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Bi 1 “Drugs and the Brain”

Lecture 17

Tuesday, May 2, 2006

From the Genome to mRNA

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1. Genomic DNA sequence as an algorithm

1. The genome contains the “parts list” PLUS the rules for parts use.

2. The major rules for “parts use” operate through differential gene expression.

Now, the goals: to understand both -the complete parts list and -the rules for use.

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2. Complete DNA sequence as scripture

basic sequence

RNA sequence

protein sequence

protein structure

RNA splicing

mutations that cause disease

single-nucleotide polymorphisms (SNPs)

orthologs in other species

protein function

proteins that bind to the sequence and regulate expression

chromosomal location

RNA abundance

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http://www.ncbi.nih.gov/Database/datamodel/index.html

2. Complete DNA sequence as scripture

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22,000 genes x 400 codons/protein x 3 bases/codon

= 26.4 million base pairs, or < 1% of the genome!

How much coding sequence is in the genome?

1. Repetitive elements (junk? selfish DNA?)

2. Regulatory regions

3. Introns

We now describe the rest

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Lander Figure 17

1. Repetitive elements

Encode proteins

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Little Alberts Fig. 8-15© Garland publishing

2. Gene activation involves regulatory regions from Lecture 14

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Gene (DNA)

protein

coding sequences noncoding sequences

3. Introns and Exons

messenger RNA (mRNA) translated sequences untranslated sequences

exon intron

translation

splicing (introns removed)

transcription (mRNA synthesis)

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Exons don’t differ much among organisms,

Lander et al Figure 35

but human introns are longer

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Humans have less than twice as many genes as worm or fly. However, human genes differ in two ways from those in worm or fly.

1. Human genes are spread out over much larger regions of genomic DNA

2. Human genes are used to construct more alternative transcripts. Result: humans have ~ 5 times as many protein products as worms or flies.

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From past lectures, some pictures of

RNA polymerase

or

Transcription factors

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in vitro RNA synthesisRNA polymerase promoter

DNA

measure

Site-Directed Mutagenesis on Ion Channels

Express by injecting into immature frog eggs

Mutate the desired codon(s)

measure

(from Lecture 7)

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RNA polymerase promoter

RNA polymerase

DNA

Step1:bind “nonspecifically”

to DNAStep 2:

bind “specifically” to promoter

(from Lecture 10)

One-dimensional diffusion: a protein bound to DNA

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Many genes have a DNA sequence called

“cAMP-Ca2+ responsive element”

(CRE)

The protein that binds to this CRE is called

“cAMP-Ca2+ responsive element binder”

(CREB).

CREB binds in its phosphorylated form, called pCREB.

pCREB is a “transcription factor”.

-O OP

O

O

kinase

phosphorylatedprotein

cAMPCa2+

intracellularmessenger

(from Lecture 14)

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Directed Mutagenesis Applied to Control Elements in DNA

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suspected control site where a protein binds

Mutate the suspected sequence in vitro

coding region of protein

Express by placing in the nucleus of an “appropriate” cell; stimulate with cAMP.

measure the amount of protein

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suspected control site

measure the amount of

fluorescence

coding region for EGFP

Express by placing in the nucleus of an “appropriate” cell; stimulate with Ca or cAMP.

(from Lecture 14)

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(from Lecture 14)and

NestlerFigure 16-5

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Proteins bind to specific but limited stretches of DNA(8 - 20 base pairs)

kinase

phosphorylatedprotein

cAMPCa2+

intracellularmessenger

http://www.its.caltech.edu/~lester/Bi-1/Transcription factor-DNA complex.pdb

(Swiss-prot viewer must be installed on your computer)

(from Lecture 14)

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Little Alberts Fig. 8-15© Garland publishing

from Lecture 14

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Viewing a single DNA-protein complex, #2Atomic Force Microscope

Sample

Cantilever with tip

Segmented photodiode

Laser

(#1 was Steve Quake’s single-molecule DNA sequencing experiment, Lecture 16)

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Tip and cantileverof an

atomic force microscope

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Single-molecule AFM image of two protein molecules bound to DNA

kinase

phosphorylatedprotein

cAMPCa2+

intracellularmessenger

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Little Alberts Fig 7-7© Garland publishing

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To build RNA (or DNA)ribonucleic acid (or deoxyribonucleic acid),

the cell begins with nucleotides, for instance ATP

hydrogen bonds to U(to T in DNA)

The BaseAU (T in DNA)CG

The phosphates4 negative charges;

2 are usually neutralized by Mg2+

The 5-carbon sugarribose

(2’-deoxyribose in DNA)

3’ 2’

5’

N

NN

N

NH2

O

OHOH

HHCH2

H

OP

O-

O

OP

O-

O

-O OP

O-

O

Mg2+

deoxyDNA

H

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OH

Little Alberts Fig 3-42© Garland publishing

ligate nick

Latin, to tie

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DNA is quite stable

A mosquito with a parasitic mite, caught in amber ~ 25 my

butRNA is quite

unstable

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How fast does the RNA chain grow? Macroscopic measurements

A. Outside the lab:

1. Build nuclear reactor

2. Irradiate [32S]sulfate to produce [32P]phosphate

3. Phosphorylate adenosine with [32P]phosphate, yielding [32P]-AMP.

4. Phosphorylate twice more to produce -[32P]ATP

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B. Inside the lab:

5. Order -[32P]ATP

6. Add -[32P]ATP to reaction

7. Separate mononucleotides from polymers (RNA)

8. Count radioactive decays in polymers

9. Result: elongation varies with nucleotide concentration, but the maximal rate is ~ 30 nt/s.

How fast does the RNA chain grow? Macroscopic measurements

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1. 32P half-life 14 d. Time constant = t/ln(2) = 20.6 d, rate constant = 0.048/d = 3.4 x 10-5 /min

2. If we would like 100 dpm (disintegrations per min), we need (100 dpm)/(3.4 x 10-5 /min) = 3.0 x 106 atoms of 32P.

3. Assume that we have an RNA molecule encoding a protein of

length 330 amino acids, or 1000 nucleotides. On the average, 25%

of these will be ATP. Therefore we need a number of RNA

molecules equal to 3.0 x 106 / 250, or 11,900 RNA molecules.

4. The least abundant mRNAs occur at abundances of one/cell; the most abundant, ~ 10,000/cell.

Therefore we need between 1 and 12,000 cells.

How much material do we need for macroscopic measurements?

291m

DNA (double-stranded) How many nucleotides (nt)?

1.5 m = 1.5 x 104 Å.(1.5 x 104 Å)/ (3.4 Å/ nt) = 4400 nt

RNA being synthesized (single-stranded)

Electron micrograph of RNA synthesis from a DNA template

~ 110 molecules of RNA polymerase(not visible)

“latest”~ 500 s

“earliest”

Little Alberts Fig 7-8

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Where does the energy go? Dynamic single-molecule measurements

E = force x distance;force is generated by viscosity

RNA polymerase

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Movie of the RNA polymerase experiments

Description of the movie

Approximately one frame every 5 seconds was selected to speed up the

movement of the transcription process.

A total of ~10 minutes of transcription is displayed and the movie is "looped" 8

times.

When a new loop starts the total shortening of the DNA tether is clearly visible.

http://alice.berkeley.edu/RNAP/

(your choice of several formats)

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RNA polymerase travels at a “constant” rate until it stalls

stall at 30% - 60% efficiency

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Gene (DNA)

protein

coding sequencesnoncoding

sequences

Components of Expression

messenger RNA (mRNA) translated sequences untranslated sequences

exon intron

translation

splicing (introns removed)

transcription (mRNA synthesis)

(from Lecture 15)

DNA has well-understood chemistry and is stable

RNA has well-understood chemistry but is unstable

Proteins have complex chemistry and a wide range of stability

Constraints on a “complete” or systems approach to biology

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1. Make a DNA array from the genome of interest

2. Extract mRNA (wear those rubber gloves!) from the organ of interest. (Control vs experiment)

3. Reverse transcribe / label with fluorescent dyes(make “complementary DNA” or cDNA)

4. Hybridize to array (use those base-pairing rules)

5. Read with scanner

Steps in Microarray Analysis

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1. One way to generate a DNA microarray: photochemistry

Unreactive, but photosensitive

moiety

spot1 spot2 spot3 spot4 spot5

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1. Another way to generate a DNA microarray: PCR + pens

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DNAsynthesis

cool tobind primers

DNAsynthesis

cool tobind primers

DNAsynthesis

cool tobind primers

fragment of DNA

to be detected

heat to separate DNA

strands

heat to separate DNA

strands

heat to separate DNA

strands

From Lecture 15:Thousands of primer pairs

Collection of standard 96-well dishes Many identical slides, each

with thousands of spots

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37Little Alberts Figure 10-25

2, 3. Generating cDNA with reverse transcriptase

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4. Next slide:An experiment to determine different gene expression in

schizophrenic vs control brains

From Dr. David LewisVisiting psychiatry lecture

Bi 1 2003

Most striking result: a decreased level of mRNA for an auxiliary protein in the G protein cycle

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Scan @ 532nm Scan @ 635nm

CONTROL SCHIZOPHRENIA

Isolate total RNA

Isolate mRNA

Reverse transcibe(make cDNA)

Cy3 Cy5Combine and Hybridize

Harvest Area 9

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Combined Image

Fulton & Weinberger, 1999 Fulton & Weinberger, 1999

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5. Microarray scanners are now about as large as a

microwave oven or boom box or desktop PC.

For example,www.genepixprofessional.com

The software for analyzing 104 to 105 spots per slide is quite advanced

Emphasizing the limitations of mRNA cDNA microarrays:

Translation rates, RNA stability, protein stability

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messenger RNA (mRNA)

Gene (DNA)

protein

coding sequences noncoding sequences

Components of Expression

translated sequences untranslated sequences

exon intron

translation (Lecture 18)

splicing (introns removed)

transcription (mRNA synthesis)

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Bi 1 “Drugs and the Brain”

End of Lecture 17