ribosomal protein biosynthesis - vanderbilt university€¦ · ribosomal protein biosynthesis: dna...
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310
Ribosomal Protein Biosynthesis:
DNA RNA Protein (genome) (transcriptome) (proteome)
transcription translation
Transcription: only one of the DNA strands is copied (codingor antisense strand). It sequence is converted to thecomplementary sequence in mRNA (template or sense strand), which codes for the amino acid sequence of a protein (or peptide)
DNA
RNApolymerase pre-mRNA mRNA
rNTPs
“splicing”
311
Pre-mRNA:
mRNA:
5'-UTR 3'-UTR
Intron Intron
Exon Exon Exon
splicing
5'-UTR 3'-UTRExon Exon Exon
Transcription:
Translation: mRNAs are transported from the nucleus to the cytoplasm, where they acts as the template for protein biosynthesis (ribosomes). A three base segment of mRNA (codon) codes for a an amino acid.
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312
Transfer RNA (tRNA): The “anticodon” region of of tRNA is complementary to the mRNA codon sequence.
The t-RNA carries an amino acid on the 3’-terminal hydroxyl (A) (aminoacyl t-RNA) and the ribosome catalyzes amide bond formation.
Although single-stranded, the are complementary sequences within tRNA that give it a defined conformation
aminoacyl t-RNA
TψC loop
D loop
variable loop
313
There are many non-standard bases found in tRNAs
. . . and alternative base-pairingN
N
N
N
O
HR
N
N
NH
H
O R
I C
N
N
N
N
O
HR
N
N
O
O R
H
I-U wobble pair
N
N
N
N
O
HR
N
N
NH
H
N
N
R
I-A Wobble pair
N
N
N
H
H
N
N
R
N
N
O
O R
HN
N
N
N
O
HR
G-U Wobble pair
N H
H
N N
O
O
RH
A-U Hoogsteen pair
N
N
N
N
O
HR
N H
H
N
N
NH
H
O R
G-C reverse pairing
dihydrouridine (D)
O N
NHHO
HO OH
O
O
O
NH
HN
HO
HO OH
O
O
pseudouridine (!)
O NHO
HO OH
N
N
NH
O
inosine (I)
O NHO
HO OH
N
N
NH
O
NH2
CH3
7-methylguanosine
O NHO
HO OH
N
N
N
NH2
CH3
1-methyladenosine
O N
NHHO
HO
O
O
thymidine (T)
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314
N
NN
NN
R
NN
O
OR
HH
H
N
N
O
OH
R
Hoogsteen pairing
W-C pairing
N
NN
NN
R
NN
O
OR
HH
H
N
N
N
N
N
H
R
H
Hoogsteen pairing
W-C pairing
. . . and triple helix formation
U • A-U A • A-U
N
N
N
N O
N
N
N
N
O
H
H
R
R
H
H
H
N
N
N
N
O
N
R
H
H
H
W-C pairing
Hoogsteen pairing
G • G-C
315
tRNA Synthetase: catalyzes the biosynthesis of specific 3’-aminoacyl tRNAs from tRNAs, amino acids, and ATP
OOP
O
O
OP
O
O
OP
O
O
O
HO OH
N
N
N
N
NH2
O
OH2N
R
OH2N
RO
OP
O
O
O
HO OH
N
N
N
N
NH2
OOP
O
O
O
O OH
N
N
N
N
NH2
tRNA
H
B:
OOP
O
O
O
O OH
N
N
N
N
NH2
tRNA
OH2N
NH2
3’-aminoacyl tRNAs
Class I: 2’-aminoacyl tRNAsClass II: 3’-aminoacyl tRNAs
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316
Ribosomal protein synthesis
U G U AA U C U CG U U
3'5'
A CU
OO
H2N
SCH3
U G U AA U C U CG U U
3'5'
A CU
OO
H2N
SCH3
A site:Aminoacyl tRNA
U AA
OO
H2N
OH
U G U AA U C U CG U U
3'5'
U AA
OO
HN
OHOH2N
CH3S
A CU
OH
U G U AA U C U CG U U
3'5'
U AA
OO
HN
OHOH2N
CH3S
A CU
OH
E site:E site:exitexit
U G U AA U C U CG U U
3'5'
U AA
OO
HN
OHOH2N
CH3S
G AC
O O
CH3H2N
P site:Peptidyl t-RNA
Peptidyl t-RNA
317
Taken from: “The New Genetic Medicines,” J. S. Cohen, M. E. Hogan Scientific American 1994 (Dec.), pp 75-82.
Oligonucleotide-Base Therapies:Inhibition of Protein Biosynthesis via theAntisense-Antigene (Triplex) Strategies
• Potentially highly selective (magic bullet) approach to therapy• ~15 bases sequence is unique in the human genome
161
318
Watson-Crickbase-pairing
Hoogsteenbase-pairing
DNA mRNA
Triple Helix mRNA•DNA
transcription translationprotein
XInhibition oftranscription
X
Inhibition oftranslation
319
Triple Helix Motifs: third strand binds in the major groovePyrimidine•Purine-Pyrimidine: third strand runs parallel to the homo-purine strand
N
NN
NN
dR
NN
O
OdR
HH
H
N
N
O
OH
dR
N
N
N
N O
N
N
N
N
O
H
H
dR
dR
H
H
H
N
N N
O
H
H
H
dRT•A-T + C
+•G-C
Hoogsteen pairing
Hoogsteen pairing
W-C pairingW-C pairing
Purine•Purine-Pyrimidine: third strand runs antiparallel to the homo-purine strand
N
NN
NN
dR
NN
O
OdR
HH
H
N
N
N
N
N
H
dR
H
N
N
N
N O
N
N
N
N
O
H
H
dR
dR
H
H
H
N
N
N
N
O
N
dR
H
H
H
A•A-T G•G-C
Hoogsteen pairing
Hoogsteen pairing
W-C pairingW-C pairing
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320
DNA Triple Helix
Major groove
Minorgroove
T• A- T
C+• G- C
pdb code: 1BWG
321
Antisense Inhibition: inhibits mRNA function
Exon ExonIntronmRNA
splicing
ExonExonAUG UAA5'-cap
initiating sequence
stopsequence
Antisense targeting: Interon-exon splice junction: interferes with slicing 5’-cap (UTR) region: interferes with binding to the ribosome Initiation and promoter sequences: protein synthesis is not initiated Coding sequence: interferes with elongation of the protein
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322
When an antisense oligonucleotide binds to mRNA, ribonuclease H is up-regulated. The mRNA of themRNA•DNA hybrid is digested.
Problems with oligonucleotide-based therapiesTransport: DNA does not cross cellular membranes very
easilyDegradation: DNA is subject to enzymatic digestion by
cellular nuclease
Synthetic Oligonucleotides: must maintain affinity and selectivityand impart nuclease resistance
• backbone replacements • modified bases
323
O
O
HO B
O
HO
O B
P-O O
O
O
HO B
O
HO
O B
P-S O
O
HN
HO T
O
HO
HN T
CH2N
O
O
HO B
O
HO
O B
PMe O+
Phosphodiester Phosphorothioate Methyl Phosphonate Deoxyribonucleic Guanidines
O
HO
HO N
NH
O
O
H3C
O
HO
HO N
N
NH2
O
H3C
O
HO
HO N
N
O
NHX
X=OX=S
Antisense Nucleosides
Triple Helix Oligonucleotides
Peptide Nucleic Acids (PNAs)
N
H2N
OH
O
O
Base
n
m
Peptide backbone forms a helical structure.Base will hybridized with ss or ds DNA or RNA with high affinity
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324
microRNA (miRNA): genetically encoded (transcribed) but non-translated RNAs (do not code for a protein or peptide)
5'-(7-methyl-G)-
3'-(A)n-
Drosha
3'
5'~ 70 nt
pri-miRNA
pre-miRNA
transport to cytoplasmthen Dicer
3'
5'
ds RNA (21-25 nt)
3'
5'
RISC (RNAi silencing complex)
5' 3' miRNA
325
Ribonucleases (RNase): enzymes that catalyze the hydrolysis of the phosphodiester bonds of RNA
single strand specific: RNase Adouble strand specific: RNase IIIspecific for DNA/RNA hybrids: RNase H
endonucleases: RNase A, RNase IIIexonucleoase: RNase II
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326
B
O
OHO
O
PO
O
B
O
OO
O
H
NH
NHis
H
N NH
His
B
O
OHO
HO
PO
-O
B
O
OHO
O
O-
NH
NHisH
N NH
His
neutralization ofcharge makes thephosphate more triester like
B
O
OHO
O
PO
O
B
O
OO
O
H
NH
NHis
H
N NH
His
Lys NH3
B
O
OHO
O
PO-O
B
O
OO
O
HLysH3N
H
N NH
His
NH
NHis
N NH
His
B
O
OHO
O
PO--O
B
O
OO
O
LysH3N
H
NH
NHisH
B
O
OHO
HO
PO O-
B
O
O
O
O
N NH
His
H
NH
NHis
H
H
O
A mechanism for ribonuclease A
327
Interference RNA (RNAi)miRNAs (or siRNAs) are important in post-trancriptional regulation of gene expression
RISC (RNAi silencing complex)- multi-protein complex withhelicase and ribonuclease activity
Andrew Fire & Craig Mello, 2006 Nobel Prize in Medicine & PhysiologyAnimation: http://www.nature.com/focus/rnai/animations/index.html
3'
5' 3'
5'
ATP
5' 3'
5'3'
target mRNA
5'
target mRNA is cleaved by RISC
Ago2
5' 3'
guide strand21-25 nt
3'
mRNA fragements degraded by RNAses;protein expression is silenced
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328
Cellular Response to DNA Damage:
DNA Damage
Cell CycleArrest
DNARepair
Apoptosis Replication Errors
CellDeath
Mutations
Cancer
329
DNA Alkylating Agent
N
NHN
N
O
N
NHN
N
NH2
NH2
Nitrogen mustards will alkylate adjacent DNA bases causing DNA-DNA cross-links, which is a severe form of DNA damage and can trigger apoptosis or cause mutations (and cancer)
HN
R
O
N
Cl
Cl
Nitrogen Mustard
HN
R
O
N
Cl
+
aziridinium ionactive alylating agent
167
330
Other simple DNA alkylating agents:
Alkyl halides (or reactive equvalents): dimethylsulfatemethylene chloride, dichloroethane, SAM (endogenous)
Enals: acrolein (industrial chemical, cigarette smoke), 4-hydroxynon-2-enal, malondialdehyde (endogenous)
Epoxides: Butadiene diepoxide (metabolism of butadiene), chlorooxirane (metabolism of vinyl chloride), benzo[a]-pyrene diol epoxide (metabolism of benzo[a]pyrene)
331
Free-radical mediated oxidative damage to DNA
Reactive oxygen species (ROS)
O O
O O molecular oxygen
super oxide
H O
O O N O peroxynitrite
hydroxyl radical
Causes oxidative cleavage of DNA in an O2 dependent manner
The oxygen paradox: oxygen is necessary for cellular metabolism;however, oxygen is transformed into highly reactive speciesthat can damage biomolecules.
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332
Oxygen metabolism
Superoxide dismutase (SOD): Zn-Cu or Mn-Fe
Catalase (heme)H2O2 O2 + H2O
O2 + e O O
superoxide
HO O
pKa ~ 4.8
O O22 H+
H2O2 + O2k= 105 - 107 M-1 • sec-1
O O22 H
+
H2O2 + O2kcat= ~ 2 x 109 M-1 • sec-1
Enz•Cu(II)Enz•Cu(I)O2
+ H2O2
+ O2+
Enz•Cu(I) Enz•Cu(II)O2+
333
Fenton reactionO2 + Fe(III)
O2 + Fe(II)
H2O2 + Fe(II)HO + HO + Fe(III)
Redox cycling
Very little free Fe(III) in cells
Haber-Weiss reaction
O2 + H2O2 HO + HO + O2slow
ONO O
superoxide
+
nitricoxide
O
NOO
peroxynitrate
ONO2 + H+
pKa ~ 7.0 O
NOHO
peroxynitrate
peroxynitrous acid
HO + NO2
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334
N N
H2N
O
HN
NH2
NH2
O
H2N
O
HN
O
HOHN
O
O
N
NH
OO
O
OH
HO
HO
O
NH2O
OH
OH
HO
NH
O
S
N
S
N
NHO
NH
NH2
H2N+
Fe Binding Domain
DNA BindingDomain
Bleomycin
Binds Fe and activates O2 giving C4 hydrogen atom abstractionmechanism of hydrogen atom abstraction may involve aFe-O•, reminiscent of P450
335
Bleomycin•Co(III)OOH - DNA complex
Solution structure of the Bleomycin•Co(III) complex
Conformation of the DNA boundBleomycin•Co(III)OOH complex
pdb code: 1MXK
pdb code: 1DEY
170
336
Abstraction of the 4’-hydrogen:
OO
B
5'-DNA-O3P
O
P_ O O
PO3-DNA-3'
O
HHO
OO
B
5'-DNA-O3P
O
P_ O O
PO3-DNA-3'
O
O2 OO
B
5'-DNA-O3P
O
P_ O O
PO3-DNA-3'
O
OO
RSH
OO
B
5'-DNA-O3P
O
P_ O O
PO3-DNA-3'
O
OH
B:
OO
O
5'-DNA-O3P
O
P_ O O
PO3-DNA-3'
O
H
H:B
OO
O
5'-DNA-O3P
O _
P_ O O
PO3-DNA-3'
O
337
Abstraction of the 5’-hydrogen
OO
B
5'-DNA-O3P
O
P_ O O
PO3-DNA-3'
O
OH
OO
B
5'-DNA-O3P
O
P_ O O
PO3-DNA-3'
O
O2 OO
B
5'-DNA-O3P
O
P_ O O
PO3-DNA-3'
O
RSH
OO
B
5'-DNA-O3P
O
P_ O O
PO3-DNA-3'
O
HH
O
O
O
H:B
O
OH
B
5'-DNA-O3P
O
P_ O O
PO3-DNA-3'
O
O
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338
Abstraction of the 1’-hydrogen
OO
B
5'-DNA-O3P
O
P_ O O
3'-DNA-O3P
O
OH
OO
B
5'-DNA-O3P
O
P_ O O
3'-DNA-O3P
O
O2O
OB
5'-DNA-O3P
O
P_ O O
3'-DNA-O3P
O
RSH
OO
B
5'-DNA-O3P
O
P_ O O
3'-DNA-O3P
O
:BO _
P_ O O
3'-DNA-O3P
O
HO O
O H
:B
OO
5'-DNA-O3P
O
P_ O O
3'-DNA-O3P
O
O
H
H
OO
5'-DNA-O3P
O
339
Formation of 8-Oxo-2’-deoxyguanosine and FormamidinopyrimidineLesions by the reaction of deoxyguanosine with ROS’s
N
N N
NH
dR
O
NH2HO
N
N N
NH
dR
O
NH2
O
H
N
NH
O
NH2
N
O
HN
dR
HH
[H]
N
N N
NH
dR
O
NH2
O
H[O]
8-oxo-dG
formamidopyrimidine (FAPY)
H
-H+
+H+
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340
O
OH
OPO2PO2PO3-
N
N
N
O
H
H
O
OR
OR
N
NN
N O
N
H
H
H
O
H
O
OH
OPO2PO2PO3-
O
OR
OR
N
N
N
N
N
H
H H
O
O
N
NN
NN
H
H
H
Alternative base-pairing of 8-oxo-dG during replication with pol T7
mutagenic
pdb code: 1TK8
non-mutagenic
pdb code: 1TKD
8-oxo-dG
dA8-oxo-dG
dC
341
DNA as a target for carcinogen:Bioactivation of pro-carcinogens:
P450
O
H2O
OH
HO
P450
OH
HO
O
benzo[a]pyrene
O
OO
O
O
OCH3
H
H
aflatoxin B1
P450O
OO
O
O
OCH3
H
H
O
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342
O
OO
O
O
OCH3
H
H
O
N
HNN
N
O
H2N
N
HNN
N
O
H2N
O
O
O
OO
OCH3
H
H
HO
+t1/2 in H2O ~ 1 sec
O
OO
O
O
OCH3
H
H
O
OO
O
O
OCH3
H
H
OO
O
acetone
T. M. Harris et al. J. Am. Chem. Soc. 1988, 110, 7929
OH
HO
O
N
NN
N
NH2
N
N
N
N
OH
HO
HO
NH
343
Adducted Phosphoramidite Approach: Benzo[a]pyrene
O
OH
HO
N
N
N
NH
NH2
O
dRN
N
N
NH
O
dR
OH
HO
HO
NH
O
O
DMTrO N
N
N
NH
O
PO N(iPr)2
NC
OAc
AcO
AcO
NH
C G G A C A G A A G
N
N
N
NH
NH
O OH
OH
OH
solid-phaseoligonucleotide
synthesis
174
344Kim, S. J.; Stone, M. P.; Harris, C. M.; Harris, T. M. J. Am. Chem. Soc. 1992, 114, 5480
O
O
DMTrO N
N
N
N
X
O
PO N(iPr)2
NC
Si(CH3)3
OH
HO
HO
NH2
C G G A C A G A A G
N
N
N
NH
NH
OOH
OH
OH
C G G A C A G A A G
N
N
N
N
X
O(H3C)3Si
solid-phaseoligonucleotide
synthesis
1)
2) Deprotection
X= -F, -OTf
Post-Synthetic Modification Strategy for N2-2'-Deoxyguanosine Adducts
345
O
O
DMTrO N
N
N
N
X
PO N(iPr)2
NC
OH
HO
HO
NH2
C G G A C A G A A G
N
N
N
N
OH
HO
HO
C G G A C A G A A G
N
N
N
N
X
solid-phaseoligonucleotide
synthesis
1)
2) Deprotection
X= -F, -Cl
NH
Post-Oligomerization Strategyfor N6-2'-Deoxyadenosine Adducts
175
346
DNA-Carcinogen AdductsDNA-PAH Adducts: benzo[c]phenanthrene
pdb code: 1HX4 pdb code: 1HWV
N
N N
NH
NH
dR
O
HO
HO
OH
N
N N
NH
NH
dR
O
HO
HO
OH
347
DNA-Carcinogen AdductsDNA-PAH Adducts: benzo[a]pyrene
pdb code: 1Y9H
benzo[c]phenanthrenebenzo[a]pyrene
176
348
DNA-Carcinogen AdductsDNA-Aflatoxin Adduct
N
N
N
NH
NH2
O
O
O
MeO
O
O
O
HO
H
H
dR
+
pdb code: 1MKL
349
DNA Repair:
1. Direct repair
2. Base excision repair (BER): repair of deglycosylation (lose of the base from the deoxyribose unit) sites, oxidation of the base, or modification by a “small” alkylating agent.
3. Nucleotide excision repair (NER): repair of “bulky” lesions
4. Mismatch Repair: repair of mis-paired DNA bases
5. Recombination: repair of double strand breaks of DNA
“Chemistry and Biology of DNA Repair” Scharer, O. D. Angew. Chem. Int. Ed. Engl. 2003, 42, 2946-2974
177
350
Direct Repair: O6-alkylguanine transferase (AGT): direct reversal by
transferring the O6-alkyl group to an active site cysteineof AGT via an SN2 reaction.
N
N N
N
O
NH2
DNA
CH3
S Cys AGT
H+
N
N N
NH
O
NH2
DNA
S Cys AGT
H3C
DNA Photolyase (bacterial): direct reversal of pyrimidine-pyrimidine photodimers (UV light induced lesion)
N
HN
N
NH
O
O O
O
h!
N
HN HN
N
O
O
O
O
DNA Photolyase: FAD dependent
light dependent repair
351
O6-Alkylguanine transferase (AGT):
N
N N
N
H2NdR
OH3C
SCys145
H
N
NHis146
H
Glu172 CO2-
H
OH
O
H
Tyr114
N
N N
N
H2NdR
OSCys145
CH3
N
NHis146
H
Glu172 CO2-
HO H
O
H
Tyr114
H
pdb code: 1T38
Glu172His146
H2O
Ser145 Tyr114
O6-Me-G
X-ray analysis of the C145S mutant
178
352
O6-Alkylguanine transferase (AGT):S
Cys145
H
N
NHis146
H
Glu172 CO2-
H
OH
N
N N
N
OdR
O
Cys145
N
NHis146
H
Glu172 CO2-
HO H
O
H
Tyr114
H
N
N N
N
dR
O
H
Tyr114
O
O Scovalent
protein-DNAcomplex
Glu172
His146
Tyr114
Cys145
353
Base-Excision Repair: HN
N N
NH
O
NH2
DNA
O
N
N N
NH
O
NH2
DNA
H3C
OGG1 AAG
N
NH
O
DNA
HOH3C
HO O N
NH
O
DNA
O
N
N N
N
NH2
DNACH3
HN
HN N
NH
O
NH2
DNA
OHC
Fpg UDGAAG Nth
Mechanism of deglycosylation:DNA glycosylase:
AP lyase: leads to DNA strand scission ala Maxim-Gilbert Chemistry
O
O
O X
DNA
DNA
O
H
H
Enzyme
OO
O
O
O
DNA
DNA
OH
Abasic (apurinic) site
O
O
O X
DNA
DNA
N
H
H
Enzyme
OO
O
O
O
DNA
DNA
HNH
Lys
Lys OH
O
O
DNA
DNA
HN Lys
OH
O
O
DNA
DNA
HN Lys
NaBH4
(chemical trap)
179
354
OH
O
PO
OH
HO
APE1
Pol !
O
PO
OH
NLys
CHO
HOOP
OP
APE1
OPHO
OH
Pol !Pol !
DNA Ligase
Base-excision repair: from DNA glycosylase from AP lyase
355
Nucleotide Excision Repair (NER): (shamefully pirated from the Scharer review)
a) A DNA lesion that causes helical distortion (red star) is initially recognized by XPC/hHR23B.
b) XPC/hHR23B recruits TFIIH to the lesion and the two helicase subunits of TFIIH, XPB, and XPD cause partial opening of the DNA around the lesion.
c) TFIIH attracts XPG and XPA/RPA to the lesion, further DNA opening takes places, and a bubble of about 25 base pairs is formed. XPC is probably no longer part of the complex at this point.
d) XPA and RPA verify the damage and ensure the proper positioning of the two endonucleases, XPG and ERCC1/XPF. XPG makes the incision 3’ to, ERCC1/XPF 5’ to the damage and an oligonucleotide of about 25–32 nucleotides in length is released.
e) The replication machinery fills in the gap and DNA ligase I seals the nick.
180
356
DNA Polymerases: Classified by Structural HomologyA (pol I)
E. coli pol I repairhuman pol γ mitochondrial DNA replicationhuman pol θ repair
B (α-like)human pol α priminghuman pol δ replicationhuman pol ζ lesion bypassE. coli pol II repairT4 DNA polymerase phage replication
C E. coli pol III replication
DDNA pol D replication
Xhuman DNA pol β repairhuman pol λ repair
Y (Umc/DinB/Rev1p/rad30 superfamily)E. coli pol IV (Din B) lesion bypassE. coli pol V (UmuDC) SOS induced lesion bypasshuman pol η lesion bypasshuman pol κ lesion bypasshuman pol ι lesion bypasshuman Rev1 lesion bypassDpo4 archaeobacteria lesion by-pass
357
DNA Replication:replicative, high-fidelity DNA polymerases
ReplicativeDNA Polymerase
high-fidelity
ReplicativeDNA Polymerase
high-fidelity
DNA lesion DNA replicationis blocked
181
358
Trans-lesion Synthesis: error prone (low fidelity), bypass polymerases (Y family)
ReplicativeDNA Polymerase
high-fidelity
DNA lesion DNA replicationis blocked
By-passDNA Polymerase
low-fidelity
ReplicativeDNA Polymerase
high-fidelity
359
Xeroderma pigmentosum (XP): • genetic predisposition to sunlight induced skin cancer,
as well as other abnormalities. • Inefficient repair of sunlight induced DNA lesions (XP-A-F) • DNA polymerase η is not expressed (XP-V)
N
HN
N
NH
O
O O
O
h!
N
HN HN
N
O
O
O
O
T T
pol !T T
A A