Basics II – Translation Extension over BSc tRNA binding sites in ribosomes E-, P-, and A-site in the structural models shown with tRNA bound to it. Alberts, Molecular Biology of the Cell 5/e © 2008 Wiley-VCH, Fig 6.64 Large subunit Compare to previous slide of the 50S sub- unit of the ribosome. Here, proteins are in blue, RNA is in orange, and the active site in red. Note that the proteins are looking like glued to the surface of the rRNA figure from: dujs.dartmouth.edu/winter- 2010/nobel-prize-in-chemistry-2009-unraveling- the-ribosome%E2%80%99s-secrets Elongation I youtube.com/watch?v=TfYf_rPWUdY youtube.com/watch?v=kmrUzDYAmEI Initiation will be covered in much detail in the following not included here Alberts, Molecular Biology of the Cell 5/e © 2008 Wiley-VCH, Fig 6.66 Elongation I Version 2. Semester (: Die Polypeptidketten-Verlängerung (Elongation) ist ein zyklischer Vorgang mit drei klar voneinander unterscheidbaren Schritten: Schritt 1 – Bindung einer neuen tRNA in Position A. Ein Aminoacyl-tRNA-Molekül wird an die leere A-Stelle neben einer besetzten P-Stelle an das Ribosom gebunden. Dort bildet es Basenpaare mit den drei mRNA-Nukleotiden, die an der A-Stelle exponiert sind. Schritt 2 – Bildung der Peptidbindung. Das Carboxy-Ende der Polypeptidkette von wird von dem an der P-Bindungsstelle liegenden tRNA-Molekül getrennt und über eine Peptidbindung an die Aminosäure gebunden, die an das tRNA-Molekül in der A-Stelle gebunden ist. Katalysiert wird diese Reaktion von der Peptidyltransferase. Diese enzymatische Aktivität wird durch einen Abschnitt des großen rRNA-Moleküls in der großen Ribosomen-Untereinheit vermittelt. Schritt 3 – Translokation und Freisetzung der tRNA aus Position B. Schließlich wird die neue Peptidyl-tRNA von der A-Stelle in die P-Stelle verschoben. Dabei bewegt sich das Ribosom um genau drei Nukleotide auf der mRNA weiter. Dieser Schritt erfordert Energie und wird durch eine Folge von Konformationsänderungen in Gang gesetzt, die durch Hydrolyse eines GTP-Moleküls getrieben werden. Während des im dritten Schritt ablaufenden Translokationsvorgangs löst sich das im zweiten Schritt an der P-Stelle gebildete freie tRNA-Molekül vom Ribosom und kehrt in den cytoplasmatischen tRNA-Vorrat zurück. Daher ist am Ende des dritten Schritts die A-Stelle wieder frei und kann eine neue tRNA auf-nehmen, an welche die nächste Aminosäure gebunden ist. Damit beginnt der ganze Vorgang von neuem. Elongation II – Peptidyltransferase peptidyl transferase is a ribozyme an adenin-base of ribosomal RNA catalyzes peptide bond formation the catalytic site is located on the large subunit relevance for our view on molecular evolution protein synthesis is still catalyzed by RNA – there are no ribosomal proteins near the actual catalytic site. under lab conditions elongation is possible without protein components of the ribosome there are still ribosomal proteins whose function is unknown Bacterial peptidyltransferase - target of several types of antibiotics Chloramphenicol (outdated; used for lab purposes) Pleuromutilins (topical; in part experimental) Macrolides (alternative to penicillin) Elongation III ‘chemical‘ representation of peptide bond formation Beringer and Rodnina, Mol Cell 26, 311ff Abstract to historical paper on next slide The Structural Basis of Ribosome Activity in Peptide Bond Synthesis Poul Nissen, Jeffrey Hansen, Nenad Ban, Peter B. Moore, and Thomas A. Steitz* Using the atomic structures of the large ribosomal subunit from Haloarcula marismortui and its complexes with two substrate analogs, we establish that the ribosome is a ribozyme and address the catalytic properties of its all-RNA active site. Both substrate analogs are contacted exclusively by conserved ribosomal RNA (rRNA) residues from domain V of 23S rRNA; there are no protein side-chain atoms closer than about 18 angstroms to the peptide bond being synthesized. The mechanism of peptide bond synthesis appears to resemble the reverse of the acylation step in serine proteases, with the base of A2486 (A2451 in Escherichia coli) playing the same general base role as histidine-57 in chymotrypsin. The unusual pKa (where Ka is the acid dissociation constant) required for A2486 to perform this function may derive in part from its hydrogen bonding to G2482 (G2447 in E. coli), which also interacts with a buried phosphate that could stabilize unusual tautomers of these two bases. The polypeptide exit tunnel is largely formed by RNA but has significant contributions from proteins L4, L22, and L39e, and its exit is encircled by proteins L19, L22, L23, L24, L29, and L31e. Steitz was awarded the 2009 Nobel Prize in Chemistry Science 289, 920-930 along with Venkatraman Ramakrishnan and Ada Yonath ’for studies of the structure and function of the ribosome’. Chemical structures of ribo- some peptidyl transferase substrates and analogs (A) The tetrahedral carbon intermediate produced during peptide bond formation; the tetrahedral carbon is indicated by an arrow. (B) The transition state analog formed by coupling the 3' OH of CCdA to the amino group of the O-methyl tyrosine residue of puromycin via a phosphate group, CCdA-p-Puro. (C) An N-amino-acylated mini-helix constructed to target the A- site. The oligonucleotide sequence 5'-CCG GCG GGC UGG UUC AAA CCG GCC CGC CGG ACC-3' puromycin should form 13 base pairs. The construct is based on a mini-helix known to be a suitable substrate for amino-acylation by Tyr-tRNA synthetase. The 3' OH of its terminal C is coupled to the 5' OH of the N6-dimethyl A moiety of puromycin by a phosphodiester bond Note: Work of R. Schroeder here at the MFPL strengthened the interpretation that the function of several classical antibiotics (which are mostly natural substances) is based on their structural similarity to peptidyltransferase intermediates From where you might know puromycin? Science 289, 920-930 Puromycin is an aminonucleoside antibiotic, from Streptomyces alboniger, that causes prema- ture chain termination during translation. Part of the molecule resembles the 3' end of aminoacylated tRNA. It enters the A site and transfers to the growing chain, cau- sing the formation of a puromycylated nascent chain and premature chain release. The 3' position contains an amide linkage instead of the normal ester linkage of tRNA. That makes the molecule more resistant to hydrolysis and stops the ribosome. Puromycin is used in cell culture as selective agent. It is toxic to prokaryotic and eukaryotic cells. Resistance to puromycin is conferred by the Pac gene encoding a puromycin N-acetyl-transferase (PAC) that was found in a Streptomyces producer strain. soluble in water (50 mg/ml); colorless; standard stock solution10 mg/ml; stable for one year as solution at -20 °C; recommended dose as a selection agent 1-10 μg/ml, can be toxic to eukaryotic cells at concentrations as low as 1 μg/ml. Puromycin acts quickly and can kill up to 99% of nonresistant cells within 2 days. Binding sites of antibiotics on bacterial ribosomes Naturally occuring antibiotics are produced by fungi. Permanent concurrence bacteria fungi for same nutrient sources Elongation IV – EFs and Proofreading In vivo tRNA binding is facilitated / accelerated by elongation factors (EFs; Tu+G in prokaryotes, EF- 1+2 in eukaryotes). Binding of EF-1 displaces the empty tRNA from the E-site under hydrolysis of GTP. The participa- tion of EF-1 greatly enhances the accuracy of codon-anticodon re- cognition. EF-2 facilitates the ~ synchronous movement of tRNAs from A+P sites to P+E, again using GTP hydrolysis as driving force. Under these conditions the rate of elongation is only 1-2 amino acids per second (~ 15 in prokaryotes). Termination I https://www.youtube.com/watch?v=kmrUzDYAmEI&t=260s Alberts, Molecular Biology of the Cell 5/e © 2008 Wiley-VCH, Fig 6.74 Termination II Die Termination verläuft bei Prokaryoten und Eukaryoten im Wesentlichen gleich. Sobald ein Stopcodon (UAA, UAG oder UGA) in die A-Stelle gelangt, kommt es zur Termination der Translation. Es gibt kein tRNA-Molekül, dessen Anticodon mit einem der Stop-Codons eine Basenpaarung eingehen könnte. Stattdessen besetzt einer von zwei Terminationsfaktoren (bei Eukaryonten eRF1) die A-Stelle des Ribosoms. Ein zweiter Terminationsfaktor (eRF3) erfüllt bei diesem Vorgang eine Hilfsfunktion. Die Bindung von eRF an die A-Stelle ändert die Aktivität der Peptidyltransferase so, dass diese ein Wassermolekül anstelle einer Aminosäure an die Peptidyl-tRNA anhängt. Dadurch wird das Carboxy-Ende der Polypeptidkette aus der Bindung an das tRNA-Molekül gelöst. Da normalerweise die wachsende Polypeptidkette ausschliesslich durch diese Bindung mit dem Ribosom verknüpft ist, wird die fertige Proteinkette ins Cytoplasma entlassen. Anschließend setzt das Ribosom auch die mRNA und die tRNA der zuletzt eingebauten Aminosäure frei und zerfällt in seine beiden Untereinheiten.Diese können sich sogleich wieder an eine mRNA anlagern. In eukaryotes, eRF1 recognizes all three termination codons, in procaryotes eRF1 recognizes UAA and UAG, eRF2 binds to UGA. high resolution animation of translation: youtube.com/watch?v=TfYf_rPWUdY Ribosome dynamics and tRNA movement by time- resolved electron cryomicroscopy Fischer N, Konevega AL, Wintermeyer W, Rodnina MV, Stark H Nature 466(#7304), 329-333, 2010 July 15 Dynamic mechanism of tRNA translocation during protein synthesis observing the classical A, P and E sites for tRNA binding as well as the hybrid A/P and P/E states during the transition. The authors hypothesize that the ribosome is a 'Brownian machine' that couples spontaneous, thermally-induced motions into directional movement. 1st ‘movie’* (20 frames GIF) showing 4 views onto a 70S procaryotic ribosome from top (upper row), from the solvent side of the 30S subunit (lower left) and onto 50S subunit and tRNAs (lower right). Note the coupling between global dynamics of the 30S subunit (yellow; left column), tRNA movement (right column), and local conformational changes of the 50S subunit (lower right). 2nd movie (11 frames GIF) showing 50S ribosomal subunit (semi-transparent grey) at larger resolution, indicating the 50S dynamics during translocation in molecular detail.