold Lys hyphen tRNA Superscript bold Lys$	${Pro-tRNA}^{Pro}$ $bold Pro hyphen tRNA Superscript bold Pro$
UUU	4.6	0	0
AAA	0	7.7	0
CCC	0	0	3.1
Information from M. Nirenberg and P. Leder, Science 145:1399, 1964. ^aEach number represents the factor by which the amount of bound ${}^{14}C$ $Superscript 14 Baseline upper C$ increased when the indicated trinucleotide was present, relative to a control with no trinucleotide.

At about this time, a complementary approach was provided by H. Gobind Khorana, who developed chemical methods to synthesize polyribonucleotides with defined, repeating sequences of two to four bases. The polypeptides produced by these mRNAs had one or a few amino acids in repeating patterns. These patterns, when combined with information from the random polymers used by Nirenberg and colleagues, permitted unambiguous codon assignments. The copolymer ${(AC)}_{n}$ $left-parenthesis AC right-parenthesis Subscript n$ , for example, has alternating ACA and CAC codons: ACACACACACACACA. The polypeptide synthesized on this messenger contained equal amounts of threonine and histidine. Given that a histidine codon has one A and two Cs (Table 27-1), CAC must code for histidine and ACA for threonine.

Consolidation of the results from many experiments permitted assignment of 61 of the 64 possible codons. The other three were identified as termination codons, in part because they disrupted amino acid coding patterns when they occurred in a synthetic RNA polymer (Fig. 27-6). Meanings for all the triplet codons (tabulated in Fig. 27-7) were established by 1966 and have been verified in many different ways. The cracking of the genetic code is regarded as one of the most important scientific discoveries of the twentieth century.

A figure shows the effect of a termination codon in a repeating tetranucleotide in three different reading frames. — FIGURE 27-6 Effect of a termination codon in a repeating tetranucleotide. Termination codons (light red) are encountered every fourth codon in three different reading frames (shown in different colors). Dipeptides or tripeptides are synthesized, depending on where the ribosome initially binds.

Three horizontal rows show three different reading frames. From top to bottom, these are labeled reading from 1, reading frame 2, and reading frame 3. All of the reading frames show the same sequence of bases, but the bases are divided up into different codons. Reading frame 1 begins with 5 prime followed by three dashes and then the following three gray boxes with three nucleotides each as follows: G U A, A G U, and A A G. It then has a red box containing U A A, then a gray box containing G U A followed by a gray box containing A G U. It ends with A A outside of a box followed by three dashes to 3 prime. Reading frame 2 begins with three dashes, then G outside of a box, then a red box containing U A A. It then has three blue boxes reading G U A, A G U, and A A G. Next, it has a red box reading U A A, then a blue box reading G U A followed by A outside of a box followed by three dashes. Reading frame 3 begins with three dashes followed by G U outside of a box, then a yellow box containing A A G, then a red box containing U A A, then yellow boxes containing G U A, A G U, and A A G, followed by a red box containing U A A followed by three dashes.

A table shows the amino acids specified by different codons. — FIGURE 27-7 “Dictionary” of amino acid code words in mRNAs. The codons are written in the $5' \to 3'$ $5 prime right-arrow 3 prime$ direction. The third base of each codon (in bold type) plays a lesser role in specifying an amino acid than the first two. The three termination codons are shaded in light red, the initiation codon AUG in green. All the amino acids except methionine and tryptophan have more than one codon. In most cases, codons that specify the same amino acid differ only at the third base.

FIGURE 27-7 “Dictionary” of amino acid code words in mRNAs. The codons are written in the $5' \to 3'$ $5 prime right-arrow 3 prime$ direction. The third base of each codon (in bold type) plays a lesser role in specifying an amino acid than the first two. The three termination codons are shaded in light red, the initiation codon AUG in green. All the amino acids except methionine and tryptophan have more than one codon. In most cases, codons that specify the same amino acid differ only at the third base.

The table has four columns and four major rows each subdivided into four sub-rows. The headings of the four columns are U, C, A, and G and an arrow pointing horizontally to the right across them reads, second letter of codon. From top to bottom, the four major rows are labeled U, C, A, and G and an arrow pointing downward from above it reads, first letter of codon (5 prime end). Each of the sixteen squares created by these four columns and four rows contains four rows that each show one codon and one nucleotide. Data in the table is as follows with each square listed with its four rows of codons followed by corresponding amino acids. Column 1 U, row 1 U: U U bolded U end bold, P h e; U U bolded C end bold, P h e; U U bolded A end bold, L e u; U U bolded G end bold, L e u. Column 2 C, row 1 U: U C bolded U end bold, S e r; U C bolded C end bold, S e r; U C bolded A end bold, S e r; U C bolded G end bold, S e r. Column 3 A, row 1 U: U A bolded U end bold, T y r; U A bolded C end bold, T y r; U A bolded A end bold in a light red box, Stop; U A bolded G end bold in a light red box, Stop. Column 4 G, row 1 U: U G bolded U end bold, C y s, U G bolded C end bold, C y s; U G bolded A end bold in a light red box, Stop; U G bolded G end bold, S e r. Column 1 U, row 2 C: C U bolded U end bold, L e u; C U bolded C end bold, L e u; C U bolded A end bold, L e u; C U bolded G end bold, L e u. Column 2 C, row 2 C: C C bolded U end bold, P r o; C C bolded C end bold, P r o; C C bolded A end bold, P r o; C C bolded G end bold, P r o. Column 3 A, row 2 C: C A bolded U end bold, H i s; C A bolded C end bold, H i s; C A bolded A end bold, G l n; C A bolded G end bold, G l n. Column 4 G, row 2 C: C G bolded U end bold, A r g; C G bolded C end bold, A r g; C G bolded A end bold, A r g; C G bolded G end bold, A r g. Column 1 U, row 3 A: A U bolded U end bold, I l e; A U bolded C end bold, I l e; A U bolded A end bold, I l e; A U bolded G end bold in a light green box, M e t. Column 2 C, row 3 A: A C bolded U end bold, T h r; A C bolded C end bold, T h r; A C bolded A end bold, T h r; A C bolded G end bold, T h r. Column 3 A, row 3 A: A A bolded U end bold, A s n; A A bolded C end bold, A s n; A A bolded A end bold, L y s; A A bolded G end bold, L y s. Column 4 G, row 3 A: A G bolded U end bold, S e r; A G bolded C end bold, S e r; A G bolded A end bold, A r g; A G bolded G end bold, A r g. Column 1 U, row 4 G: G U bolded U end bold, V a l; G U bolded C end bold, V a l; G U bolded A end bold, V a l; G U bolded G end bold, V a l. Column 2 C, row 4 G: G C bolded U end bold, A l a; G C bolded C end bold, A l a; G C bolded A end bold, A l a; G C bolded G end bold, A l a. Column 3 A, row 4 G: G A bolded U end bold, A s p; G A bolded C end bold, A s p; G A bolded A end bold, G l u; G A bolded G end bold, G l u. Column 4 G, row 4 G: G G bolded U end bold, G l y; G G bolded C end bold, G l y; G G bolded A end bold, G l y; GA G bolded G end bold, G l y.

Codons are the key to the translation of genetic information, directing the synthesis of specific proteins. The reading frame is set when translation of an mRNA molecule begins, and it is maintained as the synthetic machinery reads sequentially from one triplet to the next. If the initial reading frame is off by one or two bases, or if translation somehow skips a nucleotide in the mRNA, all the subsequent codons will be out of register; the result is usually a “missense” protein with a garbled amino acid sequence.

Several codons serve special functions (Fig. 27-7). The initiation codon AUG is the most common signal for the beginning of a polypeptide in all cells, in addition to coding for Met residues in internal positions of polypeptides. The termination codons (UAA, UAG, and UGA), also called stop codons or nonsense codons, normally signal the end of polypeptide synthesis and do not code for any known amino acids.

As described in Section 27.2, initiation of protein synthesis in the cell is an elaborate process that relies on initiation codons and other signals in the mRNA. In retrospect, the experiments of Nirenberg, Khorana, and others to identify codon function should not have worked in the absence of initiation codons. Serendipitously, experimental conditions caused the normally complex initiation requirements for protein synthesis (unknown at the time) to be relaxed. Diligence combined with chance to produce a breakthrough — a common occurrence in the history of biochemistry.

In a random sequence of nucleotides, 1 in every 20 codons in each reading frame is, on average, a termination codon. In general, a reading frame without a termination codon among 50 or more consecutive codons is referred to as an open reading frame (ORF). Long ORFs usually correspond to genes that encode proteins. In the analysis of sequence databases, sophisticated programs are used to search for ORFs in order to find genes among the often-huge background of nongenic DNA. An uninterrupted gene coding for a typical protein with a molecular weight of 60,000 would require an ORF with 500 or more codons.

A striking feature of the genetic code is that an amino acid may be specified by more than one codon, so the code is described as degenerate. This does not suggest that the code is flawed: although an amino acid may have two or more codons, each codon specifies only one amino acid. The degeneracy of the code is not uniform. Whereas methionine and tryptophan have single codons, for example, three amino acids (Arg, Leu, Ser) have six codons, five amino acids have four, isoleucine has three, and nine amino acids have two (Table 27-3).

TABLE 27-3 Degeneracy of the Genetic Code
Amino acid	Number of codons	Amino acid	Number of codons
Met	1	Tyr	2
Trp	1	Ile	3
Asn	2	Ala	4
Asp	2	Gly	4
Cys	2	Pro	4
Gln	2	Thr	4
Glu	2	Val	4
His	2	Arg	6
Lys	2	Leu	6
Phe	2	Ser	6

The genetic code is nearly universal. With the intriguing exception of a few minor variations in mitochondria, some bacteria, and some single-celled eukaryotes, amino acid codons are identical in all species examined so far. Human beings, E. coli, tobacco plants, amphibians, and viruses share the same genetic code. This suggests that all life-forms have a common evolutionary ancestor, whose genetic code has been preserved throughout biological evolution. Even the variations (Box 27-1) reinforce this theme.

Box 27-1

Exceptions That Prove the Rule: Natural Variations in the Genetic Code

In biochemistry, as in other disciplines, exceptions to general rules can be problematic for instructors and frustrating for students. At the same time, though, they teach us that life is complex and they inspire us to search for more surprises. Understanding the exceptions can even reinforce the original rule in unpredictable ways.

One would expect little room for variation in the genetic code. Even a single amino acid substitution can have profoundly deleterious effects on the structure of a protein. Nevertheless, variations in the code do occur in some organisms, and they are both interesting and instructive. The types of variation and their rarity provide powerful evidence for a common evolutionary origin of all living things.

To alter the code, changes must occur in the gene(s) encoding one or more tRNAs, with the obvious target for alteration being the anticodon. Such a change would lead to the systematic insertion of an amino acid at a codon that, according to the standard code (see Fig. 27-7), does not specify that amino acid. The genetic code, in effect, is defined by two elements: (1) the anticodons on tRNAs, which determine where an amino acid is placed in a growing polypeptide, and (2) the specificity of the enzymes — the aminoacyl-tRNA synthetases — that charge the tRNAs, which determines the identity of the amino acid attached to a given tRNA.

Most sudden changes in the code would have catastrophic effects on cellular proteins, so code alterations are more likely to persist where relatively few proteins would be affected — such as in small genomes encoding only a few proteins. The biological consequences of a code change could also be limited by restricting changes to the three termination codons, which do not generally occur within genes. This pattern is, in fact, observed.

Of the very few variations in the genetic code that we know of, most occur in mitochondrial DNA (mtDNA), which encodes only 10 to 20 proteins. Mitochondria have their own tRNAs, so their code variations do not affect the much larger cellular genome. The most common changes in mitochondria involve termination codons. These changes affect termination in the products of only a subset of genes, and sometimes the effects are minor, because the genes have multiple (redundant) termination codons.

Vertebrate mtDNAs have genes that encode 13 proteins, 2 rRNAs, and 22 tRNAs (see Fig. 19-40). Given the small number of codon reassignments, along with an unusual set of wobble rules (p. 1012), the 22 tRNAs are sufficient to decode the protein-coding genes, as opposed to the 32 tRNAs required for the standard code. In mitochondria, these changes can be viewed as a kind of genomic streamlining, as a smaller genome confers a replication advantage on the organelle. Four codon families (in which the amino acid is determined entirely by the first two nucleotides) are decoded by a single tRNA with a U residue in the first (or wobble) position in the anticodon. Either the U pairs somehow with any of the four possible bases in the third position of the codon or a “two out of three” mechanism is used — that is, no base pairing is needed at the third position. Other tRNAs recognize codons with either A or G in the third position, and yet others recognize U or C, so that virtually all the tRNAs recognize either two or four codons.

In the standard code, only two amino acids are specified by single codons: methionine and tryptophan (see Table 27-3). If all mitochondrial tRNAs recognize two codons, we would expect additional Met and Trp codons in mitochondria. And we find that the single most common code variation is UGA, usually a termination codon, specifying tryptophan. The ${tRNA}^{Trp}$ $tRNA Superscript Trp$ recognizes and inserts a Trp residue at either UGA or the usual Trp codon, UGG. The second most common variation is conversion of AUA from an Ile codon to a Met codon; the usual Met codon is AUG, and a single tRNA recognizes both codons. The known coding variations in mitochondria are summarized in Table 1.

TABLE 1 Known Variant Codon Assignments in Mitochondria
	Codons^a
	UGA	AUA	AGA AGG	CUN	CGG
Normal (cellular) code assignment	Stop	Ile	Arg	Leu	Arg
Animals
Vertebrates Drosophila	Trp Trp	Met Met	Stop Ser	+ +	+ +
Yeasts
Saccharomyces cerevisiae Torulopsis glabrata Schizosaccharomyces pombe	Trp Trp Trp	Met Met +	+ + +	Thr Thr +	+ ? +
Filamentous fungi	Trp	+	+	+	+
Trypanosomes	Trp	+	+	+	+
Higher plants	+	+	+	+	Trp
Chlamydomonas reinhardtii	?	+	+	+	?
^aN indicates any nucleotide; + indicates that a codon has the same meaning as in the cellular code; ? indicates that a codon was not observed in this mitochondrial genome.

Turning to the much rarer changes in the codes for cellular (as distinct from mitochondrial) genomes, we find that the only known variation in a bacterium is again the use of UGA to encode Trp residues, occurring in the simplest free-living cell, Mycoplasma capricolum. Among eukaryotes, rare extramitochondrial coding changes occur in a few species of ciliated protists, in which both termination codons UAA and UAG can specify glutamine. There are also rare but interesting cases in which stop codons have been adapted to encode amino acids that are not among the standard 20, as detailed in Box 27-2.

Changes in the code need not be absolute; a codon might not always encode the same amino acid. For example, in many bacteria — including E. coli — GUG (Val) is sometimes used as an initiation codon that specifies Met. This occurs only for those genes in which the GUG is properly located relative to particular mRNA sequences that affect the initiation of translation (as discussed in Section 27.2).

The most surprising alteration in the genetic code occurs in some fungal species of the genus Candida, as originally discovered for Candida albicans. C. albicans is an organism of high genomic complexity, yet its genetic code has undergone a dramatic change: the CUG codon, which usually encodes Leu residues, encodes Ser instead. The natural selection pressure for this change is completely unknown. Furthermore, Ser and Leu are quite different in chemical structure. However, even this change can be understood based on the properties of a universal code. When several codons encode the same amino acid and use multiple tRNAs, not all of the codons are used with equal frequency. In a phenomenon called codon bias, some codons for a particular amino acid are used more frequently (sometimes much more frequently) than others. The tRNAs for the frequently used codons are often present at much higher concentrations than the tRNAs required for the rarely used codons. Code degeneracy leads to the presence of six codons for Leu. In bacteria, CUG often encodes Leu. However, in fungi of genera that are very closely related to Candida but do not have the coding change, CUG only rarely encodes Leu and is often entirely absent in highly expressed proteins. A change in the coding sense of CUG would thus have a much smaller effect on fungal cell metabolism than might be expected if all codons were used equally. The coding change may have occurred by a gradual loss of CUG codons in genes and of the tRNA that recognizes CUG as a Leu codon, followed by a capture event — a mutation in the anticodon of a ${tRNA}^{Ser}$ $tRNA Superscript Ser$ that allowed it to recognize CUG. Alternatively, there may have been an intermediate stage in which CUG was recognized as encoding both Leu and Ser, perhaps with contextual signals in the mRNAs that helped one tRNA or another recognize specific CUG codons (see Box 27-2). Phylogenetic analysis indicates that the reassignment of CUG as a Ser codon occurred in Candida ancestors about 150 million to 170 million years ago.

These variations tell us that the code is not quite as universal as once believed, but that its flexibility is severely constrained. The variations are obviously derivatives of the cellular code, and no example of a completely different code has been found. The limited scope of code variants strengthens the principle that all life on this planet evolved on the basis of a single (slightly flexible) genetic code.

Wobble Allows Some tRNAs to Recognize More than One Codon

When several different codons specify one amino acid, the difference between them usually lies at the third base position (at the $3'$ $3 prime$ end). For example, alanine is encoded by the triplets GCU, GCC, GCA, and GCG. The codons for most amino acids can be symbolized by ${XY}_{G}^{A}$ $XY Subscript upper G Superscript upper A$ or ${XY}_{C}^{U} .$ $XY Subscript upper C Superscript upper U Baseline period$ The first two letters of each codon are the primary determinants of specificity, a feature that has some interesting consequences.

Transfer RNAs base-pair with mRNA codons at a three-base sequence on the tRNA called the anticodon. The first base of the codon in mRNA (read in the $5' \to 3'$ $5 prime right-arrow 3 prime$ direction) pairs with the third base of the anticodon (Fig. 27-8a). If the anticodon triplet of a tRNA recognized only one codon triplet through Watson-Crick base pairing at all three positions, cells would have a different tRNA for each amino acid codon. This is not the case, however, because the anticodons in some tRNAs include the nucleotide inosinate (designated I), which contains the uncommon base hypoxanthine (see Fig. 8-5b). Inosinate can form hydrogen bonds with three different nucleotides — A, U, and C (Fig. 27-8b) — although these pairings are much weaker than the hydrogen bonds of Watson-Crick base pairs $G \equiv C$ $upper G identical-to upper C$ and $A ═ U$ $upper A box drawings double horizontal upper U$ . In yeast, one ${tRNA}^{Arg}$ $tRNA Superscript Arg$ has the anticodon $(5') ICG$ $left-parenthesis 5 prime right-parenthesis ICG$ , which recognizes three arginine codons: $(5') CGA, (5') CGU$ $left-parenthesis 5 prime right-parenthesis CGA comma left-parenthesis 5 prime right-parenthesis CGU$ , and $(5') CGC$ $left-parenthesis 5 prime right-parenthesis CGC$ . The first two bases are identical (CG) and form strong Watson-Crick base pairs with the corresponding bases of the anticodon, but the third base (A, U, or C) forms rather weak hydrogen bonds with the I residue at the first position of the anticodon.

A two-part figure shows the pairing relationship of codon and anticodon by showing the alignment of the R N A s in part a and that three different codon pairing relationships are possible when the t R N A anticodon contains inosinate in part b. — FIGURE 27-8 Pairing relationship of codon and anticodon. (a) Alignment of the two RNAs is antiparallel. The tRNA is shown in the traditional cloverleaf configuration. (b) Three different codon pairing relationships are possible when the tRNA anticodon contains inosinate.

Part a shows a horizontal green m R N A strand that runs from its 5 prime end on the left to its 3 prime end on the right. In the center, it has a light green rectangle containing bases A U C. These bases are labeled beneath so A is 1, U is 2, and C is 3 and a bracket enclosing them indicates that they represent a codon. Sets of three short blue horizontal lines representing hydrogen bonds connect each of these bases to bases in the anticodon of t R N A. A bracket encloses the three bases of the anticodon. From left to right, the anticodon contains U A G. U bonds with A below, A bonds with U below, and G bonds with C below. The bases of the anticodon are labeled from right to left so G is 1, A is 2, and U is 3. The anticodon is at the bottom of a dark green partial circle of t R N A. The two sides of the circle come close above the anticodon, then each bends to run up vertically instead of meeting to close the circle. The left-hand side bends slight to the lower left, then back to the right, then runs horizontally left to form a loop before running back horizontally above the lower horizontal strand. This top horizontal strand then bends vertically upward and continues up until it reaches its 3 prime end. The right-hand vertical strand above the anticodon bends horizontally, forms a loop, and then runs back horizontally above the lower strand before bending vertically to run parallel to the left-hand t R N A strand. It reaches it 5 prime end below the 3 prime end of the left-hand strand. Part b shows three different codon pairing relationships arranged horizontally. The top strand of each pair is labeled anticodon and the bottom strand is labeled codon. The left-hand pair has a top half of (3 prime) G bonded to C bonded to bolded I with G numbered 3, C numbered 2, and I numbered 1. These bases are each connected by three short blue horizonal lines representing hydrogen bonds to the bases of the codon below. The bottom half of the left-hand pair, representing the codon, has (5 prime) C bonded to G bonded to bolded A with C labeled 1, G labeled 2, and A labeled 3. The center pair is similar except that the right-hand set of bases includes bolded I end bold in the anticodon and bolded U end bold in the codon. The right-hand pair is similar except that the right-hand set of bases includes bolded I end bold in the anticodon and bolded C end bold in the codon. In the right-hand pair, the anticodon ends at (5 prime) and the codon ends at (3 prime).

Examination of these and other codon-anticodon pairings led Crick to conclude that the third base of most codons pairs rather loosely with the corresponding base of its anticodon; to use his picturesque word, the third base of such codons (and the first base of their corresponding anticodons) “wobbles.” Crick proposed a set of four relationships called the wobble hypothesis:

The first two bases of an mRNA codon always form strong Watson-Crick base pairs with the corresponding bases of the tRNA anticodon and confer most of the coding specificity.
The first base of the anticodon (reading in the $5' \to 3'$ $5 prime right-arrow 3 prime$ direction; this pairs with the third base of the codon) determines the number of codons recognized by the tRNA. When the first base of the anticodon is C or A, base pairing is specific and only one codon is recognized by that tRNA. When the first base is U or G, binding is less specific and two different codons may be read. When inosine (I) is the first (wobble) nucleotide of an anticodon, three different codons can be recognized — the maximum number for any tRNA. These relationships are summarized in Table 27-4.
When an amino acid is specified by several different codons, the codons that differ in either of the first two bases require different tRNAs.

A minimum of 32 tRNAs are required to translate all 61 codons (31 to encode the amino acids, 1 for initiation).

TABLE 27-4 How the Wobble Base of the Anticodon Determines the Number of Codons a tRNA Can Recognize
1. One codon recognized: A top row is labeled anti codon and a bottom row is labeled codon. On the left, the anticodon row contains (3 prime) X bonded to Y bonded to bolded A end bold (5 prime). Beneath, the codon row shows (5 prime) X prime bonded to Y prime bonded to bolded U end bold (3 prime). Three short blue horizontal lines connect X with X prime, Y with Y prime, and A with U. A gray rectangle encloses the right-hand A and U and the region between them. To the right, a similar pair is shown.
2. Two codons recognized: A top row is labeled anti codon and a bottom row is labeled codon. On the left, the anticodon row contains (3 prime) X bonded to Y bonded to bolded U end bold (5 prime). Beneath, the codon row shows (5 prime) X prime bonded to Y prime bonded to bolded A above G end bold (3 prime). A is shown above G to indicate that either base can fill the position. Three short blue horizontal lines connect X with X prime, Y with Y prime, and A with U. A gray rectangle encloses the U above, A above G below, and the region between them. To the right, a similar pair is shown except that the right-hand base of the anticodon is bolded G end bold and the right-hand base of the codon is bolded C above U end bold, indicating that C or U can fill the position.
3. Three codons recognized: A top row is labeled anti codon and a bottom row is labeled codon. The anticodon row contains (3 prime) X bonded to Y bonded to bolded I end bold (5 prime). Beneath, the codon row shows (5 prime) X prime bonded to Y prime bonded to bolded A above U above C end bold (3 prime). A is shown above U and above C to indicate that any of these bases can fill the position. Three short blue horizontal lines connect X with X prime, Y with Y prime, and A with U. A gray rectangle encloses the I above, A above U above C below, and the region between them.
Note: X and Y denote bases complementary to and capable of strong Watson-Crick base pairing with $X'$ $upper X prime$ and $Y'$ $upper Y prime$ , respectively. Wobble bases — in the $3'$ $3 prime$ position of codons and $5'$ $5 prime$ position of anticodons — are shaded.

The wobble (or third) base of the codon contributes to specificity, but because it pairs only loosely with its corresponding base in the anticodon, it permits rapid dissociation of the tRNA from its codon during protein synthesis. If all three bases of a codon engaged in strong Watson-Crick pairing with the three bases of the anticodon, tRNAs would dissociate too slowly and this would limit the rate of protein synthesis. Codon-anticodon interactions balance the requirements for accuracy and speed.

Although only 32 tRNAs are required to translate all codons, most cells have more than that. The bacterium E. coli has 47 different tRNA genes. Many of these are present in multiple copies, such that there are 86 total tRNA genes in the E. coli genome.

The Genetic Code Is Mutation-Resistant

The genetic code plays an interesting role in safeguarding the genomic integrity of every living organism. Evolution did not produce a code in which codon assignments appeared at random. Instead, the code is strikingly resistant to the deleterious effects of the most common kinds of mutations — missense mutations, in which a single new base pair replaces another. In the third, or wobble, position of the codon, single base substitutions produce a change in the encoded amino acid only about 25% of the time. Most such changes are thus silent mutations, in which the nucleotide is different but the encoded amino acid remains the same.

Due to the types of spontaneous DNA damage that affect genomes (see Chapter 8), the most frequent missense mutation is a transition mutation, in which a purine is replaced by a purine, or a pyrimidine by a pyrimidine (for example, $G \equiv C$ $upper G identical-to upper C$ changed to $A ═ T$ $upper A box drawings double horizontal upper T$ ). All three codon positions have evolved so that there is some resistance to transition mutations. A mutation in the first position of the codon will usually produce an amino acid coding change, but the change often results in an amino acid with similar chemical properties. This is especially true for the hydrophobic amino acids that dominate the first column of the code shown in Figure 27-7. Consider the Val codon GUU. A change to AUU would substitute Ile for Val. A change to CUU would replace Val with Leu. The resulting changes in the structure and/or function of the protein encoded by that gene would often (but not always) be small.

Computational studies have shown that alternative genetic codes, delineated at random, are almost always less resistant to mutation than the existing code. The results indicate that the code underwent considerable streamlining before the appearance of LUCA, the ancestral cell.

The genetic code tells us how protein sequence information is stored in nucleic acids and provides some clues about how that information is translated into protein.

Translational Frameshifting Affects How the Code Is Read

Once the reading frame has been set during protein synthesis, codons are translated without overlap or punctuation until the ribosomal complex encounters a termination codon. The other two possible reading frames usually contain no useful genetic information. Overlap between two genes would necessarily constrain the possible amino acid sequences encoded by one or both genes in the overlap region. However, to make maximal use of limited (and expensive) genetic information, a few genes are structured so that ribosomes “hiccup” at a certain point in the translation of their mRNAs, changing the reading frame from that point on. This allows two or more related but distinct proteins to be produced from a single transcript.

One of the best-documented examples of translational frameshifting occurs during translation of the mRNA for the overlapping gag and pol genes of the Rous sarcoma virus, a retrovirus (see Fig. 26-31). The reading frame for pol is offset to the left by one base pair (−1 reading frame) relative to the reading frame for gag (Fig. 27-9).

A figure shows translational frameshifting in a retroviral transcript. — FIGURE 27-9 Translational frameshifting in a retroviral transcript. The *gag-pol* overlap region in Rous sarcoma virus RNA is shown.

The top portion is labeled italicized g a g end italics reading frame. A top horizontal strand begins at 5 prime, then there are three dashes followed by eight gray boxes. These boxes read C U A, G G G, C U C, C G C, U U G, A C A, A A U, and U U A. To the right of the eighth box, there is a light red box containing U A G. To its right, bases are shown outside of boxes. These are G G A G G G C C A followed by three dashes and then 3 prime. Amino acids are shown above the gray boxes following three dashes as follows: L e u, G l y, L e u, A r g, L e u, T h r, A s n, and L e u. The red box containing U A G is beneath the word stop. The bottom portion is labeled blue highlighted italicized p o l end italics reading frame. It begins with three dashes and then with the same sequence as above but not in boxes. From left to right, this is C U A G G G C U C C G C U U G A C A A A U U U. A blue box begins with the next base, A, which is the final base in the gray box labeled L e u above. There are four blue boxes. From left to right, these are A U A, G G G, A G G, and G C C followed by A outside of a box and three dashes to the right. From left to right, the blue boxes are labeled as I l e, G l y, A r g, and A l a followed by three dashes.

The product of the retroviral pol gene (reverse transcriptase) is translated as a larger polyprotein, on the same mRNA that is used for the Gag protein alone (see Fig. 26-30). The polyprotein, or Gag-Pol protein, is then trimmed to the mature reverse transcriptase by proteolytic digestion. Production of the polyprotein requires a translational frameshift in the overlap region to allow the ribosome to bypass the UAG termination codon at the end of the gag gene (shaded light red in Fig. 27-9).

Frameshifts occur during about 5% of translations of this mRNA, and the Gag-Pol polyprotein (and ultimately reverse transcriptase) is synthesized at about one-twentieth the frequency of the Gag protein, a level that suffices for efficient reproduction of the virus. A similar mechanism produces both the τ and γ subunits of E. coli DNA polymerase III from a single dnaX gene transcript (see footnote to Table 25-2).

Some mRNAs Are Edited before Translation

RNA editing can involve the addition, deletion, or alteration of nucleotides in the RNA in a manner that affects the meaning of the transcript when it is translated. Addition or deletion of nucleotides has been most commonly observed in RNAs originating from the mitochondrial and chloroplast genomes. The initial transcripts of the genes that encode cytochrome oxidase subunit II in some protist mitochondria provide an example of editing by insertion. These transcripts do not correspond precisely to the sequence needed at the carboxyl terminus of the protein product. A posttranscriptional editing process inserts four U residues that shift the translational reading frame of the transcript. The insertions require a special class of guide RNAs (gRNAs; Fig. 27-10) that act as templates for the editing process. The added U residues are all located in a small part of the transcript. Note that the base pairing between the initial transcript and the guide RNA includes several $G ═ U$ $upper G box drawings double horizontal upper U$ base pairs (blue dots), which are common in RNA molecules.

FIGURE 27-10 RNA editing of the transcript of the cytochrome oxidase subunit II gene from Trypanosoma brucei mitochondria. (a) Insertion of four U residues (red) produces a revised reading frame. (b) A special class of guide RNAs, complementary to the edited product, acts as templates for the editing process. Note the presence of three $G ═ U$ $upper G box drawings double horizontal upper U$ base pairs, signified by blue dots to indicate non-Watson-Crick pairing.

Part a shows a horizontal D N A coding strand above a horizontal strand labeled edited m R N A. The D N A coding strand begins with 5 prime followed by three dashes, then has a series of six gray boxes labeled as follows: A A A , G T A, G A G, A A C, C T G, and G T A followed by three dashes and then 3 prime. The amino acid sequence is shown below as six amino acids that correspond to the gray boxes. From left to right, these are three dashes followed by L y s, V a l, G l u, A s n, L e u, and V a l followed by three more dashes. There is a horizontal line connecting L y s to V a l. The edited m R N A is similar except that four red Us have been added and this shifts the reading frame. The boxes are as follows: three dashes, a gray box reading A A A and a gray box reading G U A. These are followed by five blue boxes that read as follows: G A red highlighted U end highlighting, red highlighted U end highlighting G red highlighted U end highlighting, A red highlighted U end highlighting A, C C U, and G G U, followed by three dashes. From left to right, the amino acids are as follows: three dashes followed by L y s, V al, A s p, C y s, I l e, P r o, and G l y followed by three dashes. There are lines connecting all of the amino acids. A vertical line extends down from the line between V al and A s p and joins a horizontal arrow pointing to the right. Part b shows a horizontal strand of m R N A above a horizontal strand of guide R N A. The m R N A is begins with 5 prime followed by three dashes, then has A A A G U A G A red highlighted U U end highlighted G red highlighted U end highlighting A red highlighted U end highlighting A C C U G G U, then three dashes to 3 prime. The guide R N A begins with the 3 prime end of a green strand that coils up to meet the bottom horizontal strand paired with the m R N A. It consists of U U A U A U C U A A U A U A U G G A U A U, follows by a green strand to the 5 prime end. There are blue lines joining most of the nucleotides of the top and bottom strands as follows. In the m R N A, the first two A nucleotides are joined with the first two Us below, then A in m R N A is not bonded to A in guide R N A, then G in m R N A has a blue dot between it and U in guide R N A, then there are bonds between U A G A U U in m R N A and A U C G A A in guide R N A, then G in m R N A has a blue dot between it and U in guide R N A, then there are bonds between U A U A C C U in m R N A and A U A U G G A in guide R N A, then there is a blue dot between G in m R N A and U in guide R N A, then the bases to the right are unpaired.

RNA editing by alteration of nucleotides most commonly involves the enzymatic deamination of adenosine or cytidine residues, forming inosine or uridine, respectively (Fig. 27-11), although other base changes have been described. Inosine is interpreted as a G residue during translation. The adenosine deamination reactions are carried out by adenosine deaminases that act on RNA (ADARs). The cytidine deaminations are carried out by the apoB mRNA editing catalytic peptide (APOBEC) family of enzymes, which includes the activation-induced deaminase (AID) enzymes. Both the ADAR and APOBEC groups of deaminase enzymes have a homologous zinc-coordinating catalytic domain.

FIGURE 27-11 Deamination reactions that result in RNA editing. (a) Conversion of adenosine nucleotides to inosine nucleotides is catalyzed by ADAR enzymes. (b) Cytidine-to-uridine conversions are catalyzed by the APOBEC family of enzymes.

Part a shows the conversion of adenosine into inosine. Adenosine is shown as a five-membered sugar ring with O at the top vertex, C 1 prime at the right side vertex bonded to N at the lower left vertex of the five-membered ring of a base above and bonded to H below, C 2 prime bonded to H above and O H below, C 3 prime bonded to H above and to O below further connected by a dashed line below, C 4 prime bonded to C 5 prime above and H below, and C 5 prime bonded to 2 H and to O to the left further connected by a dashed line. The base is a five-membered ring that shares its right side with an adjacent six-membered ring. The five-membered ring has N substituted for C at position 7 at the upper left vertex, a double bond between positions 7 and 8 at the left right side, and N substituted for C at position 9 at the bottom vertex and further bonded to C 1 prime of the sugar below, and a double bond between positions 4 and 5 on the right side shared with the left side of the adjacent six-membered ring. The six-membered ring has N substituted for C at position 1 at the upper right vertex, N substituted for C at position 3 at the bottom vertex, a double bond at the lower right side between positions 2 and 3, a double bond at the upper right side between positions 1 and 6, and C 6 at the top vertex bonded to N H 2. Right-and left-pointing arrows indicate a reversible reaction. The right-pointing arrow is accompanied by a curved arrow showing the addition of H 2 O and loss of N H 3. The left-pointing arrow is accompanied by a curved arrow showing the addition of N H 2 and loss of H 2 O. This yields inosine, which is a similar molecule in which the six-membered ring has N substituted for C at position 1 at the upper right vertex and bonded to H, N substituted for C at position 3 at the bottom vertex, a double bond at the lower right side between positions 2 and 3, and C 6 at the top vertex double bonded to O. Part b shows the conversion of cytidine into uridine. Cytidine is shown as a five-membered sugar ring with O at the top vertex, C 1 prime at the right side vertex bonded to N at the lower left vertex of the six-membered ring of a base above and bonded to H below, C 2 prime bonded to H above and O H below, C 3 prime bonded to H above and to O below connected by a dashed line below, C 4 prime bonded to C 5 prime above and H below, and C 5 prime bonded to 2 H and to O to the left further connected by a dashed line. The base is a six-membered ring that has N substituted for C at position 1 at the bottom vertex bonded to C 1 prime of the sugar below, C 2 at the lower right vertex double bonded to O, N substituted for C at position 3 at the upper right vertex, C 4 at the top vertex bonded to N H 2, a double bond between C 3 and C 4 at the left side, and a double bond between C 5 and C 6 at the upper right side. Right-and left-pointing arrows indicate a reversible reaction. The right-pointing arrow is accompanied by a curved arrow showing the addition of H 2 O and loss of N H 3. The left-pointing arrow is accompanied by a curved arrow showing the addition of N H 2 and loss of H 2 O. This yields uridine, which is a similar molecule except that the C at position 3 at the upper right vertex is bonded to H, there is no double bond between C 3 and C 4 at the upper left side, and C 4 at the top vertex is double bonded to O.

The ADAR-promoted A-to-I editing of RNA transcripts is particularly common in primates. Most of the editing occurs in Alu elements, a subset of short interspersed elements (SINEs), eukaryotic transposons that are particularly common in primate genomes. Human DNA contains more than a million 300 bp Alu elements, making up about 10% of the genome. These elements are concentrated near protein-coding genes, often in introns and untranslated regions at the $3'$ $3 prime$ and $5'$ $5 prime$ ends of transcripts. When it is first synthesized (before processing), the average human mRNA includes 10 to 20 Alu elements. Certain microRNAs are also targeted by ADARs. The microRNA (miRNA) alterations generally reduce expression and/or function.

The ADAR enzymes bind to and promote A-to-I editing only in duplex regions of RNA. The abundant Alu elements offer many opportunities for intramolecular base pairing within the transcripts, providing the duplex targets required by ADARs. Some of the editing affects the coding sequences of genes. Defects in ADAR function have been associated with a variety of human neurological conditions, including amyotrophic lateral sclerosis (ALS), epilepsy, and major depression.

There are six general classes of APOBEC cytidine deaminases: APOBEC1–APOBEC5 and AID. The AID proteins function in increasing antibody diversity during immunoglobulin gene maturation (see Figs. 25-42 and 25-43). APOBEC1 and some of the APOBEC3 proteins (seven APOBEC paralogs are encoded by the human genome) edit mRNAs. A well-studied example of RNA editing by APOBEC1-mediated deamination occurs in the gene for the apolipoprotein B component of low-density lipoprotein in vertebrates. One form of apolipoprotein B, apoB-100 $(M_{r} 513,000)$ $left-parenthesis upper M Subscript r Baseline 513,000 right-parenthesis$ , is synthesized in the liver; a second form, apoB-48 $(M_{r} 250,000)$ $left-parenthesis upper M Subscript r Baseline 250,000 right-parenthesis$ , is synthesized in the intestine. Both are encoded by an mRNA produced from the gene for apoB-100. An APOBEC cytidine deaminase found only in the intestine binds to the mRNA at the codon for amino acid residue 2,153 $(CAA ═ Gln)$ $left-parenthesis CAA box drawings double horizontal Gln right-parenthesis$ and converts the C to a U to create the termination codon UAA. The apoB-48 produced in the intestine from this modified mRNA is simply an abbreviated form (corresponding to the amino-terminal half) of apoB-100 (Fig. 27-12). This reaction permits tissue-specific synthesis of two different proteins from one gene.

FIGURE 27-12 RNA editing of the transcript of the gene for the apoB-100 component of LDL. Deamination, which occurs only in the intestine, converts a specific cytidine to uridine, changing a Gln codon to a stop codon and producing a truncated protein.

A top horizontal strand is labeled human liver (a p o B-100) and a bottom horizontal strand is labeled human intestine (a p o B-48). These strands are made up of boxes that are mostly gray with amino acids listed below and residue numbers listed above. Human liver (a p o B-100): 5 prime followed by three dashes is followed by 7 gray boxes, 1 blue box, and then four more gray boxes before ending with three dashes to 3 prime. From left to right, the boxes are as follows: C A A beneath 2,146, C U G, C A G beneath 2,148, A C A, U A U beneath 2,150, A U G, A U A beneath 2,152, blue C A A, U U U beneath 2,154, G A U, C A G beneath 2,156, and U A U. From left to right, the corresponding amino acids are as follows: G l n, L e u, G l n, T h r, T y r, M e t, I l e, G l n beneath the blue box, P h e, A s p, G l n, and T y r. Human intestine (a p o B-48): three dashes are followed by seven gray boxes before a light red box before four more gray boxes before ending with three dashes. From left to right, the boxes are as follows: C A A beneath 2,146, C U G, C A G beneath 2,148, A C A, U A U beneath 2,150, A U G, A U A beneath 2,152, light red U A A, U U U beneath 2,154, G A U, C A G beneath 2,156, and U A U. From left to right, the corresponding amino acids are as follows: G l n, L e u, G l n, T h r, T y r, M e t, and I l e. The word stop is beneath the red codon, then no amino acids are shown beneath the remaining four gray boxes.

APOBEC2, 4, and 5 act on DNA rather than RNA, and their functions are poorly understood. However, their ability to cause genomic mutations can make them a liability to the cell. One or more APOBEC enzymes are often overexpressed in tumor cells, and their mutagenic ability can contribute to the formation of tumors. They also provide a mechanism for introducing multiple mutations into a targeted segment of a chromosome, leading to selective and more rapid evolution of that DNA region.

SUMMARY 27.1 The Genetic Code

The particular amino acid sequence of a protein is constructed through the translation of information encoded in mRNA. This process is carried out by ribosomes. Amino acids are specified by mRNA codons consisting of nucleotide triplets. Translation requires adaptor molecules, the tRNAs, that recognize codons and insert amino acids into their appropriate sequential positions in the polypeptide.

The base sequences of the codons were deduced from experiments using synthetic mRNAs of known composition and sequence. The codon AUG signals initiation of translation. The triplets UAA, UAG, and UGA are signals for termination. The genetic code is degenerate: it has multiple codons for almost every amino acid. The standard genetic code is universal in all species, with some minor deviations in mitochondria and a few single-celled organisms. The deviations occur in a pattern that reinforces the concept of a universal code.

The third position in each codon is much less specific than the first and second positions and is said to wobble. This property allows certain tRNAs to recognize more than one codon.

The genetic code is resistant to the effects of missense mutations. The code evolved so that many nucleotide changes in a DNA codon do not alter the encoded amino acid, or they result in a very conservative alteration.

Translational frameshifting and RNA editing affect how the genetic code is read during translation.

RNA editing by ADARs (adenosine deaminases) and APOBECs (cytidine deaminases) also alters the coding sequence of some mRNAs. Many APOBEC enzymes target DNA, where they function in facilitating antibody diversity and suppression of retroviruses and retrotransposons.

Amino acid	Number of codons	Amino acid	Number of codons
Met	1	Tyr	2
Trp	1	Ile	3
Asn	2	Ala	4
Asp	2	Gly	4
Cys	2	Pro	4
Gln	2	Thr	4
Glu	2	Val	4
His	2	Arg	6
Lys	2	Leu	6
Phe	2	Ser	6

Amino acid	Number of codons	Amino acid	Number of codons
Met	1	Tyr	2
Trp	1	Ile	3
Asn	2	Ala	4
Asp	2	Gly	4
Cys	2	Pro	4
Gln	2	Thr	4
Glu	2	Val	4
His	2	Arg	6
Lys	2	Leu	6
Phe	2	Ser	6

Amino acid	Number of codons	Amino acid	Number of codons
Met	1	Tyr	2
Trp	1	Ile	3
Asn	2	Ala	4
Asp	2	Gly	4
Cys	2	Pro	4
Gln	2	Thr	4
Glu	2	Val	4
His	2	Arg	6
Lys	2	Leu	6
Phe	2	Ser	6