G C

Wild type

Mutant

Resulting progeny

Base Substitution Mutations

If one base is replaced by another, a base substitution mutation has occurred. These may be subdivided into transitions and transversions. In a transition a pyrimidine is replaced by another pyrimidine (i.e., T is replaced by C or vice versa) or a purine is replaced by another purine (i.e., A is replaced by G or vice versa). A transversion occurs when one base is replaced by another of a different type; for example, a pyrim-idine is replaced by a purine or vice versa.

DNA molecules are double stranded. If a mutation occurs and a single base is replaced with another, the DNA molecule will temporarily contain a pair of mismatched bases (Fig. 13.01). When the DNA molecule replicates, complementary bases will be incorporated into the new strands opposite the bases making up the mismatch. The result is one wild-type daughter molecule and one mutant DNA molecule.

When mutations are induced by experimental treatment, it is necessary to allow the cells time to divide after treatment before imposing any selection. This allows the original DNA strands to separate and the cell to make new DNA molecules that are either fully wild-type or fully mutant. This process is sometimes referred to as segregation, as the originally mutated cell segregates the mutation and the wild-type into separate daughter cells upon cell division.

Missense Mutations May Have Major or Minor Effects

When a change in the base sequence alters a codon so that one amino acid in a protein is replaced with a different amino acid, this is called a missense mutation. Overall, this is the most frequent outcome of changing a single base. The severity of a missense mutation depends on the location and the nature of the amino acid that was substituted.

Mutant versions of genes are numbered as described in Ch. 1.Thus genX123 refers to the 123rd mutation isolated in the gene, genX. A mutation which results in a codon for one amino acid being replaced by another may be written genX123 (Arg185Leu), missense mutation Mutation in which a single codon is altered so that one amino acid in a protein is replaced with a different amino acid segregation Replication of a hybrid DNA molecule (whose two strands differ in sequence) to give two separate DNA molecules, each with a different sequence transition Mutation in which a pyrimidine is replaced by another pyrimidine or a purine is replaced by another purine transversion Mutation in which a pyrimidine is replaced by a purine or vice versa

Missense Mutations May Have Major or Minor Effects 337

A) Conservative substitution

B) Radical replacement

DNA mutation GCA -» GGA

Original protein

DNA mutation GCA -» GGA

Amino acid change ALA -» GLY

Amino acid change ALA -» GLY

DNA mutation GCA -» GAA

Amino acid change ALA -» GLU

Mutated protein

Original protein

DNA mutation GCA -» GAA

Amino acid change ALA -» GLU

Mutated

PROTEIN (glutanine has extra negative charge and folds incorrectly)

Mutated

PROTEIN (glutanine has extra negative charge and folds incorrectly)

FIGURE 13.02 Conservative Substitution and Radical Replacement

A) A mutation resulting in DNA change from GCA to GGA will result in the conservative substitution of an alanine for a glycine. Since both amino acids have similar properties, it is likely that the mutant protein will fold similarly to the wild type. B) A mutation resulting in the substitution of a glutamate for an alanine is a radical replacement as the glutamate has an extra negative charge that will probably cause the protein to fold quite differently from the wild type.

Replacing an amino acid with a chemically similar one often has little effect on a protein.

Replacing an amino acid with one that has very different properties often causes significant damage to the protein.

or in one-letter code (R185L). This indicates that arginine at position 185 has been replaced by leucine.

Proteins must assume their correct three-dimensional structure in order to function properly. Moreover, most proteins, especially enzymes, contain an active site whose role is critical. This region contains relatively few of the many amino acids that make up a typical protein. Sequence comparison of the same protein from different organisms usually shows that only the amino acids in a few positions are invariant or nearly so. These highly conserved amino acid residues generally include those in the active site(s) plus others that are critical for correct folding of the protein. Although the protein must fold up correctly, the precise identity of the amino acids at many positions may vary substantially without causing major changes in overall structure. Thus, mutations that alter active site residues will usually have major effects. Mutations that alter residues important for structure will also have a major impact. However, mutations affecting less vital parts of the protein will often have minor effects and substantial activity may remain.

The chemical properties of the original amino acid and the one replacing it in the mutant are also important. Suppose the codon UCU, which codes for serine, is changed to ACU, which codes for threonine. Both serine and threonine are small, hydrophilic amino acids with hydroxyl groups. Replacing one amino acid with another that has similar chemical and physical properties is known as a conservative substitution. Swapping serine for threonine in the less critical regions of a protein will probably not alter its structure radically and the protein may still work, at least partially. In rare instances, the protein may actually work better. On the other hand, if the exchange is made in a critical region of the protein, such as the active site, even a conservative substitution may completely destroy activity. Nonetheless, since the critical regions of most proteins occupy only a small proportion of the total sequence, most conservative substitutions will be relatively mild and usually non-lethal (Fig. 13.02A).

Replacing one amino acid with another that has different chemical and physical properties is known as a radical replacement (Fig. 13.02B). Suppose the codon GUA, which codes for valine, is changed to GAA; this then yields a glutamic acid. This conservative substitution Replacement of an amino acid with another that has similar chemical and physical properties radical replacement Replacement of an amino acid with another that has different chemical and physical properties

Mutant proteins may sometimes be defective only under certain conditions, such as high temperature.

Mutations whose effects vary depending on a variety of environmental conditions are well known.

replaces a bulky hydrophobic residue with a smaller, hydrophilic residue that carries a strong negative charge. Under most circumstances, replacing valine with glutamic acid will seriously cripple or totally incapacitate most proteins. If the residue in question is on the surface of the protein, it is sometimes possible to get away with a radical replacement, provided that the change does not affect a critical binding site or alter the solubility of the protein too drastically.

An interesting and sometimes useful type of missense mutation is the temperature sensitive (ts) mutation. As its name indicates, the mutant protein folds properly at low temperature (the "permissive" temperature) but is unstable at higher temperatures and unfolds. Consequently, the protein is inactive at the higher or "restrictive" temperature. If a protein is essential, a missense mutation will often be lethal to the cell. However, a temperature sensitive mutant can be grown and used for genetic experiments at the lower permissive temperature, where it remains alive. To analyze the damage caused by the mutation, the temperature is then shifted upward to the restrictive temperature at which the protein is inactivated and the organism may eventually die. An example in the fruit fly, Drosophila, is the para(ts) mutation. This affects a protein that forms sodium channels necessary for transmitting nerve impulses. At high temperatures the mutant protein is inactive and the flies are paralyzed. At lower temperatures, they are capable of normal flight (Fig. 13.03).

Naturally occurring temperature sensitive mutations have given rise to the patterns of fur coloration in some animals. Many light colored animals have black tips to their paws, tails, ears and noses. This is due to a temperature-sensitive mutation in the enzyme that synthesizes melanin, the black skin pigment of mammals. In these cases, the mutant enzyme is inactive at normal mammalian body temperature, but active at the lower temperatures found at the extremities. Consequently, melanin is made only in the cooler outlying parts of the body (Fig. 13.04).

Mutations whose effects vary depending on the environment are known as conditional mutations. Cold-sensitive mutations do occur but are much rarer than high or normal temperature-sensitive mutations. Multi-subunit proteins are often held together by hydrophobic patches on the surfaces of the subunits (see Ch. 7). The hydrophobic interaction is weaker at lower temperatures. It is therefore possible to get altered proteins, whose hydrophobic bonding is weaker, that fail to assemble at low temperatures but are normal at higher temperatures. For example, microtubule proteins are temperature dependent. Microtubules are cylinders made from the helical assembly of the monomer tubulin. In Saccharomyces cerevisiae, residues whose mutation caused cold sensitivity were concentrated at the interfaces between adjacent alpha-tubulin subunits. Mutations that respond to the osmotic pressure or ionic strength of the medium are also known.

Nonsense Mutations Cause Premature Polypeptide Chain Termination

Not all codons encode amino acids. Three (UAA, UAG and UGA) are stop codons that signal the end of a polypeptide chain. A nonsense mutation occurs when the codon for an amino acid is mutated to give a stop codon. Suppose that the codon UCG for serine is changed by replacing the middle base, C, with A. This gives the stop codon UAG. When the ribosome translates the mRNA, it comes to the mutant codon that used to be serine. But this is now a stop codon, so the ribosome stops and the rest of the protein does not get made. Release factor recognizes the premature stop codon and releases the partly-made polypeptide. Hence, nonsense mutations are sometimes called chain termination mutations. Usually, the shortened polypeptide chain termination mutation Same as nonsense mutation conditional mutation Mutation whose phenotypic effects depend on environmental conditions such as temperature or pH nonsense mutation Mutation due to changing the codon for an amino acid to a stop codon temperature-sensitive (ts) mutation Mutation whose phenotypic effects depend on temperature

Nonsense Mutations Cause Premature Polypeptide Chain Termination 339

FIGURE 13.03 Temperature Sensitive Mutation

The wild-type gene encodes a protein that folds similarly at high and low temperature. The mutant protein folds normally at low temperature but unfolds at high temperature, and consequently, no longer works properly.

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