Ribosome release factor interaction

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).

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-... 

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... 

Peptidyl transferase is also involved in termination. When the final, or terminal, amino acid has been added to the peptidic sequence, that enzyme releases the neonate peptide from the 60s ribosome. Mg2+, GTP, and other release factors (RF s) are necessary for complete release. Also, at this stage, the new peptide has both at least one terminal and carboxyl function, which may interact with foreign molecules. 

The final phase of translation is termination. How does the synthesis of a polypeptide chain come to an end when a stop codon is encountered Aminoacyl-tRNA does not normally bind to the A site of a ribosome if the codon is UAA, UGA, or UAG, because normal cells do not contain tRNAs with anticodons complementary to these stop signals. Instead, these stop codons are recognized by release factors (RFs), which are proteins. One of these release factors, RFl, recognizes UAA or UAG. A second factor, RF2, recognizes UAA or UGA. A third factor, RF3, another G protein homologous to EF-Tu, mediates interactions between RFl or RF2 and the ribosome. 

In prokaryotes, two release factors have been identified, one (RFl) recognizing UAA and UAG, the other (RF2) functioning with UGA. Ribosomal binding and release of RFl and RF2 are stimulated by a third factor, RF3, which interacts with GTP and GDP. In eukaryotic cells such as reticulocytes, one release factor (eRF) has been found to function with all three termination codons, and the binding of this factor to ribosomes is stimulated by GTP but not GDP. Although the details are not entirely clear, GTP hydrolysis appears to be required for the release of the finished polypeptide chain by cleavage of the peptidyl-tRNA bond and completion of the termination process leading to dissociation of the release factor from the ribosome. 

Figure 7.5 Model of ferritin (and erythroid a-aminolaevulinate synthase) translation/ribosome binding regulation by IRP. In (a), with IRP not bound to the IRE (1) binding of the 43S preinitiation complex (consisting of the small ribosomal 40S subunit, GTP and Met-tRNAMet) to the mRNA is assisted by initiation factors associated with this complex, as well as additional eukaryotic initiation factors (elFs) that interact with the mRNA to facilitate 43S association. Subsequently (2), the 43S preinitiation complex moves along the 5 -UTR towards the AUG initiator codon, (3) GTP is hydrolysed, initiation factors are released and assembly of the 80S ribosome occurs. Protein synthesis from the open reading frame (ORF) can now proceed. In (b) With IRP bound to the IRE, access of the 43S preinitiation complex to the mRNA is sterically blocked. From Gray and Hentze, 1994, by permission of Oxford University Press.

In the protein elongation process the EF-Tu and EF-G cycles themseves interact with the mRNA-pro-gramed ribosome cyclically and since (i) EF-TU has to be released from the ribosome before EF-G can bind to it and vice versa and (ii) GTP hydrolysis is required for the release of both factors, there is a stoichiometric relationship between the number of GTPs hydrolysed and the number of amino acids incorporated into the protein synthesized. This contrasts with the other GTPases, e.g. heterotrimeric G-proteins, Ras proteins, which continue to transmit a signal as long as they remain in the E - GTP form, a key feature of the signal amplification process. 

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