Chemistry 2100

Chapter 26

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

transcription

transcription

transcription

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

TranscriptionOrganization and transcription of a split eukaryote gene.

7

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)

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

Bio-mimicry is a powerful motivation

Velcro: inspired by burrs Conformation Switching Probes

Marvin J S et al. PNAS 1997;94:4366-4371

Optical Conformation SwitchingTF Switch Sensors

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]

TF Beacon Actual Performance

Quantitative Detection in 4 easy steps

initiation

initiation

initiation

elongation

elongation

elongation

elongation

elongation

elongation

elongation

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

AU

C U

HONO

N

N

NH2

O

H

H2O

N

NO

H

OH

N

N

H

O

O

H

Pyrimidine Dimers from UV

http://highered.mcgraw-hill.com/olc/dl/120082/micro18.swf

The Genetic Code

33

ACUACCACAACG H OH

CH2OH

COOH

HH2N

Thr

UCUUCCUCAUCG

H2N H

COOH

CH2OHSer

ACUACCACAACG H OH

CH2OH

COOH

HH2N

Thr

UCUUCCUCAUCG

H2N H

COOH

CH2OHSer

ACUACCACAACG H OH

CH2OH

COOH

HH2N

Thr

UCUUCCUCAUCG

H2N H

COOH

CH2OHSer

ACUACCACAACG H OH

CH2OH

COOH

HH2N

Thr

UCUUCCUCAUCG

H2N H

COOH

CH2OHSer

ACUACCACAACG H OH

CH2OH

COOH

HH2N

Thr

UCUUCCUCAUCG

H2N H

COOH

CH2OHSer

ACUACCACAACG H OH

CH2OH

COOH

HH2N

Thr

UCUUCCUCAUCG

H2N H

COOH

CH2OHSer

ACUACCACAACG H OH

CH2OH

COOH

HH2N

Thr

UCUUCCUCAUCG

H2N H

COOH

CH2OHSer

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

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

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

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')

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')

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')

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')

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

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 . . . .

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 . . . .

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')

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 . . . .

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 . . . .

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 . . . .

PCR

PolymeraseChainReaction

DNA Fingerprinting

DNA Sequencing

DNA Sequencing

Shotgun Sequencing

Electrochemical Sequencing

http://www.youtube.com/watch?v=yVf2295JqUg

Recombinant DNA

Gene Therapy