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PROTEIN SYNTHESIS

PROTEIN SYNTHESIS. Disampaikan Oleh Mohammad Hanafi, MBBS., dr., MS. Protein Synthesis. The production (synthesis) of proteins . 3 phases : 1. Transcription 2. RNA processing 3. Translation Remember: DNA  RNA  Protein. Nuclear membrane. DNA. Transcription. Pre-mRNA.

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PROTEIN SYNTHESIS

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  1. PROTEIN SYNTHESIS Disampaikan Oleh Mohammad Hanafi, MBBS., dr., MS.

  2. Protein Synthesis • The production (synthesis) of proteins. • 3 phases: 1. Transcription 2. RNA processing 3. Translation • Remember: DNA  RNA  Protein

  3. Nuclear membrane DNA Transcription Pre-mRNA RNA Processing mRNA Ribosome Translation Protein DNA  RNA Protein Eukaryotic Cell

  4. DNA Transcription mRNA Ribosome Translation Protein DNA  RNA Protein Prokaryotic Cell

  5. Question: • How does RNA(ribonucleic acid) differ from DNA (deoxyribonucleic acid)?

  6. RNA differs from DNA 1. RNA has a sugar ribose DNA has a sugar deoxyribose 2. RNA contains uracil (U) DNA has thymine (T) 3. RNA molecule is single-stranded DNA is double-stranded

  7. Nuclear membrane DNA Transcription Pre-mRNA RNA Processing mRNA Ribosome Translation Protein 1. Transcription Eukaryotic Cell

  8. 1. Transcription • The transfer of information in the nucleus from a DNA molecule to an RNA molecule. • Only 1 DNA strand serves as the template • Starts at promoterDNA (TATA box) • Ends at terminatorDNA (stop) • When complete, pre-RNA molecule is released.

  9. Question: • What is the enzyme responsible for the production of the RNA molecule?

  10. Answer: RNA Polymerase • Separates the DNA molecule by breaking the H-bonds between the bases. • Then moves along one of the DNA strands and links RNA nucleotides together.

  11. DNA RNA Polymerase pre-mRNA 1. Transcription

  12. Question: • What would be the complementary RNA strand for the following DNA sequence? • DNA 5’-GCGTATG-3’

  13. Answer: • DNA 5’-GCGTATG-3’ • RNA 3’-CGCAUAC-5’

  14. Nuclear membrane DNA Transcription Pre-mRNA RNA Processing mRNA Ribosome Translation Protein 2. RNA Processing Eukaryotic Cell

  15. 2. RNA Processing • Maturation of pre-RNA molecules. • Also occurs in the nucleus. • Introns spliced out by splicesome-enzyme and exonscome together. • End product is a mature RNA molecule that leaves the nucleus to the cytoplasm.

  16. pre-RNA molecule exon intron exon exon intron intron intron exon exon exon splicesome splicesome exon exon exon Mature RNA molecule 2. RNA Processing

  17. Types of RNA • Three types ofRNA: A. messenger RNA (mRNA) B. transfer RNA (tRNA) C. ribosome RNA (rRNA) • Remember: all produced in thenucleus!

  18. A. Messenger RNA (mRNA) • Carries the information for a specific protein. • Made up of 500 to 1000 nucleotides long. • Made up of codons (sequence of three bases: AUG - methionine). • Each codon, is specific for an amino acid. 4 nucleotides in mRNA so theoretically there are 43 (64) possible combinations of codons Only 20 a.a.’s to encode

  19. start codon A U G G G C U C C A U C G G C G C A U A A mRNA codon 1 codon 2 codon 3 codon 4 codon 5 codon 6 codon 7 stop codon protein methionine glycine serine isoleucine glycine alanine Primary structure of a protein aa2 aa3 aa4 aa5 aa6 aa1 peptide bonds A. Messenger RNA (mRNA)

  20. Genetic Codes Codons are written 5’ to 3’ Codons for the same a.a. tend to have the nucleotides in 1st and 2nd positon Raises the possibility of 3 different reading frames depending on the start AUG is unique - encodes methionine - acts as an initiation codon

  21. B. Transfer RNA (tRNA) • Made up of 75 to 80 nucleotides long. • Picks up the appropriate amino acid floating in the cytoplasm (amino acid activating enzyme) • Transports amino acids to the mRNA. • Have anticodons that are complementary to mRNAcodons. • Recognizes the appropriate codons on the mRNA and bonds to them with H-bonds.

  22. amino acid attachment site methionine amino acid U A C anticodon B. Transfer RNA (tRNA)

  23. tRNA Coupling Aminoacyl-tRNA synthetase is required to catalyze the attachment of the specific a.a. to it’s associated tRNA

  24. C. Ribosomal RNA (rRNA) • Made up of rRNA is 100 to 3000 nucleotides long. • Important structural component of a ribosome. • Associates with proteins to form ribosomes.

  25. Ribosomes • Large and small subunits. • Composed of rRNA (40%) and proteins (60%). • Both units come together and help bind the mRNA and tRNA. • Two sites fortRNA a. P site(first and last tRNA will attach) b. A site

  26. mRNA A U G C U A C U U C G Ribosomes Large subunit P Site A Site Small subunit

  27. Ribosomes Protein formation requires orderly events to progress Ribosomes possess binding sites to achieve this mRNA binding site - holds mRNA strand in place Aminoacyl-tRNA site (A-site) - holds one tRNA molecule Peptidyl-tRNA site (P-site) - holds another tRNA molecule Exit site (E-site) - holds a third tRNA molecule

  28. Nuclear membrane DNA Transcription Pre-mRNA RNA Processing mRNA Ribosome Translation Protein 3. Translation Eukaryotic Cell

  29. 3. Translation • Synthesis of proteins in the cytoplasm • Involves the following: 1. mRNA (codons) 2. tRNA (anticodons) 3. rRNA 4. ribosomes 5. amino acids

  30. 3. Translation • Three parts: 1. initiation: start codon (AUG) 2. elongation: 3. termination: stop codon (UAG) • Let’s make a PROTEIN!!!!.

  31. mRNA A U G C U A C U U C G 3. Translation Large subunit P Site A Site Small subunit

  32. Initiation Initiation-requirements: • mRNA • Ribosome • Initiator tRNA (fMet tRNA in prokaryotes) • 3 Initiation factors (IF1, IF2, IF3) • Mg2+ • GTP (guanosine triphosphate)

  33. Initiation-steps (e.g., prokaryotes): • 30S ribosome subunit + IFs/GTP bind to AUG start codon and Shine-Dalgarno sequence composed of 8-12 purine-rich nucleotides upstream (e.g., AGGAGG). • Shine-Dalgarno sequence is complementary to 3’ 16S rRNA. • Initiator tRNA (fMet tRNA) binds AUG (with 30S subunit). All new prokaryote proteins begin with fMet (later removed). fMet = formylmethionine (Met modified by transformylase; AUG at all other codon positions simply codes for Met) mRNA 5’-AUG-3’ start codon tRNA 3’-UAC-5’ anti-codon • IF3 is removed and recycled. • IF1 & IF2 are released and GTP is hydrolysed, catalyzing the binding of 50S rRNA subunit. • Results in a 70S initiation complex (mRNA, 70S, fMet-tRNA)

  34. aa2 aa1 2-tRNA 1-tRNA G A U U A C Initiation anticodon A U G C U A C U U C G A hydrogen bonds codon mRNA

  35. Elongation of a polypeptide: Binding of the aminoacyl tRNA (charged tRNA) to the ribosome. Formation of the peptide bond. Translocation of the ribosome to the next codon.

  36. 3-1. Binding of the aminoacyl tRNA to the ribosome. • Ribosomes have two sites, P site (5’) and A site (3’) relative to the mRNA. • Synthesis begins with fMet (prokaryotes) in the P site, and aa-tRNA hydrogen bonded to the AUG initiation codon. • Next codon to be translated (downstream) is in the A site. • Incoming aminoacyl-tRNA (aa-tRNA) bound to elongation factor EF-Tu + GTP binds to the A site. • Hydrolysis of GTP releases EF-Tu, which is recycled. • Another elongation factor, EF-Ts, removes GDP, and binds another EF-Tu + GTP to the next aa-tRNA. • Cycle repeats after peptide bond and translocation.

  37. aa3 3-tRNA G A A Elongation peptide bond aa1 aa2 1-tRNA 2-tRNA anticodon U A C G A U A U G C U A C U U C G A hydrogen bonds codon mRNA

  38. aa3 3-tRNA G A A aa1 peptide bond aa2 1-tRNA U A C (leaves) 2-tRNA G A U A U G C U A C U U C G A mRNA Ribosomes move over one codon

  39. aa4 4-tRNA G C U peptide bonds aa1 aa2 aa3 2-tRNA 3-tRNA G A U G A A A U G C U A C U U C G A A C U mRNA

  40. aa4 4-tRNA G C U peptide bonds aa1 aa2 aa3 2-tRNA G A U (leaves) 3-tRNA G A A A U G C U A C U U C G A A C U mRNA Ribosomes move over one codon

  41. aa5 5-tRNA U G A peptide bonds aa1 aa2 aa4 aa3 3-tRNA 4-tRNA G A A G C U G C U A C U U C G A A C U mRNA

  42. aa5 5-tRNA U G A peptide bonds aa1 aa2 aa3 aa4 3-tRNA G A A 4-tRNA G C U G C U A C U U C G A A C U mRNA Ribosomes move over one codon

  43. aa5 aa4 Termination aa199 aa200 aa3 primary structure of a protein aa2 aa1 terminator or stop codon 200-tRNA A C U C A U G U U U A G mRNA

  44. aa5 aa4 aa3 aa2 aa199 aa1 aa200 End Product • The end products of protein synthesis is a primary structure of a protein. • A sequence of amino acid bonded together by peptide bonds.

  45. incoming large subunit 1 2 3 4 5 6 7 mRNA incoming small subunit polypeptide Polyribosome • Groups of ribosomes reading same mRNA simultaneously producing many proteins (polypeptides).

  46. Question: • The anticodon UAC belongs to a tRNA that recognizes and binds to a particular amino acid. • What would be the DNA base code for this amino acid?

  47. Answer: • tRNA - UAC (anticodon) • mRNA - AUG (codon) • DNA - TAC

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