Lecture 5

Mechanism of Elongation Michael Altmann FS 2015

Institut für Biochemie und Molekulare Medizin

Polypeptide chain elongation

tRNA charging tRNA structure, tRNA synthetases

Elongation factors

Elongation cycle

charging reaction

EFTu/Ts, eEF1

EFG, eEF2

Aa-tRNA selection

Translocation

Peptidyltransferase

EFP, eIF5A/4D

Ribosomes Subunit structure, decoding center, peptidyltransferase center

Movies www.youtube.com/watch?v=Jml8CFBWcDs www.youtube.com/watch?v=RedO6rLNQ2o

Antibiotics

Charging of tRNA

Reference: G. Löffler &PE Petrides Biochemie & Pathobiochemie, 7. Aufl., 2004

Charging of tRNA

Reference: W Müller-Esterl Biochemie, 1. Aufl., 2004

Structure of the procaryotic 70S ribosome

Reference: Schmeing & Ramakrishnan (2009) Nature 461, 1234-1242

The 3 steps during elongation of protein synthesis

Energy balance: Steps 1 and 3 consume 1 GTP (->GDP), additionally to ATP consumption during tRNA charging (2 Pi) -> at least 4 energy rich bonds have to be consumed per peptide bond formed

2. Peptidyl-transfer at A-site (catalysed by rRNA)

3. Translocation of peptidyl-tRNA to P-site which requires eEF2-GTP

1. Binding of charged tRNA which requires eEF1A-GTP and eIF1B

eIF5A/bacterial EF-P mimics a tRNA

(Grey): mammalian eIF5A / (Pink): bacterial EF-P

Essential steps in translation

Reference: Liljas (2009) Science 326, 677

EF-Tu-tRNA complex

tRNA mimicry

Reference: Y Nakamura & K Ito Trends Biochem. Sci. 28: 99-105, 2003

eEF1A and 1B

Reference: CM Abbott & CG Proud Trends Biochem. Sci. 29: 25-31, 2004

Decoding by the ribosome

Reference: Schmeing et al (2009) Science 326, 688-694

Movie S1. Animation of decoding by the ribosome and EF-Tu. The incoming aminoacyl-tRNA, shown in purple, is delivered to the ribosome as part of a ternary complex with EF-Tu, shown in red, and GTP. This animation depicts the initial binding of the TC to the ribosome, followed by sampling of the mRNA codon and codon recognition. The animation then highlights the conformation changes in the 30S subunit, EF-Tu, and the 3’ end of the aminoacyl-tRNA that provide the communication pathway between the decoding center of the 30S subunit and the GTPase center of EF-Tu. After hydrolysis of GTP and release of Pi, EF-Tu undergoes a domain rearrangement, leading to its dissociation and accommodation of the aminoacyl-tRNA into the peptidyl transferase center.

Fidelity of translation

Step 1: Hydrolytic editing by tRNA synthetases • tRNA synthetases remove their own coupling errors through hydrolytic editing of incorrectly attached amino acids • after covalent attachment, the amino acid - especially if incorrect - is „forced“ into an editing site and hydrolyzed from the tRNA

Step 2: EF-Tu & ribosome together dispose of wrong tRNAs • proper codon-anticodon formation is assessed by the 16S rRNA leading to conformational change and allowing for GTP-GDP hydrolysis. Incorrect codon-anticodon matches do not lead to conformational changes and the incorrectly bound tRNA falls of before GTP hydrolysis (1st proofreading). • after GTP-hydrolysis, there is a short time delay before the tRNA moves into position. This time is shorter por correct than incorrect codon-anticodon pairs. Incorrectly matched tRNAs dissociate more rapidly from the ribosome due to the weaker codon-anticodon interaction (2nd proofreading). This step is also known as „kinetic proofreading“.

Molecule Velocity Accuracy Correction functions

DNA 600 nt /sec (bacteria) 100-200 nt / sec (eukaryotes)

10-9 - 10-10 • DNA-pol I/II/III 3’-5’ exo; 5-3’ exo (10-7) • postreplicative correction systems (10-3) e.g. uracil-DNA-glycosylase or uvr-system (nucleotide-excision-repair)

RNA 500 nt - 50 nt / sec

10-4 No exo-activity known

Protein 20 aa / sec 10-3 – 10-4 • aa-tRNA synthetases (editing site; 10-4 -10-5!) • eEF1A-GTP-aa-tRNA (kinetic proofreading; 10-3-10-4)

Macromolecular synthesis: velocity and accuracy

Reference: TA Steitz FEBS Lett. 579: 955-958, 2005

Peptidyltransferase

Peptide-bond formation is RNA catalyzed

Reference: Schmeing & Ramakrishnan (2009) Nature 461, 1234-1242

Translocation by eEF2•GTP

Reference: S Ejiri Biosci. Biotechnol. Biochem. 66: 1-21, 2002

EF-G catalysed translocation: ratcheting

Reference: Schmeing & Ramakrishnan (2009) Nature 461, 1234-1242

Antibiotics: molecular mimicry of puromycin

• Puromycin mimics a charged tRNATyr and is delivered to the A-site. • The ribosome joins puromycin covalently to the peptidyl chain forming a modified peptide which cannot be futher extended. • Many – but not all – antibiotics affect protein synthesis. Cycloheximide is specific for eukaryotic translation.

The Resolution Revolution (2014): mitoribosome structures

Reference: Amunts et al. (2014) Science 343, 1485-1489

-  Mitoribosomes exhibit high variability among species and have diverged strongly from their bacterian & eukaryotic counterparts.

-  Several diaseases map to mitoribosomes & the toxicity of many ribosomal antibiotics is thought to be due to their interaction with the mitoribosomes.

-  Thanks to a new generation of electron detectors of unprecedented speed and sensitivity: -  Structures of macromolecular complexes such as the large 54S yeast mitoribosomal subunit can be obtained at

an near-atomic resolution (3.2 Angstroem; before, 12-14 Angstroem!) without requiring crystals. -  The central protuberance (CP) of yeast 54S lacks 5S rRNA (found in both bacterial & eukaryotic large

ribosomal subunits), instead it is much larger and consists of several unique proteins.

The mammalian mitochondrial 55S ribosome (2015)

References: Beckmann & Herrmann (2015) Science 348, 288-289; Amunts et al (2015) Science 348, 95-98; Greber et al. (2015) Science 348, 303-308

What is different from cytoplasmatic eukaryotic & bacterial ribosomes? • Though still ribozymes, mitoribosomes exhibit different morphology and greatly increased protein content: 36 proteins have no bacterial homolog, almost the entire surface is covered by proteins which in some instances replace rRNA (better shield of core RNA against reactive oxygen species). • Mitoribosomes are tethered to the matrix side of the mitochondrial inner membrane by mitoribosomal protein L45 which is located near the tunnel exit site • The central protuberance (CPT) is not formed by 5S rRNA but by a mitochondrial valine-encoding tRNA • The mechanical movements of both subunits differ substantially from those in a “canonical“ ribosome • At the subunit interface, a GTP-binding protein (mS29; function?) is detected • At the mRNA entry site a pentatricopeptide repeat (PPR) is detected (mS39; used for mRNA binding?)

Seminar 2

-  Where comes the energy for peptide bond formation from? -  What are the implications of the finding that peptidyltransferase is a ribozyme for our view

of evolution? -  Aminoglycosides: what are they and how could they affect eukaryotic cells?