Molecular Biology of the Gene

Outline: Molecular Biology of the Gene • • • • • •

Readings: Chapters 10-12 in Campbell et al Historical Genetic Material Experiments Chemical Nature of Nucleic Acids Structure of DNA DNA Replication Gene Expression – RNA and Protein Synthesis • Gene Technology

Griffith’s Bacterial Transformation Experiments

Bacteriophage (Phage) Structure

Figure 10.1A

Head DNA Tail

300,000×

Tail fiber

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Fig. 08.03

Phage/?Bacterial virus reproductive cycle – Hershey & Chase (1952) Figure 10.1C

Virus attaches to bacterial cell.

Virus injects DNA.

Hershey & Chase Phage Reproduction Experiments DNA is the Genetic material

Virus DNA directs cell to 1. make more phage DNA 2. make protein parts. New phages assemble.

Cell lyses releasing new phages.

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Figure 10.2A

DNA Structure

DNA is a Nucleotide Polymer Sugar-phosphate backbone Phosphate group

1. Nucleotides

A

2. Nitrogen Bases 3. Key Investigations 1. X-Ray Crystallography 2. Chargaff’s Rule 3. Double Helix of Watson & Crick

DNA Nucleotide

A

Nitrogenous base Sugar

C

C

T

T

Nitrogenous base N

O

C

O P O

N

O– Phosphate group

G

G

Sugar (deoxyribose) T

T

DNA polynucleotide Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings

Figure 10.2B

DNA = Nucleotide Polymer Sugar-Phosphate Linkage

Four Nitrogenous bases are in DNA

PO4 H

O H3C H

C C

C N H

N C

Thymine (T)

H

H H

O

C C

N C N H

H

H

N

N C C N H C N C N C H H

C

O

Cytosine (C)

N

H

O

Adenine (A)

Nucleotide

O

Sugar C

Guanine (G)

Phosphate Purines

Pyrimidines

Base

CH2

N C C N H H C N C N C N H H H

–O

O P O O CH2

Base O

Nucleotide

Sugar OH Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings

Fig. 14.9ab

Chargaff’s Rule

X-Ray Crystallography: Rosalind Franklin Base Pair Ratio A:T 1.1:1

G:C 1:1

1:1

1:1

1:1

1.1:1

1:1

1:1

1:1

1:1

1.1:1

1:1

DNA was helical DNA had repetitive elements 0.34 nm, 3.4 nm, 2.0nm

2

Phosphodiester bond

AT TA

Franklin’s Crystallography made sense!

G

C

2 nm

DNA Structure

Watson & Crick DNA Model

Sugar-phosphate "backbone"

Double Helix

GC C G T A G C

T

A

T A G C AT TA

C

G

Major groove A

T

Three representations of DNA

Figure 10.3D

G

C T

A A

C A C

T

G G T

–

C

–

A

T

A A

–

T T

A

O

O O O PO H2C

O

O O P O H2C

C

G

C

G

A

O

CH2 O O– P O O

O

CH2 O O– HO P O

O

O

A

T

C T

Ribbon model

CH2 O O– O P O

O CH2 O O– O P O

OH

G

DNA discovery (video)

OH

A

T

O

O O PO H2C

–

Start Monday 11/3/06

Hydrogen bond

O OH P O O H2C O O

G

T

0.34 nm

G C C G T A G C

Paired Nitrogen Bases

Base pair

3.4 nm

Chemical structure

Computer model

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Figure 10.4A

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Semiconservative DNA replication

Figure 10.5B

DNA replication – A Closer Look

DNA strands have an opposite orientation 3. Joining

2. Pairing

1. Unwinding A

T

A

C

G

C

G

C

G

A

T

A

T

A

T

T G

5′ end

A

T

A

T

A

C

G

C

G

C

G

C

G

C

G

C

T

A

T

A

T

A

T

A

T

A

C A

T

5′

HO

4′ 3′

2′ 2′

1′

Both parental strands serve as templates

Two identical daughter molecules of DNA

A

1′

3′ 4′

P C

G

G

C

T

A

P

P

P

P

OH 3′ end Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings

T

P

Nucleotides Parental molecule of DNA

3′ end

P

P

5′

5′ end

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DNA replication – A Closer Look

Figure 10.4B

Figure 10.5C

DNA replication – A Closer Look

New DNA strands are synthesized continuously & discontinuously

DNA must unwind A

T

G

C

Parental DNA 5′ 3′

G

C

A

T T C

A A

G

G C C T T

G

A

A

G

G C

G

C G

A

3′ 5′

T

C

T

A

A

T

Daughter strand synthesized in pieces

C

G C T

G T

A

A

T

T

A

C

A

T

T

A

3′ 5′ Daughter strand synthesized continuously

DNA polymerase molecule

G C

DNA ligase

5′ 3′

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10.5 DNA replication: A closer look • DNA replication – Begins at specific sites on the double helix Parental strand

Origin of replication

DNA Synthesis Animation

Daughter strand

Bubble

Two daughter DNA molecules Figure 10.5A Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings

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Genes Specify Sequences of Amino Acids

Figure 10.7

Gene Expression – The Central Dogma

Normal Hemoglobin β chain (Sanger 1953)

Gene 1

Valine

Histidine

Leucine

Threonine

Proline

Glutamic acid

Glutamic acid

Valine

Histidine

Leucine

Threonine

Proline

Valine

Glutamic acid

Gene 2

DNA molecule

Sickle cell anemia Hemoglobin β chain (Ingram 1956)

Gene 3

DNA strand A A A C C G G C A A A A

GENE = Unit of Heredity GENE = Sequence of nucleotides that determines the amino acid sequence of a protein

Transcription RNA

One DNA strand

U U U G G C C G U U U U

Translation Polypeptide Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings

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Figure 10.9B

Transcription of a gene

Figure 10.9A

Transcription of a gene – a closer look

RNA polymerase RNA nucleotides

Promoter DNA

RNA polymerase

DNA of gene

1 Initiation T C C A A

A

T

A

C C

G

T A G G T

G

C C A

G

U

C A U

2 Elongation

U

T

C T

T

A

A

Direction of transcription

3 Termination

Growing RNA

Template Strand of DNA

Newly made RNA Completed RNA

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Figure 10.6A

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Gene Expression – The Central Dogma

Figure 10.7

Gene Expression – DNA is a triplet code

RNA leaves the nucleus and enters the cytoplasm Gene 1

DNA

Gene 2

DNA molecule

Transcription Gene 3

RNA DNA strand

One DNA strand

A A A C C G G C A A A A

Transcription

Translation

U U U G G C C G U U U U

RNA

Translation

Protein

Codon Codon Codon Codon

Polypeptide

Amino acid Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings

Figure 10.8A

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The genetic code (using RNA codons)

Figure 10.8B

An exercise in translating the genetic code

Second base U

C

A

UGG Trp CGU CGC Arg CGA CGG

U C A G

AGU AAU ACU Asn Ser AGC AAC ACC Thr AAA AGA ACA Lys Arg AAG ACG AGG

U C A G

GCU GAU GGU GUU Asp GCC GAC GGC GUC Ala Val Gly GCA GUA GAA Glu GGA GCG GGG GAG GUG

U C A G

U

C

CCU CAU CUU CCC CAC His CUC Pro Leu CCA CAA CUA CCG CAG Gln CUG

First base A

AUU AUC lle AUA or AUG Met START

G

Strand to be transcribed

G U C A G

UUU UAU Phe UCU Tyr UUC UCC UAC Ser UCA UAA Stop UUA Leu UCG UUG UAG Stop

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UGU Cys UGC UGA Stop

DNA

T

A

C

T

T

C

A

A

A

A

T

C

A

T

G

A

A

G

T

T

T

T

A

G

U

A

G

Transcription Third base

RNA

A

U

Start codon

Polypeptide

Met

G

A

A

G

U

U

Translation

Lys

U

Stop codon

Phe

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Transfer RNA Structure & Function

Types of RNA

Amino acid attaches here

3’

1. Ribosomal RNA

rRNA

2. Transfer RNA

tRNA

3. Messenger RNA

mRNA

3’

Anticodon tRNA red&yellow ATP (green) Enzyme (blue)

Figure 10.12A Ribosome Structure Ribosomes build polypeptides

Ribosome Structure Subunits of a ribosome

• A ribosome consists of two subunits – Each made up of proteins and a kind of RNA called ribosomal RNA tRNA molecules

Anticodon

Hold tRNA and mRNA close together during translation

tRNA-binding sites

Large subunit

Growing polypeptide

Large subunit

Next amino acid to be added to polypeptide Growing polypeptide tRNA mRNAbinding site

mRNA

Small subunit

mRNA

Small subunit

Codons Figure 10.12B, C

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Fig. 15.2(TE Art)

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Ribosome Structure

Protein Synthesis: Initiation

P site

Large subunit

fMet

Functional Ribosome

Small subunit

E P A

E 5'

mRNA Binding site

A U A C A U G 3'

6

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Fig. 15.16a(TE Art)

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Fig. 15.16b(TE Art)

Protein Synthesis: Elongation

Protein Synthesis: Elongation Leu

Elongation factor

P site fMet

fMet

Leu

A

C

tRNA

E

G

A U A C G AA U A UGC U

5'

E 5'

U A C G A C A U G C U GA AU

3'

3'

mRNA

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Fig. 15.16c(TE Art)

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Fig. 15.16d(TE Art)

Protein Synthesis: Translocation

Protein Synthesis: Translocation fMet

fMet

Leu

Leu

E 5'

A C U A C GA A U G C U G A AU

5'

3'

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Fig. 15.16(TE Art)

C UAC GA G A A U A U G C U

3'

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Fig. 15.17b(TE Art)

Protein Synthesis: Termination Protein Synthesis Initiation Æ Elongation Æ Translocation

Val

Polypeptide chain released

Ser Ala

tRNA

5'

fMet

fMet

P site fMet

fMet Leu

Leu

Leu

tRNA A C

E site

Trp

Leu

G A site UACCUGAAU 5' AUG 3'

UACGACAAU 5' AUGCUG 3'

UAC GAC AUGCUGAAU

3'

5'

UACGAC U AUGCUGAA

Release Factor

3'

mRNA

5'

AC C UG G U A A

3'

7

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Fig. 15.17(TE Art)

Protein Synthesis: Termination Polypeptide chain released Val

Release factor

Ser Ala

tRNA

Trp tRNA

P site E site ACC UGGUAA

5'

Val Ser Ala Trp

Large ribosomal subunit

ACC

A site 3'

5'

ACC UGGUAA

3'

mRNA

Eukaryotic mRNA is processed before leaving the nucleus Intron

Exon

DNA Transcription Addition of cap and tail

Cap

RNA transcript with cap and tail

Eukaryotic RNA may be spliced in more than one way

Figure 11.7

• Alternative splicing may generate two or more types of mRNA from the same transcript

Figure 10.10

Exon Intron Exon

Protein Synthesis Animation

Small ribosomal subunit

Exons

Introns removed Tail

DNA

Exons spliced together

RNA transcript

mRNA

or

Coding sequence Nucleus

mRNA

mRNA

Cytoplasm

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Gene Expression in Prokaryotes

Gene Expression in Eukaryotes

Figure 11.1B

Protein Assemblies Control Gene Expression

Regulatory gene DNA

mRNA Translation

Intron Primary RNA transcript 5’ mRNA Cap

Operon turned off (lactose absent)

RNA polymerase cannot attach to promoter

Transcription Processing 3’ Poly-A tail

Lactose-utilization genes

repressor

mRNA

DNA Transcription

Promoter Operator

Protein

RNA polymerase bound to promoter

Operon turned on (lactose inactivates repressor)

Protein Translation Protein

DNA

mRNA Protein

Enzymes for lactose utilization

Lactose

Inactive repressor

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Figure 11.6

Protein Assemblies Control Gene Expression

Transcription Factors assist in initiating eukaryotic transcription Enhancers

Promoter Gene

DNA Activator proteins Transcription factors

END

Other proteins RNA polymerase

Gene Expression Bending of DNA

Transcription RNA polymerase

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