October 16, 2012

Termination codons are recognized by protein factors

  • A release factor (RF) is required to terminate protein synthesis to cause release of the completed polypeptide chain and the ribosome from mRNA. Individual factors are numbered. Eukaryotic factors are called eRF.
  • RF1 is the bacterial release factor that recognizes UAA and UAG as signals to terminate protein synthesis.
  • RF2 is the bacterial release factor that recognizes UAA and UGA as signals to terminate protein synthesis.
  • RF3 is a protein synthesis termination factor related to the elongation factor EF-G. It functions to release the factors RF1 or RF2 from the ribosome when they act to terminate protein synthesis.
  • Termination codons are recognized by protein release factors, not by aminoacyl-tRNAs.
  • The structures of the class 1 release factors resemble aminoacyl-tRNA·EF-Tu and EF-G.
  • The class 1 release factors respond to specific termination codons and hydrolyze the polypeptide-tRNA linkage.
  • The class 1 release factors are assisted by class 2 release factors that depend on GTP.
  • The mechanism is similar in bacteria (which have two types of class 1 release factors) and eukaryotes (which have only one class 1 release factor). 

Two stages are involved in ending translation. The termination reaction itself involves release of the protein chain from the last tRNA. The post-termination reaction involves release of the tRNA and mRNA, and dissociation of the ribosome into its subunits.
None of the termination codons is represented by a tRNA. They function in an entirely different manner from other codons, and are recognized directly by protein factors. (Since the reaction does not depend on codon-anticodon recognition, there seems to be no particular reason why it should require a triplet sequence. Presumably this reflects the evolution of the genetic code.)
Termination codons are recognized by class 1 release factors (RF). In E. coli two class 1 release factors are specific for different sequences (Scolnick et al., 1968). RF1 recognizes UAA and UAG; RF2 recognizes UGA and UAA. The factors act at the ribosomal A site and require polypeptidyl-tRNA in the P site. The release factors are present at much lower levels than initiation or elongation factors; there are ~600 molecules of each per cell, equivalent to 1 RF per 10 ribosomes. Probably at one time there was only a single release factor, recognizing all termination codons, and later it evolved into two factors with specificities for particular codons. In eukaryotes, there is only a single class 1 release factor, called eRF. The efficiency with which the bacterial factors recognize their target codons is influenced by the bases on the 3 side.
The class 1 release factors are assisted by class 2 release factors, which are not codon-specific. The class 2 factors are GTP-binding proteins. In E. coli, the role of the class 2 factor is to release the class 1 factor from the ribosome.
Although the general mechanism of termination is similar in prokaryotes and eukaryotes, the interactions between the class 1 and class 2 factors have some differences.
The class 1 factors RF1 and RF2 recognize the termination codons and activate the ribosome to hydrolyze the peptidyl tRNA. Cleavage of polypeptide from tRNA takes place by a reaction analogous to the usual peptidyl transfer, except that the acceptor is H2O instead of aminoacyl-tRNA (see Figure 6.34).
Then RF1 or RF2 is released from the ribosome by the class 2 factor RF3, which is related to EF-G (Milman et al., 1969; Mikuni et al., 1994). RF3-GDP binds to the ribosome before the termination reaction occurs. The GDP is replaced by GTP. This enables RF3 to contact the ribosome GTPase center, where it causes RF1/2 to be released when the polypeptide chain is terminated (Klaholz, Myasnikov, and Van Heel, 2004).

RF3 resembles the GTP-binding domains of EF-Tu and EF-G, and RF1/2 resemble the C-terminal domain of EF-G, which mimics tRNA. This suggests that the release factors utilize the same site that is used by the elongation factors. Figure 6.32 illustrates the basic idea that these factors all have the same general shape and bind to the ribosome successively at the same site (basically the A site or a region extensively overlapping with it) (Ito et al., 1996; for review see Nissen, Kjeldgaard, and Nyborg, 2000). 

The eukaryotic class 1 release factor, eRF1, is a single protein that recognizes all three termination codons (Frolova et al., 1994). Its sequence is unrelated to the bacterial factors. It can terminate protein synthesis in vitro without the class 3 factor, eRF3, although eRF3 is essential in yeast in vivo. The structure of eRF1 follows a familiar theme: Figure 6.33 shows that it consists of three domains that mimic the structure of tRNA (Song et al., 2000).

An essential motif of three amino acids, GGQ, is exposed at the top of domain 2. Its position in the A site corresponds to the usual location of an amino acid on an aminoacyl-tRNA. This positions it to use the glutamine (Q) to position a water molecule to substitute for the amino acid of aminoacyl-tRNA in the peptidyl transfer reaction. Figure 6.34 compares the termination reaction with the usual peptide transfer reaction. Termination transfers a hydroxyl group from the water, thus effectively hydrolyzing the peptide-tRNA bond (and see Figure 6.48 for discussion of how the peptidyl transferase center works).
Mutations in the RF genes reduce the efficiency of termination, as seen by an increased ability to continue protein synthesis past the termination codon. Overexpression of RF1 or RF2 increases the efficiency of termination at the codons on which it acts. This suggests that codon recognition by RF1 or RF2 competes with aminoacyl-tRNAs that erroneously recognize the termination codons (for review see Eggertsson and Soll, 1988). The release factors recognize their target sequences very efficiently (Freistroffer et al., 2000).

The termination reaction involves release of the completed polypeptide, but leaves a deacylated tRNA and the mRNA still associated with the ribosome. Figure 6.35 shows that the dissociation of the remaining components (tRNA, mRNA, 30S and 50S subunits) requires the factor RRF, ribosome recycling factor. This acts together with EF-G in a reaction that uses hydrolysis of GTP. Like the other factors involved in release, RRF has a structure that mimics tRNA, except that it lacks an equivalent for the 3 amino acid-binding region (Selmer et al., 1999). IF-3 is also required, which brings the wheel full circle to its original discovery, when it was proposed to be a dissociation factor! RRF acts on the 50S subunit, and IF-3 acts to remove deacylated tRNA from the 30S subunit. Once the subunits have separated, IF-3 remains necessary, of course, to prevent their reassociation.