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Biochemistry I (CHE 418 / 5418 )

Biochemistry I (CHE 418 / 5418 ). Reading Assignment Berg et. al (2007) Chapter 30. Central Dogma of Molecular Biology. Replication  DNA  RNA  Protein mRNA. tRNA, rRNA Replication - DNA directed DNA synthesis Transcription - DNA directed RNA synthesis

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Biochemistry I (CHE 418 / 5418 )

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  1. Biochemistry I(CHE 418 / 5418) Reading Assignment Berg et. al (2007) Chapter 30

  2. Central Dogma of Molecular Biology • Replication •  • DNA  RNA  Protein • mRNA. tRNA, rRNA • Replication - DNA directed DNA synthesis • Transcription - DNA directed RNA synthesis • Translation - RNA directed Protein synthesis Transcription Translation

  3. Translation RNA directed protein synthesis • Four letters of nucleic acid language translated into 20 amino acids of protein language. • mRNA, tRNA, ribosome (rRNA) • Occurs on ribosome. • Protein synthesized in N (amino) to C (carboxyl) direction • mRNA read from 5’ to 3’ N N N N 3’ 5’

  4. Translation Divided into 3 Stages Initiation Ribosome binds mRNA identifies start codon (AUG). * Met in Eukaryotes * fMet N-formylmethionine in prokaryotes Elongation Protein synthesized N to C Termination Polypeptide released upon encountering stop codons (UAA, UAG, UGA)

  5. During Translation Ribosome binds: • mRNA • Contains codon – three nucleotide sequence encoding one amino acid. • tRNAs (three sites) • Contains Anticodon – three nucleotide sequence complementary to codon. • Codon and Anticodon interact by hydrogen bonding between complementary bases.

  6. Ribosome contains three tRNA binding sites • E – Exit • Uncharged tRNA leaves ribosome. • P – Peptidyl • tRNA in this site has protein covalently attached. • A – Aminoacyl • Charged tRNA enters. H-bonding established between codon (mRNA) and anticodon of (tRNA). Sites include regions of large and small subunits of ribosome.

  7. Reaction to Add Amino Acids • Amino acids are added to the C terminus by transferring existing chain to next amino acid.

  8. Polysome (Polyribosome) • Polysome -multiple ribosomes bond to the same mRNA. • Each ribosome in a polysome is synthesizing the same protein. • In Prokayotes, transcription and translation may be coupled, since both are occurring in the same compartment at the same time.

  9. tRNA carry amino acids • Amino acid (C terminus) attached via an ester to 2’or 3’ hydroxyl of A of CCA on tRNA. • “Charged tRNA” – tRNA with correct amino acid attached. • Aminoacyl tRNA synthetases – class of enzymes that attach amino acids to tRNA.

  10. Aminoacyl tRNA synthetases • Enzyme that attaches correct amino acid to CCA of tRNA with high fidelity. (>10-4). • Binds Activation site and anticodon. • Two classes • Class I • Class II

  11. Two Classes of Aminoacyl tRNA synthetases • Two classes differ in: • Sequence alignment / comparison. • 10 tRNA per class. • Binding to tRNA on different “face” or side • Attaching amino acid to different hydroxyl • Class 1 to 2’ OH • Class 2 to 3 ‘ OH • Binding ATP in different comformation • Subunit comformation • Class 1 are monomers • Class II are dimers

  12. Aminoacyl tRNA synthetases reaction • Two step reaction • 1. AA + ATP ↔ Aminoacyl-AMP + PPi • Amino acid attached to AMP via mixed anhydride which conserves the energy of phosphodieaster bond in ATP. • Acyl adenylate • 2. Amino acid transferred to tRNA. • Ester linkage to 2’ or 3’ hydroxyl of A of CCA on 3’ end of tRNA.

  13. Threonyl-tRNA synthetase An example of Fidelity • How does Threonyl-tRNA synthetase select between similarly shaped amino acids? • Thr has almost the same shape as Ser and Val. • 1. Active site selection • 2. Editing site

  14. Threonyl-tRNA synthetase An example of Fidelity (Cont’) • 1. Active site selection by selective binding of substrate. • Zn 2+ • Coordinated to enzyme by 2 his and 1 cys residues • Binds Thr by amino group and OH of side chain • Asp of enzyme • Binds OH of thr substrate. • This interaction prohibits attachment of Val.

  15. Threonyl-tRNA synthetase An example of Fidelity (Cont’) • 2. Editing site • Removes Mischarged amino acids (Ser). • CCA with amino acid attached swings into editing site. Product does not dissociate before editing. • Increasing fidelity to > 10-4 NOT ALL AminoacyltRNAsynthetasesrequire editing.

  16. Codon hydrogen bonds to Anticodon • Codon of mRNA and Anticodon of tRNA are antiparellel.

  17. Wobble • Third base of anticodon (5’ end) allows binding with multiple bases.

  18. Codon Usage Table • mRNA are “read” three nucleotides (codon) at a time starting from a fixed point.

  19. The Genetic code is unambiguous, degenerate, non-overlapping and universal • Unambiguous • A given codon either designates a single amino acid or is a stop codon. • Degenerate • more than one codon can specify the same amino acid, so the genetic code is said to be degenerate. • Non-overlapping • the code is read sequentially, one codon after another without spacer bases, from a fixed starting point. • Universal

  20. Initiation (Prokaryotic) • Prokaryotic start codon = AUG. • Others exist (rare) • GUG = Val • UUG = Leu • AUG encodes fMET • Two important interactions • 1. Binding of mRNA to 3’ end of 16S rRNA. • 2. H bonding of initiator codon with anticodon. • tRNAf binds to P site of ribosome.

  21. Elongation (Prokaryotic) • P site is occupied by tRNA holding fMet, or later the growing polypeptide • Elongation factors bring the correct charged tRNA to the A site. • EF-Tu – brings charged tRNA to A site of ribosome. Requires bond GTP (hydrolyzed to GDP during reaction). GTP binds to p-loop. • EF-Ts – removes GDP from EF-Tu and replaces with GTP • The 23 S rRNA of the ribosome catalyzes the attachment of amino acids. • Translocation is accelerated by EF-G.

  22. Elongation (Prokaryotic)

  23. Termination (Prokaryotic) • Termination aided by RF = release factors.

  24. Castor Oil • What is Castor Oil? • Thick, yellowish or almost colorless oil extracted from castor bean (seed). • Uses of Castor Oil • Laxative • Lubricating oil • Quick-drying oil (when dehydrated) • used in paints and varnishes. • Competes with linseed and tung oil • Coating fabrics /protective coverings • Sebacic acid - basic ingredient in the production of nylon 6, 10 and other synthetic resins / fibers

  25. http://waynesword.palomar.edu/plmar99.htm#flow http://www.ansci.cornell.edu/plants/alphalist.html Castor Bean • Ricinus communis • (family Euphorbiaceae) • Source of Castor Oil

  26. Synthesis of Nylon 6, 10 • Condensation Polymerization • Sebacic acid + 1, 6-hexanediamine • Sebacic acid • 10-carbon dicarboxylic acid • carboxylic group (COOH) at each end of the molecule • 1,6-hexanediamine • 6-carbon molecule with an amino group (C-NH2) at each end. • Free carboxylic acid of Sebacic Acid reacts with amino group of 1, 6-hexamediamine • water molecule is produced at each link.

  27. What is Ricin? • Protein (64 kda) found in pulp from production of castor oil. • Heterodimer • A chain • 267 amino acid residues. • Inhibits protein synthesis. • B-chain • 262 amino acid residues. • facilitates transport into cell. • Connected by disulfide bridge.

  28. Mode of Action of Ricin • A chain • N-glycosidase which removes specific adenine (depurinates) 28 S ribosomal RNA • A-4323 in rat liver • prevents the binding of an elongation factor, leaving the ribosome incapable of protein synthesis. • Extremely toxic • 70 micrograms (equivalent to weight of grain of salt) will kill a 160 pound man. • Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals (1997)

  29. Many Antibiotics and Toxins Inhibit Translation

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