Genetic information, stored in the chromosomes and transmitted to daughter cells through DNA replication, is expressed through transcription to RNA and, in the case of mRNA, subsequent translation into proteins (Figure 9.1). The pathway of protein synthesis is called translation because the “language” of the nucleotide sequence on the mRNA is translated into the language of an amino acid sequence. The process of translation requires a genetic code, through which the information contained in the nucleic acid sequence is expressed to produce a specific sequence of amino acids. Any alteration in the nucleic acid sequence may result in an improper amino acid being inserted into the protein chain, potentially causing disease or even death of the organism. Many proteins are covalently modified following their synthesis to activate them or alter their activities.
FIGURE 9.1. Protein synthesis or translation.
THE GENETIC CODE
The genetic code is a dictionary that identifies the correspondence between a sequence of nucleotide bases and a sequence of amino acids. Each individual word in the code is composed of three nucleotide bases. These genetic words are called codons.
Codons are presented in the messenger RNA (mRNA) language of adenine (A), guanine (G), cytosine (C), and uracil (U). Their nucleotide sequences are always written from the 5? end to the 3? end. The four nucleotide bases are used to produce the three-base codons. There are, therefore, 64 different combinations of bases, taken three at a time as shown in Figure 9.2.
FIGURE 9.2. Use of genetic code table to translate the codon AUG
1. How to translate a codon: This table (or “dictionary”) can be used to translate any codon sequence and, thus, to determine which amino acids are coded for by an mRNA sequence. For example, the codon 5?-AUG-3? codes for methionine (see Figure 9.2). Sixty-one of the 64 codons code for the 20 common amino acids.
2. Termination (“stop” or “nonsense”) codons: Three of the codons, UAG, UGA, and UAA, do not code for amino acids but rather are termination codons. When one of these codons appears in an mRNA sequence, it signals that the synthesis of the protein coded for by that mRNA is complete.
Characteristics of the genetic code
Usage of the genetic code is remarkably consistent throughout all living organisms. Characteristics of the genetic code include the following:
- Specificity: The genetic code is specific (unambiguous), that is, a particular codon always codes for the same amino acid.
- Universality: The genetic code is virtually universal, that is, the specificity of the genetic code has been conserved from very early stages of evolution, with only slight differences in the manner in which that code is translated. (Note: An exception occurs in mitochondria, in which a few codons have meanings different than those shown in Figure 9.2, e.g., UGA codes for trp.)
- Degeneracy: The genetic code is degenerate (sometimes called redundant). Although each codon corresponds to a single amino acid, a given amino acid may have more than one triplet coding for it. For example, arginine is specified by six different codons (see Figure 9.2).
- Nonoverlapping and commaless: The genetic code is nonoverlapping and commaless, that is, the code is read from a fixed starting point as a continuous sequence of bases, taken three at a time. For example, ABCDEFGHIJKL is read as ABC/DEF/GHI/JKL without any “punctuation” between the codons.
Consequences of altering the nucleotide sequence
Changing a single nucleotide base on the mRNA chain (a “point mutation”) can lead to any one of three results (Figure 9.3):
FIGURE 9.3. Possible effects of changing a single nucleotide base in the coding region of the mRNA chain
- Silent mutation: The codon containing the changed base may code for the same amino acid. For example, if the serine codon UCA is given a different third base “U” to become UCU, it still codes for serine. This is termed a “silent” mutation.
- Missense mutation: The codon containing the changed base may code for a different amino acid. For example, if the serine codon UCA is given a different first base “C” to become CCA, it will code for a different amino acid, in this case, proline. The substitution of an incorrect amino acid is called a “missense” mutation.
- Nonsense mutation: The codon containing the changed base may become a termination codon. For example, if the serine codon UCA is given a different second base “A” to become UAA, the new codon causes termination of translation at that point and the production of a shortened (truncated) protein. The creation of a termination codon at an inappropriate place is called a “nonsense” mutation.
- Other mutations: These can alter the amount or structure of the protein produced by translation.
a. Trinucleotide repeat expansion: Occasionally, a sequence of three bases that is repeated in tandem will become amplified in number so that too many copies of the triplet occur. If this occurs within the coding region of a gene, the protein will contain many extra copies of one amino acid. For example, amplification of the CAG codon leads to the insertion of many extra glutamine residues in the Huntington protein, causing the neurodegenerative disorder, Huntington disease (Figure 9.4). The additional glutamines result in unstable proteins that cause the accumulation of protein aggregates. If the trinucleotide repeat expansion occurs in the untranslated portion of a gene, the result can be a decrease in the amount of protein produced as seen, for example, in fragile X syndrome and myotonic dystrophy.
FIGURE 9.4. Role of tandem triplet repeats in mRNA causing Huntington disease and other triplet expansion diseases
b. Splice site mutations: Mutations at splice sites can alter the way in which introns are removed from the pre-mRNA molecules, producing aberrant proteins.
c. Frame-shift mutations: If one or two nucleotides are either deleted from or added to the coding region of a message sequence, a frame-shift mutation occurs and the reading frame is altered. This can result in a product with a radically different amino acid sequence (Figure 9.5) or a truncated product due to the creation of a termination codon. If three nucleotides are added, a new amino acid is added to the peptide, or if three nucleotides are deleted, an amino acid is lost. In these instances, the reading frame is not affected. Loss of three nucleotides maintains the reading frame but can result in serious pathology. For example, cystic fibrosis (CF), a hereditary disease that primarily affects the pulmonary and digestive systems, is most commonly caused by a deletion of three nucleotides from the coding region of a gene, resulting in the loss of phenylalanine at the 508th position (?F508) in the protein encoded by that gene. This ?F508 mutation prevents normal folding of the CF transmembrane conductance regulator (CFTR) protein, leading to its destruction by the proteosome (see Chapter 12). CFTR normally functions as a chloride channel in epithelial cells, and its loss results in the production of thick, sticky secretions in the lungs and pancreas, leading to lung damage and digestive deficiencies. In over seventy percent of patients with CF, the ?F508 mutation is the cause of the disease.
FIGURE 9.5. Frame-shift mutations as a result of addition or deletion of a base can cause an alteration in the reading frame of mRNA.
COMPONENTS REQUIRED FOR TRANSLATION
A large number of components are required for the synthesis of a protein. These include all the amino acids that are found in the finished product, the mRNA to be translated, transfer RNA (tRNA), functional ribosomes, energy sources, and enzymes, as well as protein factors needed for initiation, elongation, and termination of the polypeptide chain.
All the amino acids that eventually appear in the finished protein must be present at the time of protein synthesis. (Note: If one amino acid is missing [e.g., if the diet does not contain an essential amino acid], translation stops at the codon specifying that amino acid. This demonstrates the importance of having all the essential amino acids in sufficient quantities in the diet to ensure continued protein synthesis.)
tRNAs are able to carry a specific amino acid and to recognize the codon for that amino acid. tRNA, therefore, functions as adaptor molecules.
At least one specific type of tRNA is required per amino acid. In humans, there are at least 50 species of tRNA, whereas bacteria contain 30 to 40 species. Because there are only 20 different amino acids commonly carried by tRNA, some amino acids have more than one specific tRNA molecule. This is particularly true of those amino acids that are coded for by several codons.
1. Amino acid attachment site: Each tRNA molecule has an attachment site for a specific (cognate) amino acid at its 3? end (Figure 9.6). The carboxyl group of the amino acid is in an ester linkage with the 3?-hydroxyl group of the ribose moiety of the adenosine nucleotide in the –CCA sequence at the 3? end of the tRNA. (Note: When a tRNA has a covalently attached amino acid, it is said to be charged; when tRNA is not bound to an amino acid, it is described as being uncharged.) The amino acid that is attached to the tRNA molecule is said to be activated.
FIGURE 9.6. Complementary antiparallel binding of the anticodon for methionyl-tRNA (CAU) to the mRNA codon for methionine (AUG)
2. Anticodon: Each tRNA molecule also contains a three-base nucleotide sequence—the anticodon—that recognizes a specific codon on the mRNA (see Figure 9.6). This codon specifies the insertion into the growing peptide chain of the amino acid carried by that tRNA.
This family of enzymes is required for the attachment of amino acids to their corresponding tRNA. Each member of this family recognizes a specific amino acid and the tRNA that corresponds to that amino acid (isoaccepting tRNA). These enzymes thus implement the genetic code because they act as molecular dictionaries that can read both the three-letter code of nucleic acids and the 20-letter code of amino acids. Each aminoacyl-tRNA synthetase catalyzes a two-step reaction that results in the covalent attachment of the carboxyl group of an amino acid to the 3? end of its corresponding tRNA.
The overall reaction requires adenosine triphosphate (ATP), which is cleaved to adenosine monophosphate (AMP) and inorganic pyrophosphate (PPi) (Figure 9.7). The extreme specificity of the synthetase in recognizing both the amino acid and its specific tRNA contributes to the high fidelity of translation of the genetic message. In addition, the synthetases have a “proofreading” or “editing” activity that can remove mischarged amino acids from the enzyme or the tRNA molecule.
FIGURE 9.7.Attachment of a specific amino acid to its corresponding tRNA by aminoacyl-tRNA synthetase (E)
The specific mRNA required as a template for the synthesis of the desired polypeptide chain must be present.
Functionally competent ribosomes
Ribosomes are large complexes of protein and ribosomal RNA (rRNA, Figure 9.8).They consist of two subunits—one large and one small—whose relative sizes are generally given in terms of their sedimentation coefficients, or S (Svedberg) values. (Note: Because the S values are determined both by shape as well as molecular mass, their numeric values are not strictly additive. The eukaryotic 60S and 40S subunits form an 80S ribosome.) Prokaryotic and eukaryotic ribosomes are similar in structure and serve the same function, namely, as the “factories” in which the synthesis of proteins occurs.
FIGURE 9.8.Eukaryotic ribosomal composition
The larger ribosomal subunit catalyzes the formation of the peptide bonds that link amino acid residues in a protein. The smaller subunit binds mRNA and is responsible for the accuracy of translation by ensuring correct base-pairing between the codon in the mRNA and the anticodon of the tRNA.
- Ribosomal RNA: Eukaryotic ribosomes contain four molecules of rRNA (see Figure 9.8). The rRNAs have extensive regions of secondary structure arising from the base-pairing of the complementary sequences of nucleotides in different portions of the molecule.
- Ribosomal proteins: Ribosomal proteins play a number of roles in the structure and function of the ribosome and its interactions with other components of the translation system.
- A, P, and E sites on the ribosome: The ribosome has three binding sites for tRNA molecules—the A, P, and E sites—each of which extends over both subunits (see Figure 9.8). Together, they cover three neighboring codons. During translation, the A site binds an incoming aminoacyl-tRNA as directed by the codon currently occupying this site. This codon specifies the next amino acid to be added to the growing peptide chain. The P-site codon is occupied by peptidyl-tRNA. This tRNA carries the chain of amino acids that has already been synthesized. The E site is occupied by the empty tRNA as it is about to exit the ribosome.
- Cellular location of ribosomes: In eukaryotic cells, the ribosomes are either “free” in the cytosol or are in close association with the endoplasmic reticulum (which is then known as the “rough” endoplasmic reticulum, or RER). The RER-associated ribosomes are responsible for synthesizing proteins that are to be exported from the cell as well as those that are destined to become integrated into plasma, endoplasmic reticulum, or Golgi membranes or incorporated in lysosomes. Cytosolic ribosomes synthesize proteins required in the cytosol itself or destined for the nucleus, mitochondria, and peroxisomes. (Note: Mitochondria contain their own set of ribosomes and their own unique, circular DNA.)