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 Translation - RNA directed Protein synthesis Transcription 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 5’5’ 3’

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) Translation Divided into 3 Stages

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.

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.

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

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.

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.

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

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

Aminoacyl tRNA synthetases reaction Two step reaction –1. AA + ATP ↔ Aminoacyl-AMP + PP i 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.

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

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.

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 > NOT ALL Aminoacyl tRNA synthetases require editing.

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

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

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

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

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. tRNA f binds to P site of ribosome.

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.

Elongation (Prokaryotic)

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

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

Castor Bean Ricinus communis –(family Euphorbiaceae) Source of Castor Oil

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- NH 2 ) at each end. –Free carboxylic acid of Sebacic Acid reacts with amino group of 1, 6- hexamediamine –water molecule is produced at each link.

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.

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)

Many Antibiotics and Toxins Inhibit Translation