BIOCHEMISTRY - L. Stryer - 1984

VOLUME 3

Part IV INFORMATION

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

26.4. The Composition of Codons for Many Amino Acids Was Determined Using Copolymers as Templates

To further investigate METABOLISM/28.html">The Genetic Code, polyribonucleotides consisting of Two Types of bases were used as templates. For example, a random copolymer of U and G contains eight different triplets: UUU, UUG, UGU, GUU, UGG, GUG, GGU, and GGG. The relative frequency of these triplets can be readily calculated from the molar ratio of U and G in the copolymer, which was 0.76:0.24 (Table 26.1). The relative incorporation of various Amino Acids in the presence of this template is given in Table 26.2. As might be expected, phenylalanine was incorporated to the greatest extent, since the UUU triplet occurred most frequently. It was followed by valine, leucine, and Cysteine. Their incorporation level was slightly over one-third of that of phenylalanine, which corresponds to the calculated frequency of occurrence of triplets containing two U's and one G. The Incorporation of Other amino acids was very low. From this, it was concluded that valine, leucine, and cysteine are encoded by codons containing 2U and 1G, whereas Tryptophan and Glycine are encoded by codons containing 1U and 2G.

Class="center">Table 26.1. Expected triplet frequencies in a random copolymer of U (0.76) and G (0.24)

Table 26.2. Amino acid incorporation in the presence of a random copolymer of U (0.76) and G (0.24)

Experiments of the same type were carried out with other random copolymers, namely UA, UC, AC, and AG, as well as UGC, AGC, UAC, and UAG. In this way, the codon composition corresponding to each of the 20 amino acids was determined in the laboratories of Nirenberg and Ochoa.

26.5. Trinucleotides Promote the Binding of Specific tRNA Molecules to Ribosomes

While The Use of mixed copolymers as templates made it possible to establish the composition of codons corresponding to specific amino acids, it did not reveal their sequence (except for UUU, AAA, and CCC). As already mentioned, valine is encoded by a triplet composed of 2U and 1G. Which specific triplet is it: UUG, UGU, or GUU? This question was answered using two entirely different experimental approaches: first, by employing synthetic polyribonucleotides with a defined sequence, and second, through codon-dependent specific binding of tRNA molecules to Ribosomes.

In 1964, Nirenberg discovered that trinucleotides promote the binding of specific tRNA molecules to ribosomes in the absence of Protein Synthesis. For example, The addition of pUpUpU triggers the binding of phenylalanine tRNA, while pApApA significantly enhances the binding of Lysine tRNA, just as pCpCpC does for Proline tRNA. Dinucleotides do not stimulate tRNA binding to ribosomes. These studies demonstrated that a trinucleotide (like a triplet in mRNA) specifically binds to a particular tRNA molecule for which it serves as the codeword. A simple and rapid binding assay was developed: ribosome-bound tRNA molecules are retained on a nitrocellulose filter, whereas unbound tRNA molecules pass through. To determine precisely which tRNA molecules adhere to the filter, tRNAs carrying a specific amino acid labeled with C were used.

Using organic chemistry and biochemical techniques, all 64 trinucleotides were synthesized. The binding of tRNA molecules corresponding to all 20 amino acids was tested for each trinucleotide. For example, it was shown that pUpUpG stimulates the binding of leucine tRNA only, pUpGpU of cysteine tRNA only, and pGpUpU exclusively promotes the binding of valine tRNA. From this, it was concluded that the UUG, UGU, and GUU codons correspond to leucine, cysteine, and valine, respectively. For a few codons, no selective binding of any tRNA was observed, whereas several others bound more than one tRNA. For the majority of codons, completely clear-cut results were obtained.

Overall, this simple and elegant approach made it possible to decipher about 50 codons.

26.6. Another Tool for Deciphering the Code: Copolymers with a Defined Sequence

Around the same time, Gobind Khorana succeeded in synthesizing polyribonucleotides with a defined repeating sequence. Combining organic chemical and enzymatic Methods, he synthesized a series of copolymers with repeating sequences of two, three, and four bases. Let us consider, for example, the synthesis strategy for poly(GUA). This ordered copolymer has the sequence

GUAGUAGUAGUAGUAGUAGUAGUA...

First, Khorana used organic synthesis methods to prepare two complementary deoxyribonucleotides, each nine NUCLEOTIDES long: d(TAC)₃ and d(GTA)₃. These two oligonucleotides were then used as templates for the synthesis of long DNA chains from four deoxynucleoside triphosphates under the action of DNA polymerase I. Neither oligonucleotide alone served as an efficient template. However, when both oligonucleotides were present, d(TAC)₃ served as the template for the synthesis of poly(dGTA), while d(GTA)₃ served as the template for poly(dTAC). These long complementary DNAs formed double-helical molecules. The next step was to produce long polyribonucleotide chains with sequences corresponding to poly(dTAC) and poly(dGTA). To achieve this, the poly(dTAC):poly(dGTA) duplex was used as a template for RNA polymerase. The DNA strand to be transcribed can be selected by adding three appropriate ribonucleoside triphosphates. If GTP, UTP, and ATP are added to the incubation mixture, the polyribonucleotide product poly(GUA) is synthesized on the poly(dTAC) template strand. The other strand is not transcribed because one of the necessary substrates, CTP, is missing. Conversely, if CTP, UTP, and ATP are added, poly(UAC) is synthesized on the opposite template strand. Thus, organic synthesis followed by template-directed synthesis using DNA polymerase and RNA polymerase made it possible to generate two long polyribonucleotides with a strictly defined repeating base sequence (Fig. 26.4).

Fig. 26.4. The strand of this double-helical template DNA to be transcribed is determined by the set of ribonucleoside triphosphates in the incubation mixture

These regular copolymers were used as templates in a Cell-free protein-synthesizing system. Let us examine some of the results of such an experiment. A copolymer consisting of an alternating sequence of two bases, A and B:

contains two types of codons—ABA and BAB. Therefore, the polypeptide product must represent an alternating sequence of Two amino acids (abbreviated as aa₁ and aa₂):

Whether ak1 or ak₂ is present at the N-terminus of the polypeptide product depends on whether the reading frame starts with A or B. When poly(UG) was used as a template, a polypeptide consisting of alternating valine (Val) and cysteine (Cvs) residues was synthesized:

This result provided unambiguous proof for the triplet Nature of the code and demonstrated that one of the triplets—UGU or GUG—codes for cysteine, and the other for valine. Combined with tRNA binding data, it was evident that UGU codes for cysteine and GUG for valine. In the presence of certain alternating copolymers containing two bases, the Synthesis of the following Polypeptides occurred:

Let us now consider a template consisting of a repeating sequence of three bases, poly(ABC). If the reading frame starts with A, the resulting polypeptide should contain only a single type of amino acid encoded by the triplet ABC:

If the reading frame starts with B, the synthesized polypeptide should contain a different amino acid encoded by the triplet BCA:

Finally, if the reading frame starts with C, a third type of polypeptide should be formed, containing The amino acid encoded by the triplet CAB:

Thus, the expected products are three different homopolypeptides. Indeed, exactly this result was obtained with most templates composed of repeating trinucleotide sequences. For example, poly(UUC) directed the synthesis of polyphenylalanine, polyserine, and polyleucine. This result, combined with data from other experiments, showed that UUC codes for phenylalanine, UCU for Serine, and CUU for leucine. Polypeptides synthesized on other templates of this type are listed in Table 26.3. Note that in the presence of poly(GUA) and poly(GAU), two rather than three homopolypeptides were synthesized. The reason for this phenomenon will become clear shortly.

Table 26.3. Homopolypeptides synthesized on templates consisting of repeating trinucleotide sequences

Khorana also synthesized a series of copolymers consisting of repeating tetranucleotides, such as poly(UAUC). This template directed the synthesis of a polypeptide with the repeating sequence Tyr-Leu-Ser-Ile regardless of the reading

frame:

From this, Conclusions could be drawn regarding the meaning of four codons.

A completely different result was obtained when poly(GUAA) was used as a template. The only products were di- and tripeptides. Why were longer chains absent? This is explained by the fact that one of the triplets occurring in this copolymer, namely UAA, does not code for an amino acid, but rather signals the termination of protein synthesis:

The poly(AUAG) template also yielded only di- and tripeptides, since UAG is a second chain termination signal:

Let us look back at Table 26.3. On the poly(GUA) template, two rather than three homopolypeptides were synthesized for the reason that the third reading frame corresponds to the sequence

i.e., it represents a repeating sequence of the termination signal. What about poly(GAU)? Only two homopolypeptides were synthesized on this template because the third reading frame corresponds to yet another termination signal—UGA:

The laboratory synthesis of polynucleotides with a defined sequence was a remarkable achievement. The use of these polymers as templates for protein synthesis, combined with Nirenberg's work on tRNA binding to ribosomes in the presence of trinucleotides, led to the complete deciphering of the genetic code by 1966. Just six years earlier, such a milestone seemed like an impossible dream. Thanks to The Development of advanced synthetic methods in Khorana's laboratory, yet another breakthrough became possible: the total synthesis of a DNA molecule corresponding to The sequence of a Transfer RNA molecule.