Protein

BI 270 CELL BIOLOGY Part III

POLYPEPTIDE SYNTHESIS Ž TRANSLATION
EVENTS in PROKARYOTES

I. Amino Acid Activation:

Aminoacyl-tRNA Synthetases [ARS] Ž 1-4 subunits [a and/or b]
        tri-functional active site
        varying sequences Ž different 3-D Ž different amino acid
        vs. conserved sequences for specific amino acid

AA + ATP + Mg⁺⁺ + (specific) ARS
Ţ {Aminoacyl Adenylate + Synthetase} + PPi
Ţ {Aminoacyl-tRNA} + Synthetase + AMP

II. Formation of the Initiation Complex:

{mRNA + soluble Initiation Factors + NfMet-tRNA + small Ribosome su}

IF3 - needed to insure 30S su binds to mRNA at the AUG Initiator codon

Temporary H-bonds between 3’ rRNA and 5’ mRNA sequences
NfMet-tRNA binds to AUG in the PEPTIDE or P site

	IF2 - binds NfMet-tRNA + GTP (to provide energy for ribosome su joining) î "BINARY COMPLEX" ["ternary complex"]
	IF1 - may promote dissociation of ribosomal su OR aid IF3 in Initiation Complex [role uncertain]
	NOTE: more than 10 eIFs in eukaryotes
	See Initiation Complex Diagram

III. Chain Initiation

INITIATOR AMINOACYL-tRNA has anticodon UAC

50S su binds to complex Ţ IFs are released See Animation
Mg++ "SALT BRIDGES" hold ribosome su together and activate specific enzymes
2^nd aminoacyl-tRNA enters AMINO ACID or A site

IV. Chain Elongation

Elongation Factors and GTP
        soluble EF-Tu (Temperature Unstable) Tu-GTP-AA-tRNA
        soluble EF-Ts (Temperature Stable) to RECYCLE EF-Tu

        Peptidyl Synthetase (Transferase) is NON-SOLUBLE

formation of PEPTIDE BONDS

part of LARGE subunit

enzymatic RNA

EF-G = Translocase (GTP used)
E site (EMPTY) č tRNA with no amino acid
Exit Channel
The universal Genetic Code
Crick : The Wobble Hypothesis

V. Chain Termination

Release Factors (when A site is empty due to ??)
Ţ release of polypeptide chain and final tRNA

R1 ž UAA, UAG

R2 ž UAA, UGA

R3 ž activates R1 & R2

use of GTP

Tetrahymena and Paramecium Ţ UGA !

VI. Ribosome Dissociation

Factor S = IF3 ! 1 ATP + 2 GTP per Amino Acid Polysomes

See Protein Synthesis overview

See more Protein Synthesis Info here

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PROTEIN STRUCTURE

NH₂

I

Hž
Cž COOH

I

R

    Amino acids
   Asymmetric Carbon
   D vs. L forms
   Amino and Carboxyl groups

PEPTIDE BOND
Dehydration Synthesis = Condensation Reaction

CONJUGATED PROTEINS
NUCLEO- GLYCO- LIPO- and CHROMOPROTEINS

Primary Structure = ??

SANGER et al. Ž sequencing of INSULIN

See INSULIN here

Find out about SANGER here

Secondary Structure Ţ H-bonds

a - helix, b - pleated sheet, etc.

Tertiary Structure = R group interactions More on Tertiary Structure

ELECTROSTATIC FORCES (Ionic Bonds)
H-BONDS
HYDROPHOBIC BONDS (van der Waals forces)
DISULFIDE BONDS -S-S- (Covalent Bonds)

Quaternary Structure = Multiple Polypeptide Chains
e.g. Insulin, Antibodies, Hemoglobin, etc.

CHAPERONE PROTEINS and efficient folding

PROTEIN DOMAINS, EXONS and the EVOLUTION of NEW PROTEINS

DENATURATION = ?? Reversible vs. Irreversible

	ANFINSEN works with RIBONUCLEASE (which is a protein!)
	124 amino acids; ONE polypeptide chain
	-S-S- contributes to 3-D
	Mercaptoethanol and Urea Urea č DENATURATION č PROVES…??

ARTIFICIAL SYNTHESIS (one amino acid at a time) č PROVES…??

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ENZYMES

I. MONOMOLECULAR – Isomerases [mutases]

II. BIMOLECULAR –

	Oxidoreductases – oxidases, dehydrogenases, etc.
	Transferases – e.g. KINASES = ??
	Hydrolases – proteinases, nucleases, etc.
	Decarboxylases – removal of … ??
	Synthetases – POLYMERASES, LIGASES, etc.

MULTI-ENZYME COMPLEXES increase the efficiency of metabolic pathways
e.g. - PYRUVATE DEHYDROGENASE

ENZYME-SUBSTRATE COMPLEX
ACTIVE SITE Ţ 3-D !!
ENERGY of ACTIVATION Ż
Koshland : Induced Fit Model See Induced Fit Illustration
Blake and LYSOZYME
COMPETITIVE INHIBITION vs. ALLOSTERIC INHIBITION
Allosteric Activation:
a) inorganic Co-factors
b) organic Co-enzymes
c) Prosthetic groups

CONSTITUTIVE vs. INDUCIBLE ENZYMES [GENES]

ISOENZYMES or ISOZYMES
Lactate Dehydrogenase variations: M₄, M₃H, M₂H₂, MH₃, H₄
EMBRYONIC Ţ M₄
HEART Ţ H₄
See the ENZYME here

COOH	NADH + H⁺	NAD⁺	COOH
ę	ë	ě	ę
C=O	Źžžžžž	žžžžžŽ	HCOH
ę	LACTATE	DEHYDROGENASE	ę
CH₃			CH₃

ZYMOGENS [proenzymes] e.g. – pepsinogen pepsin

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LIPIDS LIPIDS LIPIDS

	FATTY ACID CHAINS: saturated vs. unsaturated [cis vs. trans]
	NEUTRAL FATS: (non-polar) Ţ triglycerides and glycerol esters
	PHOSPHOLIPIDS: amphipathic Ţ hydrophilic and hydrophobic regions
	Phosphatidic Acid derivatives, e.g. lecithin = phosphatidyl choline
	Non-Glycerol Derivatives: e.g. cholesterol
	LIPID ASYMMETRY: e.g. phosphatidyl serine
	LIPID MOBILITY: lateral vs. "flip-flop" mobility Boundary Lipids Gall & Edelman - lipid immobilization
	Fluidity :TEMPERATURE, SATURATION & CHOLESTEROL

	HIRATA & AXELROD Ţ "Flip-Flop" mobility and methyltransferase
	Ca++ influx	Lysophophatidyl choline
	Arachidonic acid	Prostaglandins
	release of cAMP	Histamine (MAST CELLS)
	Lymphocyte mitogenesis	Neutrophil chemotaxis

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