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Chapter 22 - Protein Synthesis

Chapter 22 - Protein Synthesis. mRNA. The ribosome, a complex of RNA and protein, is the site where genetic information is translated into protein. protein. Exam: Tues May 4 12:00-3:00. The Genetic Code. How is the info translated from NA to protein ?.

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Chapter 22 - Protein Synthesis

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  1. Chapter 22 - Protein Synthesis mRNA The ribosome, a complex of RNA and protein, is the site where genetic information is translated into protein protein Exam: Tues May 4 12:00-3:00

  2. The Genetic Code How is the info translated from NA to protein ? • Codons - three letter genetic code (nonoverlapping) • tRNA - adapters between mRNA and proteins • Reading frame - each potential starting point for interpreting the 3 letter code • DNA – only four bases (A,T,G,C) • Must code for 20 amino acids • Two-base code: 42 = 16 combinations • Four-base code: 44 = 256 combinations • Three-base code: 43 = 64 combinations

  3. Overlapping vs nonoverlapping reading of the three-letter code What is one benefit ?

  4. Three reading frames of mRNA • Translation of the correct message requires selection of the correct reading frame tRNA ‘reads’ each codon Key: decide where to start

  5. Standard genetic code (1962 – 1965, Khorana, Nirenberg in separate laboratories) (5’-UUU-3’) (5’-UUC-3’) (START)

  6. Features of the genetic code • The genetic code is unambiguous. Each codon corresponds to only one amino acid. • 2. There are multiple codons for most amino acids (code is degenerate) 3. The first two nucleotides of a codon are often enough to specify a given amino acid (Gly = GG_ ) • 4. Codons with similar sequences specify similar amino acids • 5. Only 61 of the 64 codons specify amino acids • Termination (stop codons): UAA, UGA, UAG • Initiation codon - Methionine codon (AUG) specifies initiation site for protein synthesis Asp and Glu = GU_ single mutation chem similar AA

  7. Crack the genetic code RNA Tie Club 20 members 16 had a specific amino acid George Gamow (ALA) 4 members had specific nucleotide F. Crick (TYR) J. Watson (PRO) Richard Feynman (GLY) Combination of hard drinking and science Melvin Calvin (HIS) Edward Teller (LEU) Leslie Orgel (THR) Max Delbruck (TRY) Sydney Brenner (VAL) Erwin Chargaff (LYS) 1st meeting Woods Hole 1954

  8. Crack the genetic code RNA Tie Club Aim: “Solve the riddle of RNA structure and understand how it built proteins” George Gamow (ALA) F. Crick (TYR) J. Watson (PRO) Richard Feynman (GLY) Combination of hard drinking and science Melvin Calvin (HIS) Edward Teller (LEU) Leslie Orgel (THR) Max Delbruck (TRY) Sydney Brenner (VAL) Erwin Chargaff (LYS) 1st meeting Woods Hole 1954

  9. Crack the genetic code F. Crick (TYR) “Adapter hypothesis” George Gamow (ALA) Mathematics used to establish that 3 letter code would be enough to define all 20 amino acids

  10. Crack the genetic code Non–members of the RNA Tie Club Marshall Nirenberg 1961 Johann Matthaei ‘Cell free system’ RNA template, ribosomes, nucleotides, amino acids, ATP UUUUUUUUUUUU FFFF CCCCCCCCCCCCCC PPPP Har Gobind Khorana UCUCUCUCU Ser,Leu,Ser,Leu Robert Holley Determined the structure of tRNA 1965 Nobel prize 1968

  11. Cloverleaf structure of tRNA Amino acid • Every cell must contain at least 20 tRNA (one for every amino acid) • Each tRNA must recognize at least one codon • Watson-Crick base pairing (dashed lines) • tRNA has an acceptor stem and four arms • Conserved bases (gray) Complementary to the codon

  12. tRNA Anticodons Base-Pair with mRNA Codons • tRNA molecules are named for the amino acid that they carry (e.g. tRNAPhe) 3’ 5’ tRNAPhe • Base pairing between codon and anticodon is governed by rules of Watson-Crick Wobble position AAG A-U G-C 5’ UUC 3’ Variable position mRNA (Phe) However, the 5’ anticodon position has some flexibility in base pairing (the “wobble” position)

  13. 3’ 5’ tRNAPhe Wobble position AAG 5’ UUC 3’ Variable position mRNA (Phe) Deamination of G = I 5’ UUU 3’ (Phe)

  14. Base pairing at the wobble position * • Inosinate (I) often found at 5’ wobble position • I can form H bonds with A, C, or U • Anticodon with I can recognize more than one synonymous codon Some bacteria get by with only 31 tRNA (not 61) Weaker H-bond: speeds up prot. Syn.

  15. Protein synthesis :translation Aminoacylation of tRNA Charging of tRNA By Aminoacyl-tRNA Synthetases Initiation Chain elongation Termination

  16. Structure of the Aminoacyl-tRNA Linkage Linkage type ?

  17. * Aminoacylation of tRNA The genetic code is translated by two adaptors: The first is the aminoacyl-tRNA synthetase. The linkage is through a high energy bond created with ATP.

  18. * High energy bond The second adaptor is the tRNA itself whose anticodon forms base pairs with the appropriate mRNA codon. An error in either step causes wrong aa in peptide chain.

  19. Protein Synthesis Proceeds by the Addition of an AA to the C-terminus of a polypeptide Peptidyl Transferase mRNA 3’ 5’ Energy stored in aminoacyl-tRNA used in formation of peptide bond

  20. Aminoacyl-tRNA Synthetases • Aminoacyl-tRNA - amino acids are covalently attached to the 3’ end of each tRNA molecule (named as: alanyl-tRNAAla) • Most species have at least 20 different aminoacyl-tRNAsynthetases • (1 per amino acid) • Each synthetase specific for a particular amino acid, but may recognize isoacceptor tRNAs • Aminoacyl-tRNAs are high-energy molecules (the amino acid has been “activated”) • The activation of an amino acid by aminoacyl-tRNA synthetase requires ATP Amino acid + tRNA + ATP Aminoacyl-tRNA + AMP + PPi

  21. Synthetase binds ATP and correct AA Based on size/charge/hydrophobicity

  22. Synthetase selectively binds tRNA based on Structural features Anticodon Acceptor stem

  23. Ester linkage

  24. Specificity of Aminoacyl-tRNA Synthetase • Attachment of the correct amino acid to the corresponding tRNA is a critical step • Synthetase binds ATP and the correct amino acid (based on size, charge, hydrophobicity) • Synthetase then selectively binds specific tRNA molecule based on structural features, anticodon and acceptor stem

  25. Proofreading Activity of Aminoacyl-tRNA Synthetases • Some aa-tRNA synthetases can proofread • Isoleucyl-tRNA synthetase may bind valine instead of isoleucine and form valyl-adenylate • The valyl-adenylate is usually then hydrolyzed to valine and AMP so that valyl-tRNAIle does not form 1 in 100 1 in 10000

  26. * Ribosomes Protein synthesis by a complex composed of the ribosome Catalyzes peptide bond formation accessory protein factors Assist ribosome mRNA Carries info charged tRNA molecules Carries activated AA Initiation complex assembles at first mRNA codon, and disassembles at termination step Ribosome moves 5’ to 3’ along mRNA Peptide grows N to C direction

  27. Ribosomes Are Composed of Both rRNA and Protein

  28. Ribosomes Large subunit Small subunit 30S 50S Nobel Prize in Chemistry 2009

  29. Ribosomes Contain Two Aminoacyl-tRNA Binding Sites • Ribosome must align two charged tRNA molecules so that anticodons interact with correct codons of mRNA • Aminoacylated ends of the tRNAs are positioned at the site of peptide bond formation • Ribosome must hold both mRNA and growing polypeptide chain 30S 50S

  30. Mechanism of Translation 1. Initiation • The translation complex is assembled at the beginning of the mRNA coding sequence • Complex consists of: Ribosomal subunits mRNA template to be translated Initiator tRNA molecule Protein initiation factors IF-1 IF-2 IF-3 2. Elongation 3. Termination

  31. Initiator tRNA • First codon translated is usually AUG • Each cell contains at least two methionyl-tRNAMet molecules which recognize AUG • The initiator tRNA recognizes initiation codons • Bacteria: N-formylmethionyl-tRNAfMet • (Eukaryotes: methionyl-tRNAiMet) Second tRNAMet recognizes only internal AUG EF-Tu interaction (does not bind formyl-tRNAfMet) IF2 interacts with formyl-tRNAfMet formylmethionine

  32. Shine-Dalgarno sequences in E. coli mRNA • Ribosome-binding sites at the 5’ end of mRNA for several E. coli proteins • In prokaryotes, the 30S ribosome binds to a region of the • mRNA (Shine-Dalgarno sequence) upstream of the initiation • sequence • S-D sequence binds to a complementary base sequence at the 3’ end of the 16S rRNA

  33. Complementary base pairing of S-D sequence

  34. Initiation of Translation Initiator tRNA molecule Bacteria:N-formylmethionyl-tRNAfMet Second tRNAMet recognizes only internal AUG Protein initiation factors IF-1 IF-2 IF-3

  35. Formation of the prokaryotic 70S initiation factor * Inititation factors IF1, IF2, and IF3 are required to form the ribosomal complex

  36. Translation Initiation in Eukaryotes • Eukaryotic initiation factor 4 (eIF-4), (or cap binding protein, CBP) binds to the (5’ end) 7methylguanylate cap of eukaryotic mRNA • A preinitiationcomplex forms (40S ribosome, aminoacylated initiator tRNA, other factors) and searches the mRNA 5’ 3’ for an initiator codon • The Met-tRNAiMet binds to AUG, and the 60S ribosomal subunit binds to complete the complex

  37. Chain Elongation is a Three-Step Microcycle • The initiator tRNA is in the P site • Site A is ready to receive an aminoacyl-tRNA • Elongation is a three-step cycle: • (1) Positioning the correct aa-tRNA in site A (2) Formation of a peptide bond (3) Shifting mRNA by one codon

  38. Translating an mRNA Molecule--Elongation three-step cycle: Positioning the correct aa-tRNA in site A Formation of a peptide bond Shifting mRNA by one codon Translocation

  39. * Positioning of the aminoacyl-tRNA Insertion of aa-tRNA by EF-Tu during chain elongation

  40. * Cycling of EF-Tu-GTP *

  41. * Peptidyl Transferase Catalyzes Peptide Bond Formation • Substrate binding site in 23S rRNA and 50S ribosomal proteins • Catalyticactivity from 23S rRNA (an RNA-catalyzed reaction) Adenine: abstracts proton donates proton • Peptidyl transferase activity is contained within the large ribosomal subunit Catalyticactivity from 23S rRNA (an RNA-catalyzed reaction)

  42. A Pocket in the 23S Ribosomal RNA is the Catalyst for the Peptidyl Transferase Activity N3 of the Adenine in the catalytic pocket of 23S rRNA abstracts a proton from the aa acylated to the tRNA. The Amino N of the aa acid then attacks the carboxyl group of the peptide in the P site. The protonated Adenine donates its hydrogen to the Oxygen linked to the tRNA thus releasing the tRNA.

  43. tRNA originally attached to peptide in the P site is released New Amino Acid

  44. Translocation Moves the Ribosome by One Codon • Translocation step: the new peptidyl-tRNA is moved from the A site to the P site, while the mRNA shifts by one codon • The deaminoacylated tRNA has shifted from the P site to the E site (exit site)

  45. * EF-G-GTP GTP hydrolysis causes large conformational change that moves peptidyl tRNA to P site

  46. the mRNA shifts by one codon

  47. Translational Elongation in Prokaryotes EF-Tu is the most abundant protein in E. coli -- ~6% of total protein. It is a G protein.

  48. EF-G is another G protein and hydrolysis of its GTP powers translocation. P site A/P hybrid site E site (exit site) A site Hydroysis of GTP by EF-G causes large conformational change that moves tRNA from the A/P hybrid site to the P site.

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