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II. The Genetic Control of Protein Structure

II. The Genetic Control of Protein Structure

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and Yanofsky, 1966; Holley, 1965; Korner, 1964; Mahler and Cordes,

1966; Schweet and Heintz, 1966).

The 20 amino acids that are the usual components of protein are coded

by trinucleotide codons. As there are 4 nucleotide components of DNA,

there are 64 (43)possible codons. Three codons, UAG, UAA, and UGA,

are nonsense triplets-do not code for any amino acid -in Escherichiu

coli, the organism in which most of our information concerning codon

assignments has been obtained (Garen, 1968). The remaining 61 possible

triplets all code for amino acids. Since a given amino acid can be specified

by more than one codon, the genetic code is said to be degenerate. Sadgopal ( 1968) has reviewed the codon assignments and the evidence that the

code is universal, i.e., applies to all organisms.

The transcription of the information coded in the DNA sequence of

the gene is mediated by a DNA-dependent RNA polymerase that produces a linear RNA polymer complementary in base sequence to one of

the two DNA strands of the helix (adenosine, uridine, guanosine, and

cytidine are present in the RNA at the positions where thymidine, deoxyadenosine, deoxycytidine, and deoxyguanosine, respectively, are

present in the DNA). Not all genetic sequences are transcribed into

messenger RNA to be translated into protein. The RNA components of

the ribosomes and the transfer RNA molecules are transcribed from the

genetic information but apparently are not translated into protein sequences. They are functional RNA molecules.

The messenger RNA’s bind to the protein-synthesizing organelles, the

ribosomes. Concomitantly, free amino acids are activated and coupled to

transfer RNA molecules by amino acid-activating enzymes that are

specific both for the amino acid and the transfer RNA. A transfer RNA

charged with its activated amino acid binds to a messenger RNA trinucleotide codon with a sequence complementary to the anticodon sequence of the transfer RNA, when the codon is in the proper position

relative to the ribosome. The activated amino acid is then in proper position for the formation of a peptide bond between its amino group and the

carboxyl group of the amino acid at the end of the growing polypeptide

chain. The transfer RNA is then released from the ribosome. As the

ribosome moves down the messenger RNA, each codon sequentially

arrives in the proper position relative to the ribosome to bind the proper

transfer RNA molecule and allow the formation of the peptide bond. A

protein is thus synthesized amino acid by amino acid from its N-terminal

end toward its C-terminal end. When the synthesis of the polypeptide

chain is complete, the ribosome is at the end of the messenger RNA and

drops off. If the proper transfer RNA with its activated amino acid is not



available to match a particular codon during protein synthesis, synthesis

of the polypeptide chain ceases at that point. Thus the deficiency of a

particular amino acid may terminate the synthesis of various polypeptides

at positions where that amino acid is specified by the code.

Most of the evidence supporting this view of protein synthesis has come

from experiments with microorganisms, but cell-free protein-synthesizing

systems can be extracted from both plants and animals. The results from

heterologous cell-free protein-synthesizing systems (ribosomes and

message from one organism and transfer RN A’s from another) confirm

the universality of the code. I t is probable that protein synthesis in all

organisms follows the described course. There is little evidence to the

contrary although it has been suggested that synthesis of the storage proteins of wheat endosperm is effected by another system since a protein

body fraction obtained by relatively low speed centrifugation (50,000 g )

was found to incorporate amino acids (Morton and Raison, 1964). A

comparable fraction from developing maize endosperms incorporated

amino acids only when bacterial contamination was present (Wilson,


Since the amino acid sequence of every protein synthesized by an

organism is specified in its genetic information, conservation of a given

set of amino acid sequences is strongly favored. A change at one position

in the amino acid sequence of a protein can be effected by a point mutation as first shown by Ingram ( 1 957) for sickle cell hemoglobin in man.

Many amino acid substitutions affect the functionality of the protein in

which they occur, causing the mutation responsible for the substitution to

be sublethal or lethal. Unless a substitution (mutation) offers a selective

advantage, it is unlikely to increase in frequency except fortuitously in

genetic isolates.

The addition or deletion or a nucleotide from the DNA codescript that

is the gene shifts the “reading frame” since there are no commas or

spacers separating adjacent codons (Crick et al., 1961). Thus, from the

point of addition or deletion, a whole new set of codons is generated

resulting in a completely different amino acid sequence for the protein

produced by the mutant. Such changes have a high probability of rendering the protein nonfunctional.

A mutation usually affects the amino acid sequence of only one polypeptide chain. It can render the protein partially or completely nonfunctional, thus being lethal if the function of the protein is an essential

one. Even if the mutation is not lethal, its effect on the overall amino acid

composition of the protein coded by the locus is likely to be small, and its

effect on the overall amino acid composition of the bulked proteins of the



organism will be negligible since a given protein usually constitutes only

a small proportion by weight of the total proteins of the organism. There

are exceptions to this generalization, notably the storage proteins of

seeds which are to be discussed.

Changes that might affect the amino acid sequence of' more than one

protein can be envisioned. Assume that a transfer RNA recognizing one

or more of the codons for a particular amino acid (e.g., lysine) were to be

changed by a mutational event so that it recognized a glutamine codon, for

example. Lysine would then be inserted during protein synthesis at some

positions where glutamine was specified. Ritossa et ul. (1966) have shown

that in Drosophila rnelunoguster there is a 13-fold redundance of DNA

coding for transfer RNA. They point out that mutation to give a transfer

RNA with an altered anticodon is unlikely to be an effective suppressor

of a mutation producing an altered codon in a structural cistron because

of the apparent large number of sites coding for transfer RNA. Thus this

type of suppression operative in bacterial systems is unlikely in higher

organisms if the transfer RNA redundancy found in Drosophila is typical.

For the same reason, it is unlikely that an altered transfer RNA could

appreciably affect the amino acid composition of the proteins being


In view of the limited possibilities of affecting overall amino acid composition by a mutation in a structural gene coding for a given protein and

the further restriction that no change in sequence inactivating an essential

protein is admissible, we can understand the mechanisms tending to

conserve the sets of amino acid sequences (proteins) synthesized by an


In addition to the structural genes for proteins, there are also regulatory

genes whose function is to determine when, and to what extent, a particular protein is to be synthesized. In a multicellular organism, each tissue

or organ has its own unique spectrum of proteins appropriate to the

metabolic functions of that organ. Within an organ, not all proteins are

present in equal amounts, nor is the ratio of any two proteins invariate.

It may vary with the stage of development, degree of nutrition, hormonal

influences, etc. The synthesis of some proteins is controlled at the

transcriptional level as was first suggested for the inducible enzyme, pgalactosidase, in Escherichiu coli (Jacob and Monod, 1961). Neither for

microorganisms nor higher organisms do we understand to what extent

the control of protein synthesis lies at the level of transcription and to

what extent at the level of translation (Vogel and Vogel, 1967). The

general question as to the conditions under which specific genetic information is transcribed or translated is of paramount importance in develop-



mental biology and the study of the regulation of metabolic functions.

Instances are known in which the synthesis of a protein has been released by a mutation from the genetic controls ordinarily in force. Examples of this are the constitutive mutants of P-galactosidase and alkaline

phosphatase of E . coli (Jacob and Monod, 1961; Echols et al., 1961).

Conversely, mutations exist that severely restrict the amount of a protein

synthesized without altering the structure of the protein [e.g., the thalassemia mutations affecting hemoglobin production in man (Nance,

1963; Zuckerkandl, 196411.

The genetic control of protein synthesis has been reviewed briefly in

order to provide a background for the discussion that follows. The amino

acid sequence of each protein is genetically determined; viable mutations

result most frequently in the substitution of one amino acid for another at

a specified position in a polypeptide chain. The net effect is negligible in

terms of the overall amino acid composition of the organism. Obviously,

special circumstances are required in order to change drastically the

overall amino acid composition of an organism or a tissue. It will be

shown that such conditions probably exist only in the seeds of higher

plants where large amounts of storage proteins that have no metabolic

function are present.


The Relative Constancy of l e a f Protein Composition

The use of leaf protein concentrates as a supplemental source of protein is being intensively investigated (Stahmann, 1968). The overall

amino acid balance of leaf proteins is generally good although the content

of sulfur-containing amino acids (cysteine and methionine) is lower than


Significantly, the amino acid content of bulked leaf proteins for a

number of species has been found to be relatively constant (Lugg, 1949;

Kelley and Baum, 1953; Stahmann, 1963; Gerloff el al., 1965). This

relative constancy probably reflects the fact that most of the proteins in

leaf tissues are metabolically active, a large proportion of the leaf protein

being associated with the chloroplasts. Nonaqueously isolated chloroplasts from both bean and tobacco leaves contain approximately 70 percent of the total nitrogen of the leaves (Stocking and Ongun, 1962). The

necessity that amino acid substitutions caused by mutations not disrupt

the function of an essential enzyme or otherwise metabolically active

protein is a strongly conservative factor limiting the possible changes in

sequence. A protein with a given function can undergo changes in sequence through geological time owing to mutational events, and these

changes may be fixed in newly evolving species. Nevertheless, proteins



carrying out the same biochemical function in species as unlike as man

and baker’s yeast may still show considerable similarity in amino acid

sequence as shown for cytochrome c (Margoliash and Smith, 1965).

Where the evolutionary relationship is closer, as for man and the macaque

monkey, the cytochrome c’s differ by a single amino acid residue. Much

the same relationship is shown for the hemoglobins since the hemoglobins

of man and the gorilla differ by only a single residue (Buettner-Janusch

and Hill, 1965). The pressures of natural selection obviously tend to

conserve the sequences of functional proteins.

In tissues or organs where the majority of the proteins are functional

as in leaf tissues, there appears to be small probability of significantly

altering the overall amino acid composition.


The Storage Proteins of Seeds

In contrast to leaf tissue, seeds may contain a considerable proportion

of their protein as reserve or storage proteins that serve as a source of

nitrogen for the germinating embryo. Since a plant is capable of synthesizing all the amino acids given a source of nitrogen, there is no necessity

that the storage proteins have any particular amino acid composition.

They can vary considerably from species to species within a family. The

subject of seed proteins has been extensively reviewed (Brohult and Sandegren, 1954; Danielson, 1956; Altschul er al., 1966). In this review,

we will consider principally the cereal proteins that furnish a major portion of the protein consumed in large areas of the world and the legume

proteins because of their present and potential value as protein supplements.

T. B. Osborne initiated systematic studies of seed proteins in 1891 and

continued these studies until the 1920’s (Osborne, 1924). His classification of seed proteins according to their solubility is still used since no

functional criteria can be applied. Seed proteins are classified as albumins

(water soluble), globulins (insoluble in water but soluble in saline solutions), prolamines (soluble in relatively strong alcohol), and glutelins

(soluble in dilute alkaline solutions but not in water, saline, or alcoholic

solutions). Another method of extracting cereal proteins with an alkaline

medium containing copper, sulfate, and sulfite ions has been employed

by Mertz and Bressani (1 957). The extract can be fractionated to yield

acid-, alcohol-, and alkali-soluble fractions. Only in seeds of the Gramineae are large quantities of prolamines and glutelins found. In seeds of

other species, globulins constitute the bulk of the storage proteins

(Osborne, 1924; C. R. Smith et af., 1959).

The protein constituents of cereal grains have been intensively investi-

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II. The Genetic Control of Protein Structure

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