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Lectures 33-34GENETIC CODE and PROTEIN SYNTHESIS

Mukund Modak, Ph.D.

. Adapted from M. Mathews, Ph.D.

LecLtures 33 and 34

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Proteins are important…

~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 healthprotein 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.

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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

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POLYSOMES

E.M.

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The central dogma states that once “information” has passedinto protein it cannot get out again. The transfer of informationfrom 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 nucleicacid or of amino acid residues in the protein.

Francis Crick, 1958

DNA RNA PROTEINCENTRAL DOGMA

N- or amino-terminus

C- or carboxy-terminus

5’ 3’RNA

protein

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

C terminus ]

Not so in Eukaryotes

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UCAG

PhePheLeuLeu

SerSerSerSer

TyrTyr

STOPSTOP

CysCys

STOPTrp

LeuLeuLeuLeu

ProProProPro

HisHIsGlnGln

ArgArgArgArg

IleIleIle

Met

ThrThrThrThr

AsnAsnLysLys

SerSerArgArg

ValValValVal

AlaAlaAlaAla

AspAspGluGlu

GlyGlyGlyGly

1st position(5’ end)

2nd position 3rd position(3’ end)U C A G

UCAG

UCAG

UCAG

UCAG

GENETICCODE

NormalHb – β

Sickle cellHb – βS

CCUPro

GAGGlu

GAGGlu

CCUPro

GAGGlu

GUGVal

5 6 7codon #

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Degenerate (or redundant)

GENETIC CODE:

Nearly universal – variations in mitochondria, mycoplasma, ciliates

Unpunctuated – although some codons are signals

Non-overlapping

Co-linear triplet code

Mutations - in coding region can cause various ill-effects, such as, change in desired amino acids, early or late stop, insertion, etc.

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I. INTRODUCTION

II. TRANSLATIONAL MACHINERY Ribosomes: prokaryotic / eukaryotic

Messenger RNATransfer RNAAminoacyl-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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1. Ribosomes (large and small subunits)

2. Messenger RNA (mRNA)

3. Transfer RNAs (tRNAs)

4. Amino Acids (aa’s)

5. Enzymes (“factors”)

6. Energy (ATP, GTP)

TRANSLATIONAL COMPONENTS

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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 protuberancePT: Peptidyl tranferase centerRed, yellow, etc.: rRNABlue: Ribosomal proteinsWhite: Nascent polypeptide

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2. mRNAEukaryotic:

cap only 1 AAA ~150

5’ UTR

7-MeGpppGXY

3’ UTR poly A5’ end

3’ endMonocistronic (spliced)

5’ 3’ppp

( >1 coding region )PolycistronicProkaryotic:

# 1 # 2 # 3

( 1 coding region )

Cistron = coding region =open reading frame (ORF)

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3. tRNATranslational Adaptor

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AA + tRNA + ATP

Overall free energy change for aminoacylation of tRNA

AA ~tRNA + AMP + PPi

PPi + H2O 2 Pi

G = -6.6 Kcal/mole

G ~ -6.6 Kcal/mole

G ~0 Kcal/mole(1)

(2)

tRNAs carry “activated” amino acids:

aaRS

PPase

aaRS = aminoacyl-tRNA synthetasePPase = pyrophosphatase

4. Amino Acids

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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

61 Codons for AA’s

20 AA’s

Translation Machinery20

~50

AA – tRNA synthases ( i.e., 1 per AA )

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

CODONmRNA

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1 2 35’

5’ANTI-CODON

tRNA3’

“WOBBLE” Pairing

GAA GAG 2 codons

anti-codon stem-loopof tRNA

Wobble Positione.g. CUU 1 anti–codon

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2 tRNAs for AUG / Methionine: 2 different functions

N-formylin bacteria:

F-Met

InitiationCodon

InternalMet Codon

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5’ 3’AUG AUGUAC UAC

CCA

Met

CCA

Met

5’ 5’3’ 3’

Met – tRNA F or I Met – tRNA M

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Translation Step

Charging of tRNA

1. Initiation

2. Elongation

3. Termination

Modifications, cleavage, etc.

EnzymesProkaryotes Eukaryotes

Aminoacyl – tRNA synthetases

IF1- IF3 eIF1- eIF5 (multiple)

EF1, EF2 eEF1, eEF2

RF1- RF3 eRF1, eRF3

5. Translation Factors

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I. INTRODUCTION

II. TRANSLATIONAL MACHINERY III. MECHANISM OF TRANSLATION AND INHIBITORS OF

PROTEIN SYNTHESISInitiation

ElongationTerminationAntibioticsToxins

IV. ENERGETICS AND REGULATION OF TRANSLATION

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2. Internal ribosome entry

AUG..

HOW RIBOSOMES FIND THEIR INITIATION SITES

1. Cap - dependent scanning

cap AUG... AUG..

Shine - Dalgarno box

40S

40S

30S

S - D

eukaryotes prokaryotes

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

16S rRNA

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

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30S ribosomal subunit initiation at S-D sequence

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2. Internal ribosome entry

AUG...

HOW RIBOSOMES FIND THEIR INITIATION SITES

1. Cap - dependent scanning

cap AUG.. AUG..

Shine - Dalgarno box

40S

40S

30S

S - D

STREPTOMYCIN

eukaryotes prokaryotes

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

Streptomycin, Gentamycin, Tobramycin, Amikacin, etc.

are aminoglycosides. They also cause

miscoding during elongation

16S rRNA

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CLINDAMYCINMacrolides e.g.

ERYTHROMYCIN

TETRACYCLINESSPECTINOMYCIN

AA – tRNAbinding EF 1A, 1B (EF-Tu,

Ts)[eEF 1α, eEF1βγ ]

PeptidylTransfer

Peptidyltransferase (50S / 60S)

TranslocationEF2

[eEF2]

DIPHTHERIATOXIN

PUROMYCINCHLORAMPHENICOL

ELONGATION

RICIN -SARCIN

GTP

P Site

E Site

A Site

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Puromycin

Tyrosinyl-tRNA

Puromycin imitates AA-tRNA

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1) Diphtheria toxin inactivates eEF22) Erythromycin inhibits EF2

Inhibition of ribosome translocation

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stop codonsUAGUAAUGA

RF 1,2,3[eRF1,3]

Termination &

Release

TERMINATION

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ENERGETICS OF PROTEIN SYNTHESIS

1. Charging

2. InitiationUnwinding and scanningMet-tRNAi binding

3. Elongation AA-tRNA bindingTranslocation

4. Termination

ATP, 2~

ATP (several), 1~GTP, 1~

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

GTP (number unknown), 1~

TOTAL: 4~ per AA polymerized + initiation + termination

> 1200~ for an average protein

Compared to 36-38 ATP’s generated by Glucose CO2

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Down-regulation of the supply of initiator Met-tRNAi via eIF2

eIF2B

eIF2 • GDP

eIF2 • GTP

eIF2 • GTP • Met-tRNAi

PROTEINSYNTHESIS

eIF2 supplies Met- tRNAi to 40S subunit

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

eIF2B

eIF2 • GDP

eIF2 • GTP

eIF2 • GTP • Met-tRNAi

PROTEINSYNTHESIS

eIF2 supplies Met- tRNAi to 40S subuniteIF2 phosphorylation inhibits initiation

kinaseseIF2

eIF2B eIF2

Trapped eIF2B

INITIATION INHIBITED

P

P

eIF2 kinasesHRI: reticulocytes minus hemePKR: interferon plus virus- infection (dsRNA)PERK: ER stressGCN2: amino acid starvation

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EF-Ts

EF-Tu • GDP

EF-Tu • GTP

EF-Tu • GTP • aa-tRNA

PROTEINSYNTHESIS

GTP/GDP exchange during elongation by (e)EF1 (aka EF-Tu)

Terminology

PROK. EUK.Old New

Tu 1A 1α

Ts 1B 1βγ

aa-tRNA complex

GEF

This factor supplies aa- tRNA to ribosome during elongation.

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CYTOPLASM cytosolicprotein

“free” polysome

endoplasmicreticulumlumen

secretedprotein

membrane-boundpolysome on “rough” ER

nuclear membrane

cell membrane

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Target30S

30S50S50S

50SIle-tRNA synthase

50S, 60S

80SeEF260S60S

Action(1) Inhibits initiation(2) Causes misreadingInhibits binding of AA-tRNA to A-siteInhibits peptidyl transferaseInhibit translocation

Inhibit translocationInhibits isoleucine tRNA charging

Premature release of nascentpolypeptide

Inhibits translocationInhibits translocation¤Inhibits binding of AA-tRNA to A-site♦Inhibits binding of AA-tRNA to A-site

& translocation#

InhibitorSTREPTOMYCIN, Gentamicin, Kanamycin, Neomycin, etc.TETRACYCLINE, doxycyclineCHLORAMPHENICOLERYTHROMYCIN, Clarithromycin, AzithromycinClindamycin, LincomycinMupirocin (pseudomonic acid)

PUROMYCIN

CycloheximideDIPHTHERIA TOXINRICIN (castor beans)-Sarcin (fungus)

Aminoglycosides

Tetracylines

Macrolides

Lincosamides

Class

Inhibitors of Protein Synthesis:Antibiotics and Toxins

Catalytic activities of toxins¤ ADP ribosylation♦ 28S rRNA depurination (A)# 28S rRNA cleavageCAPITALIZED: most important

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NucleusTranscription & translation

mRNA

Ribosomes

Initiator

Site selection

PROKARYOTES EUKARYOTES

NoCoupled

Polycistronic

70S (50S, 30S)

f Met – tRNAi

Shine-Dalgarno mediated internal initiation

YesSeparated

Monocistronic, Capped & Polyadenylated80S (60S, 40S)

Met – tRNAi

1) Scanning2) IRES mediated internal

entry

Initiation factors

Order of events

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1) mRNA binding2) f Met – tRNAi binding

>12

1) Met – tRNAi binding2) mRNA binding

Antibiotics Sensitive Resistant

Toxins Resistant Sensitive

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Protein Modifications

1.Phosphorylation - (Tyr, Ser,Threo) Metabolic Regulation, Signal transduction, etc2.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