Components Required for Translation

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) for each of the amino acids, functional ribosomes, energy sources, and enzymes as well as noncatalytic protein factors needed for the initiation, elongation, and termination steps of polypeptide chain synthesis.

A. Amino acids

All the amino acids that eventually appear in the finished protein must be present at the time of protein synthesis. If one amino acid is missing, translation stops at the codon specifying that amino acid. [Note: This demonstrates the importance of having all the essential amino acids in sufficient quantities in the diet to ensure continued protein synthesis.]

B. Transfer RNA

At least one specific type of tRNA is required for each amino acid. In humans, there are at least 50 species of tRNA, whereas bacteria contain at least 30 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 3I -end (Figure 31.6). The carboxyl group of the amino acid is in an ester linkage with the 3-hydroxyl of the ribose portion of the adenine (A) nucleotide in the —CCA sequence at the 3I -end of the tRNA. [Note: When a tRNA has a covalently attached amino acid, it is said to be charged, and when it does not, it is said to be uncharged. The amino acid attached to the tRNA molecule is said to be activated.]

Figure 31.6 Complementary, antiparallel binding of the anticodon for methionyl-tRNA (CAU) to the messenger RNA (mRNA) codon for methionine (AUG), the initiation codon for translation.

2. Anticodon: Each tRNA molecule also contains a three-base nucleotide sequence, the anticodon, that pairs with a specific codon on the mRNA (see Figure 31.6). This codon specifies the insertion into the growing peptide chain of the amino acid carried by that tRNA.

C. Aminoacyl-tRNA synthetases

This family of enzymes is required for attachment of amino acids to their corresponding tRNAs. Each member of this family recognizes a specific amino acid and all the tRNAs that correspond to that amino acid (isoaccepting tRNAs, up to five per amino acid). Aminoacyl-tRNA synthetases catalyze a two-step reaction that results in the covalent attachment of the carboxyl group of an amino acid to the 3I -end of its corresponding tRNA. The overall reaction requires adenosine triphosphate (ATP), which is cleaved to adenosine monophosphate (AMP) and inorganic pyrophosphate (PPi) as shown in Figure 31.7. The extreme specificity of the synthetases in recognizing both the amino acid and its cognate tRNA contributes to the high fidelity of translation of the genetic message. In addition to their synthetic activity, the aminoacyl-tRNA synthetases have a “proofreading” or “editing” activity that can remove an incorrect amino acid from the enzyme or the tRNA molecule.

Figure 31.7 Attachment of a specific amino acid to its corresponding tRNA by aminoacyl-tRNA synthetase. PPi = pyrophosphate; Pi = inorganic phosphate; A = adenine; C = cytosine; ATP = adenosine triphosphate; AMP = adenosine monophosphate.

D. Messenger RNA

The specific mRNA required as a template for the synthesis of the desired polypeptide chain must be present. [Note: In eukaryotes, mRNA is circularized for use in translation.]

E. Functionally competent ribosomes

Ribosomes are large complexes of protein and ribosomal RNA ([rRNA], Figure 31.8), in which rRNA predominates. They consist of two subunits (one large and one small) whose relative sizes are 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. For example, the prokaryotic 50S and 30S ribosomal subunits together form a 70S ribosome. 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 macromolecular complexes in which the synthesis of proteins occurs.

The small ribosomal 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 in the tRNA. The large ribosomal subunit catalyzes formation of the peptide bonds that link amino acid residues in a protein.

Figure 31.8 Ribosomal composition. [Note: The number of proteins in the eukaryotic ribosomal subunits varies somewhat from species to species.] S = Svedberg unit.

1. Ribosomal RNA: As discussed, prokaryotic ribosomes contain three size species of rRNA, whereas eukaryotic ribosomes contain four (see Figure 31.8). The rRNAs are generated from a single pre-rRNA by the action of ribonucleases, and some bases and riboses are modified.

2. Ribosomal proteins: Ribosomal proteins are present in greater numbers in eukaryotic ribosomes than in prokaryotic ribosomes. These proteins play a variety of roles in the structure and function of the ribosome and its interactions with other components of the translation system.

3. 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. 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. (See Figure 31.13 for an illustration of the role of the A, P, and E sites in translation.)

Figure 31.13 Steps in prokaryotic protein synthesis (translation), and their inhibition by antibiotics. [Note: EF-Ts is a guanine nucleotide exchange factor. It facilitates the removal of GDP, allowing its replacement by GTP. The eukaryotic equivalent is EF-1βγ.] fMet = formylated methionine; S = Svedberg unit; GTP = guanine nucleoside triphosphate; Phe = phenylalanine.

4. 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 incorporated into plasma, endoplasmic reticulum, or Golgi membranes or imported into lysosomes. Cytosolic ribosomes synthesize proteins required in the cytosol itself or destined for the nucleus, mitochondria or peroxisomes. [Note: Mitochondria contain their own set of ribosomes and their own unique, circular DNA. Most mitochondrial proteins, however, are encoded by nuclear DNA, synthesized in the cytosol, and posttranslationally targeted to mitochondria.]

F. Protein factors

Initiation, elongation, and termination (or release) factors are required for peptide synthesis. Some of these protein factors perform a catalytic function, whereas others appear to stabilize the synthetic machinery. [Note: A number of the factors are monomeric G proteins, and thus are active when bound to guanosine triphosphate (GTP) and inactive when bound to guanosine diphosphate (GDP).]

G. ATP and GTP are required as sources of energy

Cleavage of four high-energy bonds is required for the addition of one amino acid to the growing polypeptide chain: two from ATP in the aminoacyl-tRNA synthetase reaction—one in the removal of PPi, and one in the subsequent hydrolysis of the PPi to inorganic phosphate by pyrophosphatase —and two from GTP—one for binding the aminoacyl-tRNA to the A site and one for the translocation step (see Figure 31.13). [Note: Additional ATP and GTP molecules are required for initiation in eukaryotes, whereas an additional GTP molecule is required for termination in both eukaryotes and prokaryotes.]