PROTEIN SYNTHESIS GENETIC CODE and

Proteins are important Bcos They are

~44% of the dry wt. of the human body. ~5% of human caloric intake goes for protein synthesis.

catalyze most of the reactions in living organisms.

serve many roles (enzymatic, structural, transport, regulation, ...)

in sickness and in health

protein synthesis is tightly regulated by environmental stimuli as well as intrinsic processes (e.g., hormonal, developmental). dysregulation can cause disease. many antibiotics act at the level of protein synthesis.

I.

INTRODUCTION Central Dogma Ribosomes and polysomes Genetic Code Mutations with effects at the translation level

II. TRANSLATIONAL MACHINERY III. MECHANISM OF TRANSLATION AND INHIBITORS OF PROTEIN SYNTHESIS IV. ENERGETICS AND REGULATION OF TRANSLATION

POLYSOMES

E.M.

CENTRAL DOGMA
DNA RNA PROTEIN
The central dogma states that once information has passed into protein it cannot get out again. The transfer of information from nucleic acid to nucleic acid, or from nucleic acid to protein, may be possible, but transfer from protein to protein, or from protein to nucleic acid is impossible. Information means here the precise determination of sequence, either of bases in the nucleic acid or of amino acid residues in the protein. Francis Crick, 1958

5
N- or aminoterminus

RNA

3
C- or carboxyterminus
5

protein

Coupled transcription & translation in bacteria [ N terminus to [ 5 to 3 ] C terminus ]

Not so in Eukaryotes
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1st position (5 end)

2nd position

U
Phe Phe Leu Leu Leu Leu Leu Leu Ile Ile Ile Met Val Val Val Val

C
Ser Ser Ser Ser Pro Pro Pro Pro Thr Thr Thr Thr Ala Ala Ala Ala

A
Tyr Tyr STOP STOP His HIs Gln Gln Asn Asn Lys Lys Asp Asp Glu Glu

G
Cys Cys STOP Trp Arg Arg Arg Arg Ser Ser Arg Arg Gly Gly Gly Gly

3rd position (3 end)

GENETIC CODE

U C A G

U C A G U C A G U C A G U C A G

codon # 5 6 7 CCU GAG GAG

Pro Glu Glu

Normal Hb Sickle cell Hb S

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CCU GUG GAG

Pro Val Glu

GENETIC CODE:
Co-linear triplet code Nearly universal variations in mitochondria, mycoplasma, ciliates Degenerate (or redundant) Non-overlapping Unpunctuated although some codons are signals Mutations - in coding region can cause various ill-effects, such as, change in desired amino acids, early or late stop, insertion, etc.

I.

INTRODUCTION

II. TRANSLATIONAL MACHINERY Ribosomes: prokaryotic / eukaryotic Messenger RNA Transfer RNA Aminoacyl-tRNA synthetases; Met-tRNA forms (m, f, i) Initiation, elongation and termination enzymes III. MECHANISM OF TRANSLATION AND INHIBITORS OF PROTEIN SYNTHESIS IV. ENERGETICS AND REGULATION OF TRANSLATION
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TRANSLATIONAL COMPONENTS
1. Ribosomes (large and small subunits) 2. Messenger RNA (mRNA) 3. Transfer RNAs (tRNAs) 4. Amino Acids (aas) 5. Enzymes (factors) 6. Energy (ATP, GTP)

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1. Ribosome Structure

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Section through 50S ribosomal subunit

Peptidyl transferase is RNA Polypeptide exit tunnel is 40~50 aa long

C: Central protuberance PT: Peptidyl tranferase center Red, yellow, etc.: rRNA Blue: Ribosomal proteins White: Nascent polypeptide

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2. mRNA
Eukaryotic: Monocistronic (spliced)
5 end
cap 7-MeGpppGXY ( 1 coding region ) 5 UTR only 1 3 UTR 3 end poly A AAA ~150

Cistron = coding region = open reading frame (ORF)

Prokaryotic: Polycistronic
5 ppp #1 ( >1 coding region ) #2 #3
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3. tRNA
Translational Adaptor

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4. Amino Acids
tRNAs carry activated amino acids:
aaRS

(1) AA + tRNA + ATP

PPase

AA ~tRNA + AMP + PPi G ~0 Kcal/mole 2 Pi G = -6.6 Kcal/mole

(2)

PPi + H2O

Overall free energy change for aminoacylation of tRNA G ~ -6.6 Kcal/mole

aaRS = aminoacyl-tRNA synthetase PPase = pyrophosphatase

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Formation of aminoacyl-tRNA
The amino acid is first activated by reacting with ATP

The activated amino acid is transferred from aminoacyl-AMP to tRNA

These enzymes are vital for the fidelity of protein synthesis: 2 steps allow proofreading

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Genetic Code 20 AAs 61

Codons for AAs

Translation Machinery 20 AA tRNA synthases

( i.e., 1 per AA )

~50

tRNA species (at least 1 per AA, but less than 1 per codon)

WOBBLE Pairing
anti-codon stem-loop of tRNA

Wobble Position e.g. CUU 1 anticodon tRNA GAA GAG 2 codons

5
ANTI-CODON

3 2 1 5 mRNA 1 2 3 CODON

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2 tRNAs for AUG / Methionine: 2 different functions

N-formyl in bacteria: F-Met

Met

Met CCA Met tRNA M

CCA

Met tRNA F or I

3 5

UAC AUG 1

UAC AUG

5 3

Initiation Codon

Internal Met Codon

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5. Translation Factors
Translation Step Enzymes
Prokaryotes Eukaryotes

Charging of tRNA

Aminoacyl tRNA synthetases

1. Initiation

IF1- IF3

eIF1- eIF5 (multiple)

2. Elongation

EF1, EF2

eEF1, eEF2

3. Termination

RF1- RF3

eRF1, eRF3

Modifications, cleavage, etc.

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I.

INTRODUCTION

II. TRANSLATIONAL MACHINERY III. MECHANISM OF TRANSLATION AND INHIBITORS OF PROTEIN SYNTHESIS Initiation Elongation Termination Antibiotics Toxins IV. ENERGETICS AND REGULATION OF TRANSLATION
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HOW RIBOSOMES FIND THEIR INITIATION SITES

eukaryotes
1. Cap - dependent scanning
40S
16S rRNA

prokaryotes

30S

cap

AUG...

S-D

AUG..

Shine - Dalgarno box

2. Internal ribosome entry

40S

AUG..

---------------IRES-----------

Next step: large subunit 50S/60S subunit joining

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30S ribosomal subunit initiation at S-D sequence

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HOW RIBOSOMES FIND THEIR INITIATION SITES

eukaryotes
1. Cap - dependent scanning
40S
16S rRNA

prokaryotes
STREPTOMYCIN
30S

cap

AUG..

S-D

AUG..

Shine - Dalgarno box

2. Internal ribosome entry

40S Streptomycin, Gentamycin, Tobramycin, Amikacin, etc. are aminoglycosides. They also cause miscoding during elongation

AUG...

---------------IRES-----------

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ELONGATION
P Site

A Site

AA tRNA binding

E Site

TETRACYCLINES SPECTINOMYCIN EF 1A, 1B (EF-Tu, Ts) [eEF 1, eEF1 ]

Peptidyl Transfer CLINDAMYCIN Macrolides e.g. ERYTHROMYCIN Translocation RICIN -SARCIN

GTP

PUROMYCIN CHLORAMPHENICOL Peptidyl transferase (50S / 60S)

EF2 [eEF2] DIPHTHERIA TOXIN

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Puromycin imitates AA-tRNA

Puromycin

Tyrosinyl-tRNA

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Inhibition of ribosome translocation

1) Diphtheria toxin inactivates eEF2 2) Erythromycin inhibits EF2

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TERMINATION

stop codons
UAG UAA UGA

Termination & Release

RF 1,2,3 [eRF1,3]

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ENERGETICS OF PROTEIN SYNTHESIS

1. 2. Charging Initiation Unwinding and scanning Met-tRNAi binding Elongation AA-tRNA binding Translocation ATP, 2~

ATP (several), 1~ GTP, 1~

3.

GTP, 1~ (see later) GTP, 1~

4.

Termination

GTP (number unknown), 1~

TOTAL: 4~ per AA polymerized + initiation + termination > 1200~ for an average protein Compared to 36-38 ATPs generated by Glucose CO2
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Down-regulation of the supply of initiator Met-tRNAi via eIF2

eIF2 GDP eIF2B eIF2 GTP

eIF2 GTP Met-tRNAi

PROTEIN SYNTHESIS

eIF2 supplies Met- tRNAi to 40S subunit

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Control : Down-regulation of the supply of initiator Met-tRNAi via eIF2 kinases

kinases eIF2

eIF2 kinases
P
HRI: reticulocytes minus heme PKR: interferon plus virusinfection (dsRNA) PERK: ER stress GCN2: amino acid starvation

eIF2 GDP eIF2B eIF2 GTP

eIF2B

eIF2

Trapped eIF2B eIF2 GTP Met-tRNAi INITIATION INHIBITED

PROTEIN SYNTHESIS

eIF2 supplies Met- tRNAi to 40S subunit eIF2 phosphorylation inhibits initiation
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GTP/GDP exchange during elongation by (e)EF1 (aka EF-Tu)

Terminology
EF-Tu GDP EF-Ts EF-Tu GTP

PROK. Old New aa-tRNA complex Tu 1A GEF Ts 1B

EUK. 1 1

EF-Tu GTP aa-tRNA

PROTEIN SYNTHESIS

This factor supplies aa- tRNA to ribosome during elongation.

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nuclear membrane

membrane-bound polysome on rough ER

endoplasmic reticulum lumen

secreted protein free polysome

CYTOPLASM

cytosolic protein cell membrane

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Inhibitors of Protein Synthesis: Antibiotics and Toxins

Inhibitor STREPTOMYCIN, Gentamicin, Kanamycin, Neomycin, etc. TETRACYCLINE, doxycycline CHLORAMPHENICOL ERYTHROMYCIN, Clarithromycin, Azithromycin Clindamycin, Lincomycin Mupirocin (pseudomonic acid) PUROMYCIN Class Aminoglycosides Tetracylines Macrolides Target 30S 30S 50S 50S Action (1) Inhibits initiation (2) Causes misreading Inhibits binding of AA-tRNA to A-site Inhibits peptidyl transferase Inhibit translocation

Lincosamides

50S Ile-tRNA synthase 50S, 60S

Inhibit translocation Inhibits isoleucine tRNA charging Premature release of nascent polypeptide

Cycloheximide DIPHTHERIA TOXIN RICIN (castor beans) -Sarcin (fungus)

80S eEF2 60S 60S

Inhibits translocation Inhibits translocation Inhibits binding of AA-tRNA to A-site Inhibits binding of AA-tRNA to A-site & translocation#

CAPITALIZED: most important

Catalytic activities of toxins ADP ribosylation 28S rRNA depurination (A) # 28S rRNA cleavage

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PROKARYOTES
Nucleus No Transcription & translation Coupled mRNA Polycistronic 70S (50S, 30S) f Met tRNAi Shine-Dalgarno mediated internal initiation

EUKARYOTES
Yes Separated Monocistronic, Capped & Polyadenylated 80S (60S, 40S) Met tRNAi 1) Scanning 2) IRES mediated internal entry >12 1) Met tRNAi binding 2) mRNA binding Resistant Sensitive
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Ribosomes
Initiator Site selection

Initiation factors Order of events

3 1) mRNA binding 2) f Met tRNAi binding Sensitive Resistant

Antibiotics Toxins

Protein Modifications
1. Phosphorylation - (Tyr, Ser,Threo) Metabolic Regulation, Signal transduction, etc 2. Hydroxylation - (Proline) in collagen, Endoplasmic Reticulum

3. Glycosylation (O-linked as with Ser/Threo- OH or N-Linked as in lysine) 4. Other - biotinilation, farnesyl, etc

Protein Degradation - Mostly thru specific proteases and ubiquitin-proteosome system

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