BIOCHEMISTRY - L. Stryer - 1984

VOLUME 3

Part IV INFORMATION

CHAPTER 26. THE GENETIC CODE AND THE RELATIONSHIP BETWEEN GENES AND PROTEINS

26.2. Amino Acids Are Coded by Triplets of Bases Starting from a Fixed Point

1. What is the coding ratio? Since DNA contains four types of bases, a singlet code (one base per amino acid) could specify only four Amino Acids. A doublet code (two bases per amino acid) would specify 16 amino acids (4 • 4 = 16), whereas a triplet code (three bases per amino acid) yields 64 amino acids (4 • 4 • 4 = 64). Proteins are built from a standard set of 20 amino acids. This simple calculation makes it obvious that at least three bases are required to specify a single amino acid. Genetic experiments subsequently proved that Amino acids are indeed encoded by groups of three bases. Such a group of bases is called a codon.

2. Is the code overlapping? In a non-overlapping triplet code, each group of three bases specifies

only one amino acid, whereas in a fully overlapping triplet code, bases ABC would specify the first amino acid, BCD the second, CDE the third, and so on.

This dilemma was resolved by determining the Amino acid sequences of mutants. Suppose that base B mutates to B'. In a non-overlapping code, only a single amino acid will change. In a fully overlapping code, however, a mutation of B to B' would alter amino acids 1, 2, and 3. An Analysis of the coat protein sequence in tobacco mosaic virus mutants revealed that such Mutations typically altered only a single amino acid. Furthermore, as discussed earlier in relation to abnormal Hemoglobins in Chapter 5, the majority of mutants also exhibited A change in just one amino acid. These findings led to the Conclusion that METABOLISM/28.html">The Genetic Code is non-overlapping:

Class="center">

3. How are triplets read correctly? A priori, one possibility is that one of the four bases (designated as Q) acts as a "punctuation mark" separating the triplets:

. . . QABCQDEFQGHIQJKLQ. . .

It turned out this is not the case. The base sequence is read sequentially, starting from a strictly defined point:

There are no commas or punctuation marks in the code. Suppose that a mutation results in the deletion of base G:

The first Two amino acids in the polypeptide chain remain normal, but the downstream base sequence is misread because the deletion of G causes a frameshift. Now suppose that base X is inserted between E and G:

This insertion mutation also disrupts the reading frame starting from the codon for amino acid 3. In fact, genetic studies of insertion and deletion mutants have elucidated many Fundamental properties of the genetic code.

4. As noted above, there are 64 possible base triplets and 20 amino acids. Does each of the 20 amino acids correspond to only a single triplet, or are Some amino acids encoded by more than one triplet? Genetic studies showed that most of the 64 triplets encode amino acids. Subsequent biochemical investigations established that 61 of the 64 triplets specify particular amino acids. Thus, Most amino acids are specified by more than one codon; in other words, the genetic code is degenerate.

26.3. Deciphering the Genetic Code: Synthetic RNAs Can Serve as Messenger RNAs

What is the relationship between the 64 codons and the 20 amino acids? In principle, this question could be answered directly by comparing The amino acid sequence of a protein with the base sequence of its Gene or mRNA. In 1961, however, this approach was entirely out of reach because the base sequences of genes and mRNA molecules were completely unknown. It seemed at the time that Structure/149.html">The problem of the genetic code might not be solved for the foreseeable future, but the situation suddenly changed. Marshall Nirenberg discovered that The addition of polyuridylate [poly(U)] to a Cell-free protein-synthesizing system led to the synthesis of polyphenylalanine. Evidently, poly(U) acted as a Messenger RNA. The first code word was thus deciphered: UUU codes for phenylalanine. This remarkable experiment paved the way for the complete cracking of the genetic code.

Let us examine this epoch-making experiment in greater detail. The two main components used were a cell-free system capable of active Protein Synthesis AND a synthetic polyribonucleotide that functioned as an mRNA. The Cell-free protein-synthesizing system was prepared from E. coli as follows. Bacterial Cells were gently disrupted by grinding with a finely powdered alumina abrasive to obtain a cell juice. Cell wall debris and cell membranes were then removed by centrifugation, yielding an extract containing DNA, mRNA, tRNA, Ribosomes, Enzymes, and other cellular constituents. Upon the addition of ATP, GTP, and amino acids, this cell-free system synthesized protein. At least one of the added amino acids was radioactive, making it possible to detect its incorporation into protein. The mixture was incubated at 37°C for approximately 1 hour. Trichloroacetic acid was then added to stop the reaction and precipitate the proteins, leaving free amino acids in the supernatant fraction. The precipitate was washed, and its radioactivity was measured to determine The amount of labeled amino acid incorporated into the newly synthesized protein. A crucial feature of this system is that protein synthesis can be halted by adding deoxyribonuclease, which degrades the templates required for new mRNA synthesis. Because the mRNA already present in the mixture at the time of deoxyribonuclease addition is labile, protein synthesis ceases within a few minutes. Nirenberg then discovered that protein synthesis could be restarted by adding an unpurified mRNA fraction. Thus, Nirenberg had in his hands a cell-free protein-synthesizing system whose activity depended on added mRNA.

The other vital component in this experiment was the synthetic polyribonucleotide poly(U). Poly(U) was synthesized using polynucleotide phosphorylase, an enzyme discovered in 1955 by Marianne Grunberg-Manago and Severo Ochoa. This enzyme catalyzes the synthesis of polyribonucleotides from ribonucleoside diphosphates:

(РНК)_n + Рибонуклеозиддифосфат ⇄ (РНК)_n+1 + Рi

Polynucleotide phosphorylase and RNA polymerase catalyze entirely different reactions. In the reaction shown above, the activated precursors are ribonucleoside diphosphates rather than triphosphates. The reaction product is orthophosphate, not pyrophosphate. Consequently, the reaction equilibrium cannot be driven to the right by Pyrophosphate Hydrolysis. In fact, in vivo the equilibrium strongly favors RNA degradation rather than its synthesis. A fundamental difference is that polynucleotide phosphorylase does not use a template. The COMPOSITION OF THE RNA synthesized by this enzyme is determined by the relative concentrations of ribonucleotides in the incubation mixture, and its sequence is close to random. Because of this, polynucleotide phosphorylase proved to be an invaluable tool in experiments aimed at deciphering the genetic code. For instance, poly(U) was synthesized by incubating a high-concentration solution of UDP in the presence of the enzyme. Copolymers of two ribonucleotides, such as U and A, with random sequences were prepared similarly by incubating UDP and ATP with this enzyme.

Fig. 26.3. Protein synthesis in a cell-free system halts a few minutes after the addition of deoxyribonuclease and resumes upon the addition of mRNA

Various synthetic ribonucleotides were introduced into a cell-free protein-synthesizing system, and the incorporation of ¹⁴C-labeled L-phenylalanine was measured. The results were striking:

The same experiment was performed using various ¹⁴C-labeled amino acids in each incubation mixture. It turned out that poly(A) directs the synthesis of polylysine, and poly(C) directs the synthesis of polyproline. In this way, three codons were deciphered:

The GGG codon could not be deciphered in this manner because poly(G) fails to function as a template. This is likely due to its ability to form a triple-stranded helical structure. Polyribonucleotides that form extended ordered structures are ineffective as templates for protein synthesis.