Releasing factors eukaryotic

Competition with release factors can be reduced by deactivating a release factor in a cell-free lysate prepared from a prokaryote. This option is not available in rabbit reticulyte lysate since mammals have only two release factors (RFs), termed eukaryotic release factors 1 and 3 (eRFl and eRF3). In eukaryotes, eRFl recognizes all three stop codons and eRF3 stimulates eRFl activity in the presence of GTP [41]. Deactivation of either RF would effectively deactivate all three stop codons, and at least one termination codon must function properly for the suppression method to be useful. 

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... 

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). 

Frolova L, Le Goff X, Rasmussen HH, Cheperegin S, Drugeon G, Kress M, Arman 1, Haenni AL, Cells JE, Philippe M (1994) A highly conserved eukaryotic protein family possessing properties of polypeptide chain release factor. Nature 372 701-703... 

Frolova L, Le Goff X, Zhouravleva G, Davydova E, Philippe M, Kisselev L (1996) Eukaryotic polypeptide chain release factor eRF3 is an eRFl- and ribosome-dependent guanosine triphosphatase. RNA 2 334-341... 

Frolova LY, Simonsen JL, Merkulova Tl, Litvinov DY, Martensen PM, Rechinsky VO, Camonis JH, Kisselev LL, Justesen J (1998) Functional expression of eukaryotic polypeptide chain release factors 1 and 3 by means of baculovirus/insect cells and complex formation between the factors. Eur J Biochem 256 36 4... 

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... 

Inge-Vechtomov S, Zhouravleva G, Philippe M (2003) Eukaryotic release factors (eREs) history. Biol Cell 95 195-209... 

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... 

Song H, Mugnier P, Das AK, Webb HM, Evans DR, Tuite ME, Hemmings BA, Barford D (2000) The crystal structure of human eukaryotic release factor eRFl—mechanism of stop codon recognition and peptidyl-tRNA hydrolysis. Cell 100 311-321... 

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... 

It is not essential to life for E. coli, but it accelerates the release of RF1 or RF2 and is needed for rapid growth.417 418 Eukaryotes contain one release factor eRFl, which recognizes all three termination codons, and a second release factor eRF3, which binds and hydrolyzes GTP.413/413b/418a 419... 

A single eukaryotic release factor recognizes all three termination codons and requires ATP for activity. 

Termination in eukaryotes is carried out by a single eukaryotic release factor... 

The coding region ends with the protein sequence—that is, there is no stop codon present. In the prokaryotic system the presence of a stop codon would result in the binding of the release factors (Grentzmann et al, 1995 Tuite and Stansfield, 1994) and the ribosome recycling factor (Janosi et al., 1994) to the mRNA-ribosome-protein complexes. This would then lead to the release of the protein by hydrolysis of the peptidyl-tRNA (Tate and Brown, 1992), thereby dissociating the ribosomal complexes (Fig. 4A). A similar mechanism exists in eukaryotic systems (Frolova et al., 1994 Zhouravleva et al., 1995). 

There is a single release factor in eukaryotes, and a GTP-GDP is involved. It is not absolutely universal, being slightly different in human mitochondria. 

The elongation reaction described earlier for the prokaryotic system is essentially the same as that found in the eukaryotic system. The final difference between the two systems lies in the mechanism of translational termination. Recall that the prokaryotic system utilizes two different release factor proteins. The eukaryotic system relies on a single release factor protein, eRF. 

The structure resembles that of a tRNA by molecular mimicry. The sequence Gly-Gly-Gln, present in both eukaryotes and prokaryotes, occurs at the end of the structure corresponding to the acceptor stem of a tRNA. This region binds a water molecule. Disguised as an aminoacyl-tRNA, the release factor may carry this water molecule into the peptidyl transferase center and, assisted by the catalytic apparatus of the ribosome, promote this water molecule s attack on the ester linkage, freeing the polypeptide chain. The detached polypeptide leaves the ribosome. Transfer RNA and messenger RNA remain briefly attached to the 70S ribosome until the entire complex is dissociated in a GTP-dependent fashion by ribosome release factor (RRF) and EF-G. Ribosome release factor is an essential factor for prokaryotic translation. 

Figure 29.31. Structure of a Release Factor. The structure of a eukaryotic release factor reveals atRNA-like fold. The acceptor-stem mimic includes the sequence Gly-Gly-Gln at its tip. This region appears to bind a water molecule, which may be brought into the peptidyl transferase center. There it can participate in the cleavage of the peptidyl-tRNA ester bond, with the aid of the glutamine residue and the ribosomal catalytic apparatus. 

Elongation and termination. Eukaryotic elongation factors EFla and EFip y are the counterparts of prokaryotic EF-Tu and EF-Ts. The GTP form of EFla delivers aminoacyl-tRNA to the A site of the ribosome, and EFip y catalyzes the exchange of GTP for bound GDP. Eukaryotic EF2 mediates GTP-driven translocation in much the same way as does prokaryotic EF-G. Termination in eukaryotes is carried out by a single release factor, eRFl, compared with two in prokaryotes. Finally, eIF3, like its prokaryotic counterpart IF3, prevents the reassociation of ribosomal subunits in the absence of an initiation complex. 

Figure 2 In ribosome display, mRNA (A) extracted from a cell is converted into a cDNA library (B) is transcribed back into mRNA with no stop codons. Prokaryotic or eukaryotic proteosomes are added and the ribosome then travels down the mRNA (C) translating until it reaches the end of the mRNA molecule (D), where the ribosome halts. With no stop codon, the release factor proteins cannot bind and so the protein, ribosome, and mRNA are physically associated and can be stabilized by high Mg2+ and low temperatures. This complex could then be bound directly to an immobilized natural product (E), the nonbinding library members washed away and the bound members eluted with EDTA (F), which destabilizes the ribosomal complexes by removing Mg2+. The purified sublibrary is converted into cDNA by reverse transcription (RT-PCR) and amplified by regular PCR (B). The/n vitro transcription and translation can be repeated for another round of selection or the cDNA can be analyzed by agarose electrophoresis and/or sequencing.

TERMINATION In eukaryotic cells two releasing factors, eRF-1 and eRF-3 (a GTP-binding protein), mediate the termination process. When GTP binds to eRF-3, its GTPase activity is activated. eRF-1 and eRF-3-GTP form a complex that bind in the A site when UAG, UGA, or UAA enter. Then GTP hydrolysis promotes the dissociation of the releasing factors from the ribosome. This step is soon followed by the release of mRNA and the separation of the functional ribosome into its subunits. As described, the release of the newly synthesized polypeptide is catalyzed by peptidyl transferase. 

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