Biochemistry - Chemical Reactions in Living Cells, Volume 3 - D. Metzler 1980

Biochemical Genetics and the Synthesis of Nucleic Acids and Proteins
Origins of the Current Concept
The Genetic Code

The assumption regarding the universal nature of METABOLISM/28.html">The Genetic Code stemmed from the very Structure of DNA. Both DNA and Proteins are linear polymers. Consequently, it seemed entirely logical to hypothesize that The base sequence in DNA encodes the Amino Acid Sequence. However, DNA contains only four types of bases, whereas twenty different Amino Acids are found in proteins (at the time of their synthesis). Therefore, each amino acid must be specified by a combination of multiple bases. Sixteen possible Base Pairs are likewise insufficient to encode twenty distinct amino acids. Thus, it is evident that each amino acid must correspond to at least a triplet—that is, a group consisting of three NUCLEOTIDES [16]. There are 64 (4³) such triplet codons (Tables 15-2 and 15-3).

Class="center">Table 15-2 The Genetic Code^a

^a The codons for each amino acid are presented as a base sequence in mRNA, read from 5' to 3' from left to right. The letter X denotes any of the four RNA bases. Thus, each codon containing X actually represents a group of four codons.

¹⁾ These numerical data are not entirely precise. For instance, the chromosome map shown in Fig. 15-1 was constructed assuming a total chromosome length of 4100 kb and a Molecular Weight of 2.7×10⁹ [15].

Table 15-3 The Sixty-Four Codons of the Genetic Code

^a Initiation codons. The methionine codon AUG is the most prevalent initiation codon; however, GUG can also function in this capacity. In such cases, it typically specifies methionine rather than valine.

The simplest hypothesis to envision was that The amino acid sequence in proteins is uniquely determined by sequential, nonoverlapping triplets. However, in the absence of initial data supporting this assumption, other possibilities were actively debated. Nevertheless, genetic experiments conducted over several years (some of which are described in Section D), alongside purely chemical studies discussed in the following section, conclusively proved that the code is nonoverlapping.

Deciphering the Code

Even after the triplet Nature of the genetic code became apparent, numerous questions remained unresolved. Do Cells utilize all 64 possible codons? If so, are all of them employed to encode amino acids, or do certain codons serve other purposes? How many codons specify a single amino acid? Is the code "universal" across all organisms, or does each Organism employ its own code? How can the code be deciphered? Despite The complexity of these questions, definitive Answers were eventually obtained for each of them.

An important experiment was conducted in 1961 by Nirenberg¹⁾ and Matthaei [17].

¹⁾ In 1968, Nirenberg, Khorana, and Holley—who were the first to determine The nucleotide sequence in Transfer RNA—were awarded the Nobel Prize.

Using standard biochemical methodology, Nirenberg isolated Ribosomes from *E. coli* and mixed them with crude soluble extracts derived from the same *E. coli* cells. These extracts contained tRNA molecules and amino acid-activating Enzymes; twenty amino acids, ATP, and an ATP-generating system (phosphoenolpyruvate + Pyruvate kinase) were also added to the system. Nirenberg demonstrated that under these conditions, The addition of RNA stimulated Protein Synthesis on the ribosomes. Notably, tobacco mosaic virus RNA (Chapter 4, Section D, 2) was exceptionally effective in promoting protein synthesis. However, a pivotal role was played by experiments (originally set up merely as "controls") in which synthetic poly(U)—a polynucleotide consisting solely of uridylate residues—was added in place of mRNA. This polynucleotide essentially served as a synthetic mRNA composed of repeating UUU codons. To Nirenberg's surprise, the ribosomes "read" the encoded information and synthesized a peptide consisting exclusively of phenylalanine. Thus, it was established that poly(U) directs the synthesis of polyphenylalanine and that the UUU triplet is the codon specifying phenylalanine. Consequently, the first nucleotide triplet had been identified! Employing this same approach, it was shown that CCC serves as the codon for proline and AAA for lysine. The investigation of mixed copolymers containing two different nucleotides arranged in random sequence provided clues regarding The Significance of other codons. However, the remaining codons were identified only several years later, after Khorana introduced Methods for synthesizing oligonucleotides and polymers with well-defined, regularly repeating nucleotide sequences.

A breakthrough in this methodology arose from the discovery that synthetic trinucleotides can induce the binding of specific tRNA molecules—charged with their cognate amino acids—to ribosomes [18, 19]. For example, the trinucleotides UpUpU and ApApA stimulated the binding of ¹⁴C-labeled phenylalanyl-tRNA and lysyl-tRNA, respectively. Conversely, the corresponding dinucleotides failed to elicit this effect. These findings not only permitted the identification of two codons but also provided direct Evidence for the triplet nature of the genetic code. Another effective approach involved The Use of synthetic RNA polymers generated through a combination of chemical and enzymatic methods [20]. For instance, the polynucleotide CUCUCUCUCU directed the ribosomal synthesis of a polypeptide composed of alternating leucine and serine residues.

Table 15-2 lists the codons now known for each of the 20 amino acids. In Table 15-3, these same 64 codons are arranged differently. Note that In addition to codons specifying particular amino acids, there are three codons—namely, UAA, UAG, and UGA—designated as termination codons. They are also frequently referred to as nonsense codons. The termination codons UAA and UAG are likewise termed ochre and amber codons, respectively¹⁾ [21]. It was discovered that the AUG (methionine) codon and, much less frequently, the GUG (valine) codon act as initiation codons in bacterial protein synthesis. Thus, the N-terminal amino acid in newly synthesized bacterial proteins is most commonly methionine, or more specifically, N-formylmethionine. N-formylmethionine-tRNA specifically binds to initiation sites containing the AUG codon within the mRNA-ribosome complex. This process is described in detail in Section B.

¹⁾ THE ORIGIN OF these terms is explained by the notion that "it is always safer to give a new discovery a meaningless name than a speculatively descriptive one." The term amber was proposed for a class of Mutations discovered with the participation of an undergraduate student named Bernstein, which translates to amber in German [21].

The results of numerous studies indicate that the genetic code established for *E. coli* is universal. For example, in the laboratories of Wittmann and Fraenkel-Conrat, RNA extracted from tobacco mosaic virus was treated with nitrous acid; this Treatment is known to deaminate many cytosine residues, converting them to uracil residues and thereby transforming the UCU (serine) codons into UUU (phenylalanine). Similarly, the CCC (proline) codon can be converted to CUC (leucine). It was demonstrated that when tobacco plants are infected with nitrous acid-treated viral RNA, the amino acid sequence of the coat protein isolated from mutant strains indeed changes [22]. Furthermore, many of these observed alterations could be accurately predicted using the data presented in Table 15-3. Analogously, Amino Acid Substitutions in defective Hemoglobin molecules (Fig. 4-17) can, in most cases, be attributed to a single base change. For instance, hemoglobin S may arise from either of the following alterations in the seventh codon: GAA(Glu)→GUA(Val) or GAG(Glu)→GUG(Val). A further argument supporting the universality of the genetic code is the capacity of ribosomes and tRNA molecules from *E. coli* to translate an mRNA chain encoding hemoglobin and thereby synthesize functional hemoglobin [23].