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Review Protein Synthesis 1 tmRNA – in bacteria 2 Eukaryotic and Prokaryotic Protein Synthesis Differences • • • • 1. Ribosomes. Eukaryotic ribosomes are larger. (Slide 29, lecture 4) 2. Initiator tRNA. In eukaryotes, the initiating amino acid is methionine rather than N-formylmethionine. However, as in prokaryotes, a special tRNA participates in initiation. 3. Initiation. The initiating codon in eukaryotes is always AUG. Eukaryotes, in contrast with prokaryotes, do not use a specific purinerich sequence (RBS) on the 5′ side to distinguish initiator AUGs from internal ones. Instead, the AUG nearest the 5′ end of mRNA is usually selected as the start site. A 40S ribosome attaches to the cap at the 5′ end of eukaryotic mRNA and searches for an AUG codon by moving step-by-step in the 3′ direction. The 5′ cap provides an easily recognizable starting point. 4. Elongation and termination. Eukaryotic elongation factors EF1α and EF1βγ are the counterparts of prokaryotic EF-Tu and EF-Ts. The GTP form of EF1α delivers aminoacyl-tRNA to the A site of the ribosome, and EF1βγ catalyzes the exchange of GTP for bound GDP. Eukaryotic EF2 mediates GTP-driven translocation in much the same way as does prokaryotic EF-G. Termination in eukaryotes is carried out by a single release factor, eRF1, compared with two in prokaryotes. Finally, eIF3, like its prokaryotic counterpart IF3, prevents the 3 reassociation of ribosomal subunits in the absence of an initiation complex. Eukaryotic Initiation Binding of 5’ cap of mRNA Ratchet search of AUG – dependent on ATP Association of ribosome 4 Antibiotics inhibit protein synthesis Antibiotic Action Streptomycin and other aminoglycosides Inhibit initiation and cause misreading of mRNA (prokaryotes) Tetracycline Binds to the 30S subunit and inhibits binding of aminoacyl-tRNAs (prokaryotes) Chloramphenicol Inhibits the peptidyl transferase activity of the 50S ribosomal subunit (prokaryotes) Cycloheximide Inhibits the peptidyl transferase activity of the 60S ribosomal subunit (eukaryotes) Erythromycin Binds to the 50S subunit and inhibits translocation (prokaryotes) Puromycin Causes premature chain termination by acting as an analog of aminoacyltRNA (prokaryotes and eukaryotes) 5 6 Proteins • Building blocks (amino acids) and properties • Primary, Secondary, Tertiary, and Quaternary Structure • Stabilizing forces of a protein fold • Functions • Enzymes • Mechanisms of Catalysis 7 Amino Acids Building Blocks of Proteins 8 Amino Acids Can Join Via Peptide Bonds 9 20 Common Amino Acids • • • • • Non-polar amino acids Polar, uncharged amino acids Acidic amino acids Basic amino acids When looking at these structures I want you to think about potential interactions that could stabilize the three dimensional structure of proteins 10 Long haul to identifying the 20 aa 1819 1820 1846 1865 1866 1869 1873 (1932) 1873 (1932) 1875 1881 Leucine Glycine Tyrosine Serine Glutamic Acid Aspartic Acid Asparagine Glutamine Alanine Phenylalanine 1889 1890 1895 1896 1901 1901 1901 1903 1922 1936 Lysine Cysteine Arginine Histidine Valine Proline Trytophan Isoleucine Methionine Threonine 11 Hydrophobic Amino Acids 12 Hydrophobic Amino Acids 13 Polar, Uncharged Amino Acids 14 Polar, Uncharged Amino Acids 15 Acidic Amino Acids 16 Basic Amino Acids 17 Uncommon Amino Acids We may see some of these in later chapters • • • • Hydroxylysine, hydroxyproline - collagen Carboxyglutamate - blood-clotting proteins Pyroglutamate - bacteriorhodopsin Phosphorylated amino acids - signaling device 18 Amino Acids are Weak Polyprotic Acids 19 pKa Values of the Amino Acids • Alpha carboxyl group - pKa = 2 • Alpha amino group - pKa = 9 • These numbers are approximate, and can be perturbed by the local protein environment. 20 pKa Values of the Amino Acid Side Chains • Arginine, Arg, R: pKa(guanidino group) = 12.5 • Lysine, Lys, K: pKa = 10.5 • • • • Aspartic Acid, Asp, D: pKa = 3.9 Glutamic Acid, Glu, E: pKa = 4.3 Cysteine, Cys, C: pKa = 8.3 Histidine, His, H: pKa = 6.0 • Serine, Ser, S: pKa = 13 • Threonine, Thr, T: pKa = 13 • Tyrosine, Tyr, Y: pKa = 10.1 21 Titration of Glycine pI = (pK1 + pK2)/2 pH where the net charge of the molecule is zero 22 Titration of Glutamic Acid 23 Titration of Lysine 24 Stereochemistry of Amino Acids • All but glycine have chiral Cα • L-amino acids predominate in nature • R,S-nomenclature system is superior, since amino acids like isoleucine and threonine (with two chiral centers) can be named unambiguously SH > OH> NH2 > COOH > CHO > CH2OH > CH3 All amino acids have the Cα configuration of S except for cysteine because of the thiol group 25 Spectroscopic Properties • All amino acids absorb in infrared region • Only Phe, Tyr, and Trp absorb UV • Absorbance at 280 nm can be used to quantify the concentration for amino acids The ε is proportional to the number of F, W, and Y in the protein Therefore you can calculate a theoretical ε and use Beer’s law to calculate the concentration of the protein 26