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1
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
123
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
1
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
29
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.
32
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
3
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