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Chemistry 2100
Chapter 26
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The Central Dogma
The central dogma of molecular biology:– Information contained in DNA molecules is expressed
in the structure of proteins.– Gene expression is the turning on or activation of a
gene.
2
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transcription
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transcription
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transcription
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TranscriptionThe architecture of yeast RNA polymerase II. Transcription of DNA (helical structure) into RNA (red) is shown. The template strand of DNA (blue) and the coding strand (green) and also shown.
6
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TranscriptionOrganization and transcription of a split eukaryote gene.
7
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Why detect Transcription Factor targets?
• Transcription factors are medically relevant– ~10% of human genes– Crucial roles in
development and cell life cycle
– Misregulation and mutation cause disease
– Critically, most cancers involve TF overactivity
Darnell, Nature Reviews Cancer 2, 740 (2002)
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Traditional methods for Transcription Factor detection
Expression Microarrays Gel Shift Assays
The challenge: Most of these methods are indirect, slow
(hours), or can’t differentiate active and inactive protein.
Western Blots
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Bio-mimicry is a powerful motivation
Velcro: inspired by burrs Conformation Switching Probes
Marvin J S et al. PNAS 1997;94:4366-4371
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Optical Conformation SwitchingTF Switch Sensors
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Rationally Tuning TF Sensors
% s
wit
ches
open
KS = 10
KS = 1
KS = 0.1
KS
= 0
.01
KS
= 0
.001
KS = [ ]
[ ]
KS [target]
KD (1+ KS) + KS [target] % switches open =
KD = [ ]
[ ]
[ ]
Target [M]
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TF Beacon Actual Performance
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Quantitative Detection in 4 easy steps
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initiation
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initiation
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initiation
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elongation
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elongation
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elongation
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elongation
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elongation
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elongation
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elongation
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A – U – G – U – U – U – G – U – C – A – G – U . . . . . mRNA
tRNA
peptide Met –––– Phe –––– Val –––– Ser . . . .
(5')
U – A – C – A – A – A – C – A – G – U – C – A
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AU
C U
HONO
N
N
NH2
O
H
H2O
N
NO
H
OH
N
N
H
O
O
H
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Pyrimidine Dimers from UV
http://highered.mcgraw-hill.com/olc/dl/120082/micro18.swf
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The Genetic Code
33
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ACUACCACAACG H OH
CH2OH
COOH
HH2N
Thr
UCUUCCUCAUCG
H2N H
COOH
CH2OHSer
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ACUACCACAACG H OH
CH2OH
COOH
HH2N
Thr
UCUUCCUCAUCG
H2N H
COOH
CH2OHSer
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ACUACCACAACG H OH
CH2OH
COOH
HH2N
Thr
UCUUCCUCAUCG
H2N H
COOH
CH2OHSer
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ACUACCACAACG H OH
CH2OH
COOH
HH2N
Thr
UCUUCCUCAUCG
H2N H
COOH
CH2OHSer
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ACUACCACAACG H OH
CH2OH
COOH
HH2N
Thr
UCUUCCUCAUCG
H2N H
COOH
CH2OHSer
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ACUACCACAACG H OH
CH2OH
COOH
HH2N
Thr
UCUUCCUCAUCG
H2N H
COOH
CH2OHSer
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ACUACCACAACG H OH
CH2OH
COOH
HH2N
Thr
UCUUCCUCAUCG
H2N H
COOH
CH2OHSer
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ACUACCACAACG H OH
CH2OH
COOH
HH2N
Thr
UCUUCCUCAUCG
H2N H
COOH
CH2OHSer
Val
H2N H
COOH
CH(CH3)2
GUAGUG
Glu
H2N H
COOH
CH2CH2COOH
GAAGAG
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ACUACCACAACG H OH
CH2OH
COOH
HH2N
Thr
UCUUCCUCAUCG
H2N H
COOH
CH2OHSer
Val
H2N H
COOH
CH(CH3)2
GUAGUG
Glu
H2N H
COOH
CH2CH2COOH
GAAGAG
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ACUACCACAACG H OH
CH2OH
COOH
HH2N
Thr
UCUUCCUCAUCG
H2N H
COOH
CH2OHSer
Val
H2N H
COOH
CH(CH3)2
GUAGUG
Glu
H2N H
COOH
CH2CH2COOH
GAAGAG
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DNAT – A – C – A – A – A – C – A – G – T – C – A . . . . .
A – T – G – T – T – T – G – T – C – A – G – T . . . . .
(3')
(5')
peptide Met –––– Phe –––– Val –––– Ser . . . .
A – U – G – U – U – U – G – U – C – A – G – U . . . . . mRNA (5')
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DNAT – A – C – A – A – A – C – A – G – T – C – A . . . . .
A – T – G – T – T – T – G – T – C – A – G – T . . . . .
(3')
(5')
peptide Met –––– Phe –––– Val –––– Ser . . . .
A – U – G – U – U – U – G – U – C – A – G – U . . . . . mRNA (5')
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DNAT – A – C – A – A – A – C – A – G – T – C – A . . . . .
A – T – G – T – T – T – G – T – C – A – G – T . . . . .
(3')
(5')
peptide Met –––– Phe –––– Val –––– Ser . . . .
A – U – G – U – U – U – G – U – C – A – G – U . . . . . mRNA (5')
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DNAT – A – C – A – A – A – C – A – G – T – C – A . . . . .
A – T – G – T – T – T – G – T – C – A – G – T . . . . .
(3')
(5')
peptide Met –––– Phe –––– Val –––– Ser . . . .
A – U – G – U – U – U – G – U – C – A – G – U . . . . . mRNA (5')
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peptide Met –––– Phe –––– Val –––– Ser . . . .
A – U – G – U – U – U – G – U – C – A – G – U . . . . . mRNA (5')
peptide Met –––– Phe –––– Asp –––– Ser . . . .
A – U – G – U – U – U – G – A – C – A – G – U . . . . . mRNA (5')
Mutations
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point substitution mutation
peptide Met –––– Phe –––– Val –––– Ser . . . .
mRNA (5')
peptide Met –––– Phe –––– Asp –––– Ser . . . .
A – U – G – U – U – U – G – A – C – A – G – U . . . . . mRNA (5')
A – U – G – U – U – U – G – U – C – A – G – U – A . . . .
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point substitution mutation
peptide Met –––– Phe –––– Val –––– Ser . . . .
mRNA (5')
peptide Met –––– Phe –––– Asp –––– Ser . . . .
A – U – G – U – U – U – G –A– C – A – G – U – A . . . . mRNA (5')
A – U – G – U – U – U – G – U – C – A – G – U – A . . . .
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point substitution mutation
peptide Met –––– Phe –––– Val –––– Ser . . . .
A – U – G – U – U – U – G – U – C – A – G – U – A . . . . mRNA (5')
peptide Met –––– Phe ––– Asp ––– Ser . . . .
A – U – G – U – U – U – G –A– C – A – G – U – A . . . . mRNA (5')
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peptide Met –––– Phe –––– Val –––– Ser . . . .
mRNA (5')
frameshift mutation
peptide Met –––– Phe –––– Val –––– Val . . . .
A – U – G – U – U – U – G – U – C – A – G – U – A . . . .mRNA (5')
A – U – G – U – U – U – G – U – C – A – G – U – A . . . .
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peptide Met –––– Phe –––– Val –––– Ser . . . .
mRNA (5')
frameshift mutation
peptide Met –––– Phe –––– Val –––– Val . . . .
A – U – G – U – U – U – G – U – C – A – G – U – A . . . .mRNA (5')
A – U – G – U – U – U – G – U – C – A – G – U – A . . . .
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peptide Met –––– Phe –––– Val –––– Ser . . . .
mRNA (5')
frameshift mutation
peptide Met –––– Phe –––– Val –––– Val . . . .
A – U – G – U – U – U – G – U – C – A – G – U – A . . . .mRNA (5')
A – U – G – U – U – U – G – U – C – A – G – U – A . . . .
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PCR
PolymeraseChainReaction
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DNA Fingerprinting
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DNA Sequencing
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DNA Sequencing
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Shotgun Sequencing
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Electrochemical Sequencing
http://www.youtube.com/watch?v=yVf2295JqUg
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Recombinant DNA
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Gene Therapy