Translation release factors

Abbreviations aa-tRNA Amino-acyl tRNA eLF Eukaryotic translation initiation factor IF Prokaryotic translation initiation factor eEF Eukaryotic translation elongation factor EF Prokaryotic translation elongation factor eRF Eukaryotic translation termination factor (release factor) RF Prokaryotic translation release factor RRF Ribosome recycling factor Rps Protein of the prokaryotic small ribosomal subunit Rpl Protein of the eukaryotic large ribosomal subunit S Protein of the prokaryotic small ribosomal subunit L Protein of the prokaryotic large ribosomal subunit PTC Peptidyl transferase center RNC Ribosome-nascent chain-mRNA complex ram Ribosomal ambiguity mutation RAC Ribosome-associated complex NMD Nonsense-mediated mRNA decay... 

Figure 10 Alteration of the genetic code for incorporation of non-natural amino acids, (a) In nonsense suppression, the stop codon UAG is decoded by a non-natural tRNA with the anticodon CUA. In vivo decoding of the UAG codon by this tRNA is in competition with termination of protein synthesis by release factor 1 (RFl). Purified in vitro translation systems allow omission of RF1 from the reaction mixture, (b) A new codon-anticodon pair can be created using four-base codons such as GGGU. Crystal structures of these codon-anticodon complexes in the ribosomal decoding center revealed that the C in the third anticodon position interacts with both the third and fourth codon position (purple line) while the extra A in the anticodon loop does not contact the codon.(c) Non-natural base pairs also allow creation of new codon-anticodon pairs. Shown here is the interaction of the base Y with either base X or (hydrogen bonds are indicated by red dashes).

During the normal process of termination of translation, stop codons are recognized by protein release factors (RF). Although the details of the process are not fully understood, it is believed that when a termination codon reaches the ribosomal A-site, the RF associates with the ribosomal-mRNA complex, inducing the peptidyl-transferase center to hydrolyze the ester bond of the pepti-... 

These experiments make it clear that removing competition with release factors leads to more efficient incorporation of the desired amino acid. Unfortunately, the technology to incorporate nonstandard nucleotides into mRNAs coding for full-length proteins is not yet available. Alternatives that have been tested include using (i) a 4-nucleotide codon-anticodon pair, dubbed frame-shift suppression (Sect. 6.1), (ii) a rare codon, and (iii) cell-free extracts from organisms that are either deficient in a release factor (Sect. 5.1) or unable to translate one or more codons (Sect. 6.2). 

In eukaryotes, translation termination is mediated by two essential release factors eRFl (in yeast encoded by SUP45) and eRF3 (in yeast encoded by SUP35), which act as class I and II factors respectively (Frolova et al. 1994 Stansfield et al. 1995b Zhouravleva et al. 1995). eRFl and eRF3 interact both in vitro and in vivo and form a heterodimeric complex (Stansfield et al. 1995b Paushkin et al. 1997 Frolova et al. 1998 Ito et al. 1998 Eurwilaichitr et al. 1999). 

Accurate selection of translation termination factors to ribosomes containing a stop codon in the A-site is less well understood. A picture is only beginning to emerge as the bacterial 708 ribosome and class I release factor RF2 atomic models have recently been fitted into cryo-EM stmctures. Via multiple interactions RF2 connects the ribosomal decoding site with the PTC and functionally mimics a tRNA molecule in the A-site. In the complex RF2 is close to helices 18, 44, and 31 of the 168 rRNA, small subunit ribosomal protein 812, helices 69, 71, 89, and 92 of the 238 rRNA, the L7/L12 stalk, and protein LI 1 of the large subunit (Arkov et al. 2000 Klaholz et al. 2003 Rawat et al. 2003). The L7/L12 stalk inter-... 

Recent studies have shown that proteins interacting with the release factors can modulate the efficiency of stop codon readthrough. Physical and functional interaction with the translation termination factors was demonstrated for different components of the translational machinery. 

Bertram G, Bell HA, Ritchie DW, Fullerton G, Stansfield I (2000) Terminating errkaryote translation domain 1 of release factor eRFl functions in stop codon recognition. RNA 6 1236-1247 Bidou L, Hatin I, Perez N, Allamand V, Panthier JJ, Rousset JP (2004) Premature stop codons involved in muscular dystrophies show a broad spectrum of readthrough efficiencies in response to gentamicin treatment. Gene Ther 11 619-627... 

Hoshino S, Imai M, Kobayashi T, Uchida N, Katada T (1999) The eukaryotic pol>peptide chain releasing factor (eRF3/GSPT) carrying the translation termination signal to the 3 -poly(A) tail of mRNA. J Biol Chem 274 16677-16680... 

Kim SY, Craig EA (2005) Broad sensitivity of Saccharomyces cerevisiae lacking ribosome-associated chaperone Ssb or Zuol to cations, including aminoglycosides. Eukaryot Cell 4 82-89 Kisselev L, Ehrenberg M, Frolova L (2003) Termination of translation interplay of mRNA, rRNAs and release factors . EMBO J 22 175-182... 

Kobayashi T, Funakoshi Y, Hoshino SI, Katada T (2004) The GTP-binding release factor eRF3 as a key mediator coupling translation termination to mRNA decay. J Biol Chem 279 45693 5700 Kong C, Ito K, Walsh MA, Wada M, Liu Y, Kumar S, Barford D, Nakamura Y, Song H (2004) Crystal structure and functional analysis of the eukaryotic class II release factor eRF3 from S. pombe. Mol Cell 14 233-245... 

Orlova M, Yueh A, Leung J, Goff SP (2003) Reverse transcriptase of Moloney murine leukemia vims binds to eukaryotic release factor 1 to modulate suppression of translational termination. Cell 115 319—331 Palmer E, Wilhelm JM, Sherman F (1979) Phenotypic suppression of nonsense mutants in yeast by aminoglycoside antibiotics. Nature 277 148-150... 

Stansfield 1, Jones KM, Kushnirov VV, Dagkesamanskaya AR, Poznyakovski Al, Paushkin SV, Nierras CR, Cox BS, Ter-Avanesyan MD, Tuite ME (1995) The products of the SUP45 (eRFl) and SUP35 genes interact to mediate translation termination in Saccharomyces cerevisiae. EMBO J 14 4365 373 Stansfield 1, Eurwilaichitr L, Akhmaloka, Tuite ME (1996) Depletion in the levels of the release factor eRFl causes a reduction in the efficiency of translation termination in yeast. Mol Microbiol 20 1135-1143 Stansfield 1, Kushnirov VV, Jones KM, Tuite ME (1997) A conditional-lethal translation termination defect in a sup45 mutant of the yeast Saccharomyces cerevisiae. Fur J Biochem 245 557-563 Stark H (2002) Three-dimensional electron cryomicroscopy of ribosomes. Curr Protein Pept Sci 3 79-91... 

Yusupov MM, Yusupova GZ, Baucom A, Lieberman K, Earnest TN, Cate JH, Noller HF (2001) Crystal structure of the ribosome at 5.5A resolution. Science 292 883—896 Zhouravleva G, Frolova L, Le Goff X, Le Guellec R, Inge-Vechtomov S, Kisselev L, Philippe M (1995) Termination of translation in eukaryotes is governed by two interacting polypeptide chain release factors, eRFl and eRF3. EMBO J 14 4065-4072... 

After many such elongation cycles, synthesis of the polypeptide is terminated with the aid of release factors. At least four high-energy phosphate equivalents (from ATP and GTP) are required to generate each peptide bond, an energy investment required to guarantee fidelity of translation. 

Termination of Translation Requires Release Factors and Termination Codons Ribosomes Can Change Reading Frame during Translation... 

The last step in translation involves the cleavage of the ester bond that joins the now complete peptide chain to the tRNA corresponding to its C-terminal amino acid (fig. 29.19). Termination requires a termination codon, mRNA and at least one protein release factor (RF). The freeing of the ribosome from mRNA during this step requires the participation of a protein called ribosome releasing factor (RRF). 

The appearance of a UAA or UAG termination (stop) codon in the A site causes release factor RF1 to bind whereas RF2 recognizes UGA. RF3 assists RF1 and RF2. The release factors trigger peptidyl transferase to transfer the polypeptide to a water molecule instead of to aminoacyl-tRNA. The polypeptide, mRNA, and free tRNA leave the ribosome and the ribosome dissociates into its subunits ready to begin a new round of translation. 

Eventually, one of three termination codons (also called Stop codons) becomes positioned in the A site (Fig. 7). These are UAG, UAA and UGA. Unlike other codons, prokaryotic cells do not contain aminoacyl-tRNAs complementary to Stop codons. Instead, one of two release factors (RF1 and RF2) binds instead. RF1 recognizes UAA and UAG whereas RF2 recognizes UGA. A third release factor, RF3, is also needed to assist RF1 or RF2. Thus either RF1 + RF3 or RF2 + RF3 bind depending on the exact termination codon in the A site. RF1 (or RF2) binds at or near the A site whereas RF3/GTP binds elsewhere on the ribosome. The release factors cause the peptidyl transferase to transfer the polypeptide to a water molecule instead of to aminoacyl-tRNA, effectively cleaving the bond between the polypeptide and tRNA in the P site. The polypeptide, now leaves the ribosome, followed by the mRNA and free tRNA, and the ribosome dissociates into 30S and 50S subunits ready to start translation afresh. 

Three of the 64 codons, UAG, UAA, and UGA, do not specify any amino acid. When a translating ribosome encounters such a stop codon, no amino acid is inserted. Instead, one of two release factors... 

Fig. 4. Role of the stop codon and lOSa-RNA in E. coli translation. (A) When a stop codon is encountered, a complex of two release factors, RF-1 and RF-3 or RF-2 and RF-3, binds instead of the tRNA. The release factor RF-1 recognizes the stop codons UAA and UAG, while RF-2 recognizes UAA and UGA. The binding of the release factor complex results in hydrolysis of the peptidyl-tRNA and release of the peptide. (B) The role of lOSa-RNA. If truncated mRNA without a stop codon is translated in E. coli, the ribosome stops at the end of the mRNA. lOSa-RNA can then bind to the ribosomal A site and lOSa-RNA can act as tRNA by transferring an alanine to the truncated protein. Subsequently, lOSa-RNA acts as mRNA and a peptide tag with the indicated sequence is added to the truncated protein. lOSa-RNA encodes a stop codon and therefore the protein is released and then degraded by proteases specifically recognizing this C-terminal tag.

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