protein synthesis and the ribosome

protein synthesis and the ribosome

Central dogma of biology DNA codes for DNA DNA codes for RNA RNA codes for proteins not surprisingly, many points for regulation of the process

RNA codes for proteins genetic code is very redundant 64 combinations of codons for 20 amino acids + 1 stop codon redundancy typically in third base of codon start codon is also Met - all proteins begin with this amino acid

Translation by the ribosome Figure reproduced from: Schmeing and Ramakrishnan (2009) What recent ribosome structures have revealed about the mechanism of translation. Nature. 461:1234.

Peptide bond formation -process takes place at the heart of the ribosome in its peptidyl transferase center (PTC) Ribosome (not to scale! more on this now)

ribosome structure composed of two subunits, large and small large+small: 50S+30S = 70S??? (bacteria) or 60S+40S = 80S??? (eukaryotes) large:23s + 5S rrna + 31 proteins (bacteria) small: 16S rrna + 21 proteins (bacteria) 2009 Nobel Prize (chemistry) catalytic core formed at center, RNA only (ribozyme?)

polypeptide exit tunnel large subunit contains exit tunnel for polypeptide as it s being made some nascent proteins target the ribosome to the membrane, where they insert directly from tunnel to channel ribosome exit tunnel exit tunnel prevents polypeptide from looping back on itself during synthesis SecY it s also the site where a large class of antibiotics bind (macrolides) membrane

Increase in ribosome complexity Another demonstration of evolution Although main elements of ribosome are conserved (particularly the catalytic core), new features are layered on top in higher-order species for additional regulation additions include: RNA expansion segments longer existing proteins more ribosomal proteins Human Fruit fly Structures of the human and Drosophila 80S ribosome Andreas M. Anger, Jean-Paul Armache, Otto Berninghausen, Michael Habeck, Marion Subklewe, Daniel N. Wilson & Roland Beckmann Nature 497, 80 85 (02 May 2013)

Increase in ribosome complexity what defines a human? Having the most complex ribosome! (according to Loren Williams) Petrov,, Williams. Evolution of the ribosome at atomic resolution. (2014) PNAS 111:10251-10256.

Elongation cycle DNA codes for DNA Steitz. Nat Rev Mol Cell Biol (2008) 9:242-253.

transfer RNA (trna) trnas bring each amino acid to the ribosome amino acid anticodon three sites: A, P, and E; peptidebond formation happens at P wobble base pairing (noncanonical) allows ~45 trnas to match 61 codons

molecular basis of fidelity (ribosome) R+T corr RT corr elongation (trna) k c + k c - d[rt corr ] dt = k c +[R][T corr ] k c [RT corr ]=0 Similar form for wrong trna (error), just with different rates p err = [RT err] p corr [RT corr ] Kcorr d K err d thermodynamic specificity correct vs. incorrect trna different by a single hydrogen bond (~ 2-4 kt), giving an error rate of 0.13 however, the known error rate is 0.0001, a factor of 1000 less! PBoC 21.5.1

high specificity requires energy additional proofreading step R+T corr RT corr RT corr elongation kd pd for Terr R+T corr energy-consuming proofreading step causes a conformational change that alters off rates, mainly by delaying accommodation of trna f = [RT err] [RT corr] f 0 k d p d error rate is equal to original rate scaled by new off rates proofreading carried out by GTP hydrolyzing EF-Tu, an elongation factor PBoC 21.5.1

The ribosome rocks Ratchet-like intersubunit rotation was imaged by cryo-em [1,4], smfret [3] P-site peptidyl-trna locks the ribosome [2] ratcheting moves trnas through A, P, and E sites (classical states); intermediate states known as hybrid ratcheting requires energy from GTP hydrolysis [1] Frank and Agrawal. Nature (2000) 406:318-322. [2] Valle et al. Cell (2001) 114:123-134. [3] Cornish et al. Mol Cell (2008) 30:578-588. [4] Agirrezabala et al. Mol Cell (2008) 32:190-197.

Regulatory nascent chains Several nascent chains have been found to regulate their own translation at the ribosome Most examples identified thus far involve nascent chains that cause ribosome stalling under certain conditions There are known regulatory nascent chains in prokaryotes, eukaryotes, and viruses Some examples: cat, cmia, ermc (regulation of expression of antibiotic resistance genes) SecM (sensor of protein translocation) TnaC (regulation of Trp degradation) Stalling through binding of erythromycin + nascent peptide ErmCL in exit tunnel Vasquez-Laslop et al. (2010) EMBO. 29:3108-3117.

Regulation of the tna operon Transcription terminates resumes initiates pauses CAP = RNA catabolite polymerase activating protein CAP mrna tnac Low [Trp]: TnaC Transcription termination by Rho factor Rho binding site No expression of structural genes (tnaa/tnab) High [Trp]: Stop Translational stalling leads to transcription antitermination Induction of expression of structural genes (tnaa/tnab) TnaA Rho factor TnaB 50S 30S Rho-dependent transcription termination sites Translation initiates TnaC + Trp stall the ribosome Translation Ribosomal terminates subunits tnaa/tnab Recognition of the regulatory nascent chain TnaC by the ribosome. L G. Trabuco, C B Harrison, E Schreiner, and K Schulten. Structure, 18:627-637, 2010. High Low [Trp] Discovered by Yanofsky and colleagues

Role of SecM in bacteria SecM regulates expression of translocase SecA via a negative feedback loop secm repressor helix seca SecM pause sequence ribosomes SecM SecA secm is translated until stalling point is reached Transcription pauses, repressor helix unfolds, and seca is translated SecA SecA pulls(?) on SecM, permits translation to continue, and repressor helix refolds

Closeup of SecM in the exit tunnel (MDFF-fitted structure) hydrophobic interaction between R163 and A2062 Gumbart et al. (2012) Biophys. J. 103: 331-341. base stacking between W155 and A751 at L4/L22 constriction site Structure is static! We need dynamics to determine importance of observed interactions

Stability of interactions in WT and mutants interactions (hydrophobic and/or philic) quantified over time (2x 20-ns sims) A2503 -Q158A has no effect (as expected from experiment) -all other mutants notably disrupt multiple interactions (>25% change) Gumbart et al. (2012) Biophys. J. 103: 331-341.

Dynamic interaction relays signal SecM s presence known to be required for SecM stalling Relay between R163 and the P-site trna Relay between R163 and the A-site trna -first relay similar to earlier proposals, robust; second relay is less stable, some interactions missing in repeated simulations -result of the relay is to shift the position of the P-tRNA away from PTC Gumbart et al. (2012) Biophys. J. 103: 331-341.

R163 acts to restrain A2062 w/secm in tunnel w/tnac in tunnel Empty tunnel SecM compresses A2062 against tunnel wall, reducing its fluctuations Gumbart et al. (2012) Biophys. J. 103: 331-341.

trna Phe shifts downward when placed where A-site trna Pro is in stalled ribosome, reduces N-C bonding distance significantly Gumbart et al. (2012) Biophys. J. 103: 331-341. Role of A-site trna Pro RMSD vs. time trna Phe trna Pro trna Pro trna Pro trna Phe trna Phe peptide-bonding distance

Role of W155 and how SecA relieves stalling Constant force vs. time Force vs. extension -W155 stacking with A751 leads to compaction of SecM, localizing R163 -applied force (predicted to be the action of SecA on SecM) breaks this interaction, extends SecM, and disrupts relay Gumbart et al. (2012) Biophys. J. 103: 331-341.

Novel Azithromycin Deriva3ves Mimic SecM 6 Interac3ons Overlap of azithromycin and SecM after 16S alignment Azithromycin A751 Novel azithromycin derivatives N N N Linker Tryptophan side chain analogs COMP-2 N W155 SecM backbone HN HO HO OH HO NH O O O O O O O OH Azithromycin COMP-NO 2, Cl, H R=NO 2, Cl, H N R Indole derivatives mimic π-stacking between W155 and A751 Surprisingly, improved activity was only observed for the less polar derivatives Washington et al. ACS Chem. Biol. 9, 2014, 2621 2631. Gumbart et al. Biophys. J. 103, 2012, 331 341

Summary Protein synthesis, translation, is done by the ribosome During elongation process, codons of mrna are matched with anti-codons of trna, carrying the correct amino acid The matching is very accurate due to additional proofreading by EF-Tu Some peptides can stall the ribosome during their translation: another way to control gene expression