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PROTEIN SYNTHESIS

PROTEIN SYNTHESIS. POINTS ABOUT TRANSCRIPTION. NEED RNA POLYMERASE CODES FOR 20 AMINO ACIDS CODON:SERIES OF TRIPLET BASE PAIRS. 64 CODONS, 60 FOR AA, OTHERS FOR STARTS/STOPS. INTRONS=NON-CODING EXONS= CODING FOR RNA. PROTEIN TRANSCRIPTION. NUCLEUS RNA POLYMERASE CODES TO DNA

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PROTEIN SYNTHESIS

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  1. PROTEIN SYNTHESIS

  2. POINTS ABOUT TRANSCRIPTION • NEED RNA POLYMERASE • CODES FOR 20 AMINO ACIDS • CODON:SERIES OF TRIPLET BASE PAIRS. • 64 CODONS, 60 FOR AA, OTHERS FOR STARTS/STOPS. • INTRONS=NON-CODING • EXONS= CODING FOR RNA

  3. PROTEIN TRANSCRIPTION • NUCLEUS • RNA POLYMERASE CODES TO DNA • DNA TRANSCRIBES TO m-RNA • INTRONS SNIPPED OUT • EXONS KEPT IN CODE

  4. (snipped out) (snipped out) unit of transcription in a DNA strand exon intron exon intron exon 3’ 5’ transcription into pre-mRNA poly-A tail cap 5’ 3’ 5’ 3’ mature mRNA transcript

  5. transcribed DNA winds up again DNA to be transcribed unwinds sugar-phosphate backbone of one strand of nucleotides in a DNA double helix Newly forming RNA transcript The DNA template at the assembly site sugar-phosphate backbone of the other strand of nucleotides part of the sequence of base pairs in DNA 5’ 3’ RNA polymerase growing RNA transcript 3’ 5’ 3’ 5’ direction of transcription

  6. m-RNA GOES THRU RIBOSOME. RIBOSOME IS r-RNA,CODE THREADS THRU RIBOSOME. AREA OF RIBOSOME BOUND TO tRNA 20 TYPES OF AA ANTICODON ON ONE END OF t-RNA. AA ON OTHER END OF t-RNA AA ATTACH TO EACH OTHER IN PEPTIDE BOND FORM PROTEINS PROTEIN TRANSLATION

  7. Binding site for mRNA P (first binding site for tRNA) A (second binding site for tRNA)

  8. Unwinding of gene regions of a DNA molecule TRANSCRIPTION Pre mRNA Transcript Processing mRNA rRNA tRNA protein subunits Mature mRNA transcripts ribosomal subunits mature tRNA Convergence of RNAs TRANSLATION Cytoplasmic pools of amino acids, tRNAs, and ribosomal subunits Synthesis of a polypetide chain at binding sites for mRNA and tRNA on the surface of an intact ribosome FINAL PROTEIN Destined for use in cell or for transport

  9. VALINE VALINE PROLINE THREONINE LEUCINE HISTIDINE GLUTAMATE GLUTAMATE VALINE PROLINE THREONINE LEUCINE HISTIDINE GLUTAMATE

  10. mRNA transcribed from the DNA PART OF PARENTAL DNA TEMPLATE resulting amino acid sequence ARGININE GLYCINE TYROSINE TRYPTOPHAN ASPARAGINE ARGININE GLYCINE LEUCINE LEUCINE GLUTAMATE altered message in mRNA A BASE INSERTION (RED) IN DNA the altered amino acid sequence

  11. Overview: the roles of transcription and translation in the flow of genetic information

  12. The triplet code

  13. TRANSCRIPTION AND TRANSLATION • C DNA. ATC-GCG-TAT • m-RNA. UAG-CGC-AUA • t-RNA. AUC-GCG-UAU • AMINO ACID ISO-ALA-TYR • PEPTIDE BONDS/POLYPEPTIDES/PROTEINS

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

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

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

  17. 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.

  18. 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)

  19. 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.

  20. anticodon codon in mRNA anticodon amino acid attachment site amino acid OH tRNA MOLECULE amino acid attachment site

  21. The structure of transfer RNA (tRNA)

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

  23. 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.

  24. 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

  25. Ribosomes

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

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

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

  29. Translation • Initiation • The inactive 40S and 60S subunits will bind to each other with high affinity to form inactive complex unless kept apart • This is achieved by eIF3, which bind to the 40S subunit • mRNA forms an initiation complex with a ribosome • A number of initiation factors participate in the process.

  30. Translation • Cap sequence present at the 5’ end of the mRNA is recognized by eIF4 • Subsequently eIF3 is bound and cause the binding of small 40S subunit in the complexes • The 18S RNA present in the 40 S subunit is involved in binding the cap sequence • eIF2 binds GTP and initiation tRNA, which recognize the the start codon AUG • This complex is also bound to 40S subunit

  31. Translation • Driven by hydrolysis of ATP, 40S complex migrate down stream until it finds AUG start codon • The large 60S subunit is then bound to the 40S subunit • It is accompanied by the dissociation of several initiation factor and GDP • The formation of the initiation complex is now completed • Ribosome complex is able to translate

  32. Translation • Extrachromosomal mRNAs have no cap site • Plastid mRNA has a special ribosome binding site for the initial binding to the small subunit of the ribosome (shine-Dalgarno sequence) • This sequence is also found in bacterial mRNA, but it is not known in the mitochondria • In the prokaryotic, the initiation tRNA is loaded with N-formylmethionine • After peptide formation, the formyl residue is cleaved from the methionine

  33. 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

  34. Translation • Elongation • A ribosome contains two sites where the tRNAs can bind to the mRNA. • P (peptidyl) site allows the binding of the initiation tRNA to the AUG start codon. • The A (aminoacyl) site covers the second codon of the gene and the first is unoccupied • On the other side of the P site is the exit (E) site where empty tRNA is released

  35. Translation • Elongation • The elongation begins after the corresponding aminoacyl-tRNA occupies the A site by forming base pairs with the second codon • Two elongation factors (eEF) play an important role • eEF1 binds GTP and guides the corresponding aminoacyl-tRNA to the A site, during which GTP is hydrolized to GDP and P. • The cleavage of the energy-rich anhydride bond in GTP enables the aminoacyl-tRNA to bind to codon at the A site

  36. Translation • Elongation • Afterwards the GDP still bound to eEF1, is exchange for GTP as mediated by the eEF1 • The eEF1 -GTP is now ready for the next cycle • Subsequently a peptide linkage is form between the carboxyl group of methionine and the amino group of amino acid of the tRNA bound to A site • Peptidyl transferase catalyzing the reaction. It facilitates the N-nucleophilic attack on the carboxyl group, whereby the peptide bond is formed with the released of water

  37. Translation • Elongation • Accompanied by the hydrolysis of one molecule GTP to form GDP and P, the eEF2 facilitates the translocation of the ribosome along the mRNA to three bases downstream • Free tRNA arrives at site E is released, and tRNA loaded with the peptide now occupies the P Site • The third aminoacyl-tRNA binds to the vacant A site and a further elongation cycle can begin

  38. 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

  39. 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

  40. 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

  41. 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

  42. 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

  43. 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

  44. 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

  45. Translation • Release • When A site finally binds to a stop codon (UGA, UAG, UAA) • Stop codons bind eRF accompanied by hydrolysis GTP to form GDP and P • Binding of eRF to the stop codon alters the specificity the peptidyl transferase • Water instead amino acid is now the acceptor for the peptide chain • Protein released from the tRNA

  46. Ribosome Amino Acids forming Peptide chain P Site A Site E Site tRNA anti-codon AUG GGA codon Translation Met His Tyr Val Pro 3’ CAU UAC GUA CCU 5’ mRNA strand

  47. Translation • The difference • Eukaryotic and prokaryotic translation can react differently to certain antibiotics Puromycin an analog tRNA and a general inhibitor of protein synthesis  Cycloheximide only inhibits protein synthesis by eukaryotic ribosomes  Chloramphenicol, Tetracycline, Streptomycin inhibit protein synthesis by prokaryotic ribosome

  48. 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.

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