The Genetic Code:-The Genetic Code Was Cracked Using Artificial mRNA Templates
By the 1960s it had long been apparent that at least three nucleotide residues of DNA are necessary to en code each amino acid. The four code letters of DNA (A, T, G, and C) in groups of two can yield only 42=16 different combinations, insufficient to encode 20 amino acids. Groups of three, however, yield 43=64 different combinations. Several key properties of the genetic code were established in early genetic studies (Figs 1, 2). A codon is a triplet of nucleotides that codes for a specific amino acid. Translation occurs in such a way that these nucleotide triplets are read in a successive, nonoverlapping fashion. 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.3), 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? In 1961 Marshall Nirenberg and Heinrich Matthaei reported the first breakthrough. They incubated synthetic polyuridylate, poly(U), with an E. 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 the triplet UUU. A radioactive polypeptide was indeed formed in only one of the 20 tubes, the one containing radioactive phenylalanine. Niren berg and Matthaei therefore concluded that the triplet codon UUU encodes phenyl alanine. The same approach revealed that polycytidylate, poly(C), encodes a polypep tide containing only proline (polyproline), and polyadenylate, poly(A), encodes polylysine. Polyguanylate did not generate any polypeptide in this experiment because it spontaneously forms tetraplexes that cannot be bound by ribosomes. The synthetic polynucleotides used in such experiments were prepared with polynucleotide phosphory lase (p. 1020), which catalyzes the formation of RNA polymers starting from ADP, UDP, CDP, and GDP. This enzyme requires no template and makes polymers with a base composition that directly reflects the relative concentrations of the nucleoside 5-diphosphate pre cursors in the medium. If polynucleotide phosphorylase is presented with UDP only, 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 are 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, investigators 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 could not reveal the sequence of the bases. 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. (By convention, the identity of a tRNA is indicated by a superscript, such as tRNAAla, and the aminoacylated tRNA by a hyphenated name: alanyl tRNAAla or Ala-tRNAAla.) For example, ribosomes incubated with poly(U) and phenylalanyl-tRNAPhe (Phe tRNAPhe) 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 about 50 of the 64 possible triplet codons. For some codons, either no amino acyl-tRNA or more than one would bind. Another method was needed to complete and confirm the entire genetic code.
At about this time, a com plementary approach was provided by H. Gobind Khorana, who developed chemical methods to synthesize poly ribonucleotides 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 H. Gobind Khorana (AC)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 the 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. 5). Meanings for all the triplet codons 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. Codons are the key to the translation of genetic in formation, 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. There are a few unusual but interesting exceptions to this rule (Box 27–1). Several codons serve special functions . The initiation codon AUG is the most common signal for the beginning of a polypeptide in all cells (some rare alternatives are discussed in Box 27–2), in addition to coding for Met residues in internal positions of polypep tides. 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 and Khorana to identify codon function should not have worked in the absence of initiation codons. Serendipitously, experi mental conditions caused the normal initiation requirements for protein synthesis 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 codons is referred to as an open reading frame (ORF). Long open reading frames usually correspond to genes that encode proteins. In the analysis of sequence databases, sophisticated programs are used to search for open reading frames in order to find genes among the of tenhuge background of nongenic DNA. An uninterrupted gene coding for a typical protein with a molecular weight of 60,000 would require an open reading frame 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 (Leu, Ser, Arg) have six codons, five amino acids have four, isoleucine has three, and nine amino acids have two (Table 27–3). The genetic code is nearly universal. With the intriguing exception of a few minor variations in mitochondria, some bacteria, and some single-celled eukaryotes (Box 27–2), amino acid codons are identical in all species examined so far. Human beings, E. coli, to bacco plants, amphibians, and viruses share the same genetic code. Thus, it would appear that all life forms have a common evolutionary ancestor, whose genetic code has been preserved throughout biological evolution. Even the variations (Box 27–2) reinforce this theme.
FIGURE 1 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 maxi mum overlap, many nucleotides, such as the third nucleotide from the left (A), are shared by three codons. Note that in an overlapping code, the triplet sequence of the first codon limits the possible sequences for the second codon. 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 genetic code used in all living systems is now known to be nonoverlapping.
FIGURE 2The 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 triplets in a nonoverlapping code; all amino acids coded by the mRNA following the change are affected. 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.
FIGURE 3 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.
FIGURE 4 Effect of a termination codon in a repeating tetranucleotide. Termination codons (pink) 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.
FIGURE 5” Dictionary” of amino acid code words in mRNAs. The codons are written in the 5→3 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 pink, the initiation codon AUG in green. All the amino acids except me thionine and tryptophan have more than one codon. In most cases, codons that specify the same amino acid differ only at the third base.
