Ribosome release factor

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 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 29.32. Structure of Ribosome Release Factor (RRF). RRF is another protein that resembles tRNA. The a helices of this protein mimic the tRNA structure. In contrast, in EF-G, P strands are the mimics, revealing an independent evolutionary origin.

V. Heurgue-Hamard, R. Karimi, L. Mora, J. MacDougall, C. Leboeuf, G. Grentzmann, M. Ehrenberg, and R.H. Buckingham. 1998. Ribosome release factor RF4 and termination factor RF3 are involved in dissociation of peptidyl-tRNA from the ribosome EMBO J. 17 808-816. (PubMed)... 

Abbreviations IFl, initiation factor 1 IF2, initiation factor 2 IF3, initiation factor 3 EFTu, elongation factor Tu EFG, elongation factor G RFl, release factor 1 RF2, release factor 2 RF3, release factor 3 RR, ribosome release factor. 

RF3)" " with the ribosome. However, the maximum resolution that can currently be obtained by cryo-EM is about 10 nm (8-12 A), far from the desired atomic resolution. Therefore, the crystal structures of the 30S subunit with initiation factors 1 and 3 (IFl IF3 ) and of the 70S subunit with release factors 1 and 2 (RFl/2 " ) as well as RRF have been important milestones toward understanding the interaction of the ribosome with protein factors. 

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).

The principal drawbacks to the technique are its labor-intensive nature and low protein yields, which in the best cases reach only a milligram or less. Competition with release factors at the amber stop codon often results in truncated protein as the primary product [16, 17]. Suppression efficiency rates are also affected by the character of the amino acid, which determines whether it is a good substrate for the ribosome and protein elongation factors. In addition, context effects variously ascribed to the influence of neighboring mRNA... 

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-... 

Termination Three codons (UAA, UAG and UGA) are stop codons which do not code for any amino acid but, instead of attaching to a tRNA molecule, they bind a protein release factor. When one of these factors is encountered by the ribosome, peptidyl transfer is aborted, the completed polypeptide chain released by hydrolysis and the ribosome subunits separate. The N-terminal methionine unit is then removed from the polypeptide chain. 

There are no complementary tRNAs for the stop codons. Instead, two releasing factors bind to the ribosome. One of these factors (RF-1) catalyzes hydrolytic cleavage of the ester bond between the tRNA and the C-ter-minus of the peptide chain, thereby releasing the protein. 

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... 

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-... 

Bulygin KN, Repkova MN, Ven yaminova AG, Grarfer DM, Karpova GG, Frolova LY, Kisselev LL (2002) Positioning of the mRNA stop signal with respect to polypeptide chain release factors and ribosomal proteins in SOS ribosomes. FEBS Lett 514 96-101... 

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... 

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... 

Klaholz BP, Pape T, Zavialov AV, Myasnikov AG, Orlova EV, Vestergaard B, Ehrenberg M, van Heel M (2003) Structure of the Escherichia coli ribosomal termination complex with release factor 2. Nature 421 90-94... 

Klaholz BP, Myasnikov AG, Van Heel M (2004) Visualization of release factor 3 on the ribosome during termination of protein synthesis. Nature 427 862-865... 

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... 

The ribosome can carry two aminoacyl-tRNAs simultaneously. In the chain elongation stage, the growing polypeptide is carried on one of these tRNAs. The chain is transferred to the second tRNA, which adds its amino acid to the growing peptide, and displaces the first tRNA. The ribosome then moves one codon along the mRNA to allow the next to be read. Termination of protein synthesis involves the release of the completed polypeptide, expulsion of the last tRNA, and dissociation of the ribosome from the mRNA. This is signaled by specific termination codons (UAA, UAG, or UGA) in the mRNA and requires the participation of various release factors. 

The last stage of peptide chain synthesis is termination. The genetic code specifies three stop codons, indicating the termination of a coding sequence. When the ribosome encounters one of these stop codons on the mRNA, certain release factors... 

Stags 4 Termination and Release Completion of the polypeptide chain is signaled by a termination codon in the mRNA. The new polypeptide is released from the ribosome, aided by proteins called release factors. 

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