10.3.2.2—
Elongation of the Polypeptide Chain
The elongation process starts once the 80s plant ribosome containing mRNA and methionyl-tRNAi , has been assembled and takes place by the following repeated cycle of events, each cycle being separated into:
(a) codon-directed binding of aminoacyl-tRNA;
(b) peptide bond formation; and
(c) translocation.
The ribosome has two binding sites; a 'P' site for the binding of methionyl-tRNAi and an 'A' site where all other aminoacyl-tRNAs bind. After the initiation complex has been formed, the next aminoacyl-tRNA, i.e. the one carrying the anticodon to the next codon of the mRNA, binds at the 'A' site. Once the aminoacyl-tRNA has been bound in the 'A' site, a peptide bond is formed by a peptidyl-transferase enzyme, which is part of the large subunit, such that the 'A' site now carries the aminoacyl-tRNA joined to methionine by a peptide bond. The 'P' site now carries the deacylated tRNAiMet , which is subsequently removed leaving an open 'P' site, and the peptidyl-tRNA moves from the 'A' to the 'P' site, so moving the message relative to the ribosome. The 'A' site is now empty and is ready for the next aminoacyl-tRNA to enter according to the next codon in the message. This cycle of reactions requires K+ , Mg2+ , GTP and various elongation factors, proteins, which are found in the soluble fraction of cell homogenates.
Our knowledge of polypeptide chain elongation is most complete with E. coli, where three elongation factors are known, EF-Tu, EF-Ts and EF-G (Fig. 10.7). Factor EF-Tu binds stepwise with GTP and aminoacyl-tRNA and this ternary complex transfers the aminoacyl-tRNA to the 'A' site of the ribosome, releasing an EF-Tu•GDP complex and inorganic phosphate. Factor EF-Ts displaces GDP to form the complex EF-Ts•EF-Tu, from which GTP displaces EF-Ts to regenerate EF-Tu•GTP. The third elongation factor, EF-G,
and free GTP, are responsible for the translocation of the peptidyl tRNA from the A' to the 'P' site with the prior removal of the deacylated tRNA from the 'P' site; GTP is hydrolyzed during the process. The elongation factors, like the initiation factors, recycle during protein synthesis (Fig, 10.7). The protein chain grows from the N-terminal end, and probably starts to fold into its three-dimensional conformation whilst the process of elongation is proceeding.
Figure 10.7
Polypeptide chain elongation in prokaryotes. Ribosomes cover more
than two codons on mRNA. Reaction I ® II is catalysed by peptidyl transferase.
(
(Modified from Haselkorn & Rothman-Denes (1973).)
Fewer details of the elongation factors are available in eukaryotic systems. However, two elongation factors, designated as the binding enzyme EF-1 and the translocase EF-2 (Hardesty et al., 1963; Gasior & Moldave, 1965) have been purified from ungerminated wheat embryos (Golinska & Legocki 1973; Twardowski & Legocki, 1973; Legocki, 1973; Allende et al., 1973). The mechanism of binding of aminoacyl-tRNA to wheat ribosomes is on the whole similar to
that in E. coli, the main difference being the absence in the eukaryotic system of a factor with the properties of EF-Ts, i.e. EF-1 = EF-Tu and EF-2 = EF-G.
Elongation factor EF-I, which requires the presence of Mg2+ at low concentration, K+ and GTP, is responsible for the binding of aminoacyl-tRNA to wheat ribosomes. It contains three active forms differing in molecular weight. The larger EF-1 forms dissociate to a single species of molecular weight 6 × 104 daltons during gel electrophoresis in the presence of SDS. The three forms observed may represent a monomer, trimer and tetramer of a single protein unit, of which the trimer seems to be the most stable form. Some experimental evidence supports the view that the light species is the active form of the EF-1 enzyme. For example, in calf brain and wheat embryo, it has been shown that the interaction of EF-1 with GTP and aminoacyl-tRNA yields a ternary complex which contains the light form of the enzyme, i.e. molecular weight 5 × 104 to 6 × 104 daltons (Moon et al., 1972; Legocki, 1973). It is suggested that the amino group of the esterified amino acid plays an important role in aminoacyl-tRNA binding (Jerez et al., 1969) and this differs from the bacterial system where EF-Tu can react with the deaminated product of phenylalanyl-tRNA, phenyl-lactyl-tRNA (Fahnestock et al., 1972).
The second elongation factor, EF-2, is involved in translocation of the peptidyl-tRNA from the 'A' site to the 'P' site during elongation. It is a protein of molecular weight 7 × 104 daltons and requires thiol compounds for activity (Twardowski & Legocki, 1973).
Ribosomes and elongation factors from the cytoplasm of eukaryotic organisms are not functionally interchangeable with their E. coli counterparts (Krisko et al., 1969). Furthermore, Perani et al., (1971) have shown absolute ribosome specificity, either for 70s or 80s ribosomes, of two sets of elongation factors, T and G (EF-1 and EF-2), isolated from yeast, and for factor EF-2 isolated from the alga, Prototheca zopfii. It is generally accepted that elongation factors within each ribosomal type (70s or 80s) are exchangeable between eukaryotes (Ciferri, 1972).