BIOLOGY Chapter 12: 10th Edition Molecular Biology of the Gene · Green fluorescent protein (GFP) used as a marker A jellyfish gene codes for GFP The jellyfish gene is isolated and

Sylv

ia S

. Ma

der

Copyright © The McGraw Hill Companies Inc. Permission required for reproduction or display

PowerPoint® Lecture Slides are prepared by Dr. Isaac Barjis, Biology Instructor

BIOLOGY 10th Edition

Molecular Biology of the Gene

Chapter 12: pp. 211 - 232

1

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

b.

3.4 nm

2 nm

0.34 nm

G C

A

T

T A

P

P

P

P

C G

G

C

Sugar hydrogen bonds

sugar-phosphate

backbone

2

Outline

Genetic Material

Transformation

DNA Structure

Watson and Crick

DNA Replication

Prokaryotic versus Eukaryotic

Replication Errors

Transcription

Translation

Structure of Eukaryotic Chromosome

3

Genetic Material

Frederick Griffith investigated virulence of

Streptococcus pneumoniae

Concluded that virulence passed from the

dead strain to the living strain

Transformation

Further research by Avery et al.

Discovered that DNA is the transforming

substance

DNA from dead cells was being incorporated

into genome of living cells

4

Griffith’s Transformation Experiment

Mice were injected with two strains of

pneumococcus: an encapsulated (S) strain

and a non-encapsulated (R) strain.

The S strain is virulent (the mice died); it has a

mucous capsule and forms “shiny” colonies.

The R strain is not virulent (the mice lived); it

has no capsule and forms “dull” colonies.

5

Griffith’s Transformation Experiment


capsule

Injected live

S strain has

capsule and

causes mice

to die.

a. b.

Injected live

R strain has

no capsule

and mice

do not die.

c.

Injected heat-

killed S strain

does not cause

mice to die.

d.

Injected heat-killed

S strain plus live

R strain causes

mice to die. Live S strain is

withdrawn from

dead mice.

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7

Transformation of Organisms Today

Result the so-called genetically modified

organisms (GMOs)

Invaluable tool in modern biotechnology today

Commercial products that are currently much used

Green fluorescent protein (GFP) used as a marker

A jellyfish gene codes for GFP

The jellyfish gene is isolated and then transferred to a

bacterium, or the embryo of a plant, pig, or mouse.

When this gene is transferred to another organism, the

organism glows in the dark

8

Transformation of Organisms

A normal canola plant (left) and a transgenic canola

plant expressing GFP (right) under a fluorescent light.


(Bacteria): © Martin Shields/Photo Researchers, Inc.; (Jellyfish): © R. Jackman/OSF/Animals Animals/Earth Scenes; (Pigs): Courtesy Norrie Russell, The Roslin Institute;

(Mouse): © Eye of Science/Photo Researchers, Inc.; (Plant): © Dr. Neal Stewart

9

Structure of DNA

DNA contains:

Two Nucleotides with purine bases

Adenine (A)

Guanine (G)

Two Nucleotides with pyrimidine bases

Thymine (T)

Cytosine (C)

Animation













11

Chargaff’s Rules

The amounts of A, T, G, and C in DNA: Identical in identical twins Varies between individuals of a species Varies more from species to species

In each species, there are equal amounts of: A & T G & C

All this suggests DNA uses complementary base pairing to store genetic info

Human chromosome estimated to contain, on average, 140 million base pairs

Number of possible nucleotide sequences, 4,140,000,000

12

Nucleotide Composition of DNA


O

N

N

CH

CH

C

C

NH 2

cytosine (C)

3 C C

2

C 1

O HO P O

O

H

H H

H H

OH

CH 3

O

HN

N

C

CH

C

C

O HO P O

O

H

H H

H H

OH

HN

N

N

C CH

O

C

C C

N

H 2 N

C 2

C 2

C 1

C 1

O HO P O

O

guanine (G)

phosphate

H

H H

H H

OH

N

N

N

HC CH

NH 2

C

C C

N

4

3 C

2

C 1

5 O

O

O

O

O

O

H

H H

H H

OH

c. Chargaf f ’ s data

DN A Composition in V arious Species (%)

Species

Homo sapiens (human)

Drosophila melanogaster (fruit fly)

Zea mays (corn)

Neurospora crassa (fungus)

Escherichia coli (bacterium)

Bacillus subtilis (bacterium)

31.0

27.3

25.6

23.0

24.6

28.4

31.5

27.6

25.3

23.3

24.3

29.0

19.1

22.5

24.5

27.1

25.5

21.0

18.4

22.5

24.6

26.6

25.6

21.6

A T G C

a. Purine nucleotides b. Pyrimidine nucleotides

nitrogen-containing base

sugar = deoxyribose

thymine (T)

adenine (A)

HO P O CH 2

5 CH 2

5 CH 2

5 CH 2

C

4 C

4 C

4 C

C

3 C

3 C

Animation













Animation

14













15

Watson and Crick Model

Watson and Crick, 1953

Constructed a model of DNA

Double-helix model is similar to a twisted

ladder

Sugar-phosphate backbones make up the sides

Hydrogen-bonded bases make up the rungs

Received a Nobel Prize in 1962

16

Watson/Crick Model of DNA


a: © Kenneth Eward/Photo Researchers, Inc.; d: © A. Barrington Brown/Photo Researchers, Inc.

P

P

P

P

c.

b.

complementary

base

sugar-phosphate backbone

3.4 nm

2 nm

0.34 nm

P

P

S S

4

5 end 3 end

1 1

2 3

2 3

5

4

5

C G

G

C

C

G

T

T

A

A

C

G

a.

d. d.

5 end

sugar

hydrogen bonds

17

X-Ray Diffraction of DNA

Rosalind Franklin studied the structure of DNA

using X-rays.

She found that if a concentrated, viscous solution

of DNA is made, it can be separated into fibers.

Under the right conditions, the fibers can produce

X-ray diffraction pattern

She produced X-ray diffraction photographs.

This provided evidence that DNA had the following

features:

DNA is a helix.

Some portion of the helix is repeated.

18

X-Ray Diffraction of DNA


© Photo Researchers, Inc.; c: © Science Source/Photo Researchers, Inc.

X-ray beam

b. c.

Rosalind Franklin

diffraction pattern

Crystalline

DNA

diffracted

X-rays

a.

19

Replication of DNA

DNA replication is the process of copying a

DNA molecule.

Replication is semiconservative, with

each strand of the original double helix

(parental molecule) serving as a template

(mold or model) for a new strand in a

daughter molecule.

Animation













Animation

21













Animation













23

Replication: Eukaryotic

DNA replication begins at numerous points along linear chromosome

DNA unwinds and unzips into two strands

Each old strand of DNA serves as a template for a new strand

Complementary base-pairing forms new strand on each old strand

Requires enzyme DNA polymerase

Animation













Animation













26

Replication: Eukaryotic

Replication bubbles spread bi-directionally until they meet

The complementary nucleotides join to form new strands. Each daughter DNA molecule contains an old strand and a new strand.

Replication is semiconservative:

One original strand is conserved in each daughter molecule i.e. each daughter double helix has one parental strand and one new strand.

Animation













28

Semiconservative Replication of DNA


region of parental

DNA double helix

G

G

G

T

A

A

C

C

3' 5'

A T

C G

A T

A

G

G C

C G

A

region of

replication:

new nucleotides

are pairing

with those of

parental strands

region of

completed

replication

daughter DNA double helix

old

strand

new

strand daughter DNA double helix

old

strand

new

strand

C C

A

A

T T

G

G

T A

T A

C

G

A

T

A

T

A

C G A

T A T

A

T A

C G

C G

A

G

T

A

C

G

C

G

A

Animation

29













Animation













31

Science Focus: Aspects of DNA

Replication

G C

A T

G C

G C

P

P

P

P

P

P

P

P

P

P

P

P is attached here OH

CH2

C

C C

C

H H

H

H H

OH

OH O

base is attached here

5 end

3 end 5 end

template strand

Direction of replication new strand

Deoxyribose molecule

RNA primer

3

3

5

3

5

5 parental DN A helix

helicase at replication fork

leading new strand

template strand

template strand

lagging strand

DN A polymerase

DNA polymerase DNA ligase

Okazaki fragment

Replication fork introduces complications

5

7

6

4

3

2

1

DNA polymerase

attaches a new

nucleotide to the 3

carbon of the

previous nucleotide.


5

4

3 2

1

3 end

3

32

Replication: Prokaryotic

Prokaryotic Replication

Bacteria have a single circular loop

Replication moves around the circular DNA

molecule in both directions

Produces two identical circles

Cell divides between circles, as fast as every

20 minutes

33

Replication: Prokaryotic vs. Eukaryotic


origin

replication is

occurring in

two directions

replication is

complete

replication fork replication bubble

a. Replication in prokaryotes

parental strand

daughter strand

new DNA

duplexes

b. Replication in eukaryotes

34

Replication Errors

Genetic variations are the raw material for evolutionary change

Mutation:

A permanent (but unplanned) change in base-pair sequence

Some due to errors in DNA replication

Others are due to to DNA damage

DNA repair enzymes are usually available to reverse most errors

35

Function of Genes

Genes Specify Enzymes Beadle and Tatum:

Experiments on fungus Neurospora crassa Proposed that each gene specifies the synthesis of

one enzyme One-gene-one-enzyme hypothesis

Genes Specify a Polypeptide A gene is a segment of DNA that specifies the

sequence of amino acids in a polypeptide Suggests that genetic mutations cause

changes in the primary structure of a protein

36

Protein Synthesis: From DNA to RNA to

Protein

The mechanism of gene expression

DNA in genes specify information, but information is not structure and function

Genetic info is expressed into structure & function through protein synthesis

The expression of genetic info into structure & function:

DNA in gene controls the sequence of nucleotides in an RNA molecule

RNA controls the primary structure of a protein

Animation













38

The Central Dogma of Molecular Biology


nontemplate strand

3' 5'

A G G G A C C C C

T C G C T G G G G

5' 3'

template strand transcription

in nucleus

3' 5'

mRN

DNA

A G G G A C C C C

codon 1 codon 2 codon 3

polypeptide

translation

at ribosome

N N N C C C C C

R1 R2 R3

Serine Aspartate Proline

O O O

39

Types of RNA

RNA is a polymer of RNA nucleotides

RNA Nucleotides are of four types: Uracil, Adenine, Cytosine, and Guanine

Uracil (U) replaces thymine (T) of DNA

Types of RNA Messenger (mRNA) - Takes genetic message from

DNA in nucleus to ribosomes in cytoplasm

Ribosomal (rRNA) - Makes up ribosomes which read the message in mRNA

Transfer (tRNA) - Transfers appropriate amino acid to ribosome when “instructed”

40

Structure of RNA

G

U

A

C

S

S

S

S

P

P

P

P

ribose

G

U base is

uracil instead

of thymine

A

C

one nucleotide


41

RNA vs. DNA structure

42

The Genetic Code

Properties of the genetic code: Universal

With few exceptions, all organisms use the code the same way

Encode the same 20 amino acids with the same 64 triplets

Degenerate (redundant) There are 64 codons available for 20 amino acids Most amino acids encoded by two or more codons

Unambiguous (codons are exclusive) None of the codons code for two or more amino acids Each codon specifies only one of the 20 amino acids

Contains start and stop signals Punctuation codons Like the capital letter we use to signify the beginning of a

sentence, and the period to signify the end

43

The Genetic Code

The unit of a code consists of codons, each of which is a unique arrangement of symbols

Each of the 20 amino acids found in proteins is uniquely specified by one or more codons The symbols used by the genetic code are the mRNA bases

Function as “letters” of the genetic alphabet Genetic alphabet has only four “letters” (U, A, C, G)

Codons in the genetic code are all three bases (symbols) long Function as “words” of genetic information Permutations:

There are 64 possible arrangements of four symbols taken three at a time

Often referred to as triplets

Genetic language only has 64 “words”

44

Messenger RNA Codons


Second Base Third

Base

First

Base U C G A

U

C

A

G

UUU

phenylalanine

UCU

serine

UAU

tyrosine

UGU

cysteine

UUC

phenylalanine UCC

serine

UAC

tyrosine

UGU

cysteine

UCA

serine UUA

leucine

UCG

serine

UUG

leucine UGG

tryptophan

UGA

stop

UAA

stop

UAG

stop

U

C

A

G

CUU

leucine

CUC

leucine

CUA

leucine

CUG

leucine

CCU

proline

CCC

proline

CCA

proline

CCG

proline

CAC

histidine

CAA

glutamine

CAG

glutamine

CAU

histidine

CGA

arginine

CGG

arginine

CGU

arginine

CGC

arginine

U

C

A

G

AUG (start)

methionine

AUU

isoleucine

AUC

isoleucine

AUA

isoleucine

ACU

threonine

ACC

threonine

ACA

threonine

ACG

threonine

AAU

asparagine

AAC

asparagine

AAA

lysine

AAG

lysine

AGU

serine

AGC

serine

AGA

arginine

AGG

arginine

U

C

A

G

GUU

valine

GUC

valine

GUA

valine

GUG

valine

GCU

alanine

GCC

alanine

GCA

alanine

GCG

alanine

GAU

aspartate

GAC

aspartate

GAA

glutamate

GAG

glutamate

GGU

glycine

GGC

glycine

GGA

glycine

GGG

glycine

U

C

A

G

45

Steps in Gene Expression: Transcription

Transcription

Gene unzips and exposes unpaired bases

Serves as template for mRNA formation

Loose RNA nucleotides bind to exposed DNA

bases using the C=G & A=U rule

When entire gene is transcribed into mRNA,

result is a pre-mRNA transcript of the gene

The base sequence in the pre-mRNA is

complementary to the base sequence in DNA

Animation













47

Transcription of mRNA

A single chromosomes consists of one very long molecule encoding hundreds or thousands of genes

The genetic information in a gene describes the amino acid sequence of a protein The information is in the base sequence of one side (the “sense” strand)

of the DNA molecule

The gene is the functional equivalent of a “sentence”

The segment of DNA corresponding to a gene is unzipped to expose the bases of the sense strand The genetic information in the gene is transcribed (rewritten) into an

mRNA molecule

The exposed bases in the DNA determine the sequence in which the RNA bases will be connected together

RNA polymerase connects the loose RNA nucleotides together

The completed transcript contains the information from the gene, but in a mirror image, or complementary form

Animation













49

Transcription


nontemplate

strand template

strand

5'

C

C G

T A

A T

G

G

G

A

C

C

A

G

RNA

polymerase

DNA

template

strand

mRNA

transcript

C

A T

C G

T A

to RNA processing

3'

3'

5'

Animation













Animation

51













52

RNA Polymerase

© Oscar L. Miller/Photo Researchers, Inc.


a. 200m

b.

spliceosome

DNA

RNA

polymerase

RNA

transcripts

53

Processing Messenger RNA

Pre-mRNA, is modified before leaving the

eukaryotic nucleus.

Modifications to ends of primary transcript:

Cap of modified guanine on 5′ end

The cap is a modified guanine (G) nucleotide

Helps a ribosome where to attach when translation begins

Poly-A tail of 150+ adenines on 3′ end

Facilitates the transport of mRNA out of the nucleus

Inhibits degradation of mRNA by hydrolytic

enzymes.

54

Processing Messenger RNA

Pre-mRNA, is composed of exons and introns. The exons will be expressed,

The introns, occur in between the exons. Allows a cell to pick and choose which exons will go into a

particular mRNA

RNA splicing:

Primary transcript consists of:

Some segments that will not be expressed (introns)

Segments that will be expressed (exons)

Performed by spliceosome complexes in nucleoplasm

Introns are excised

Remaining exons are spliced back together

Result is mature mRNA transcript

55

RNA Splicing

In prokaryotes, introns are removed by

“self-splicing”—that is, the intron itself has

the capability of enzymatically splicing itself

out of a pre-mRNA

Animation

56













57

Messenger RNA Processing


exon

intron intron

exon exon

DNA

transcription

exon

intron intron

exon exon

5' 3'

pre-mRNA

exon exon exon

intron intron cap poly-A tail 5' 3'

exon exon exon

spliceosome

cap poly-A tail

pre-mRNA

splicing

intron RNA

5' 3'

cap poly-A tail

mRNA

nuclear pore

in nuclear envelope

nucleus

cytoplasm

5' 3'

Animation













59

Functions of Introns

As organismal complexity increases; Number of protein-coding genes does not keep pace

But the proportion of the genome that is introns increases

Humans: Genome has only about 25,000 coding genes

Up to 95% of this DNA genes is introns

Possible functions of introns: More bang for buck

Exons might combine in various combinations

Would allow different mRNAs to result from one segment of DNA

Introns might regulate gene expression

Exciting new picture of the genome is emerging

60

Steps in Gene Expression: Translation

tRNA molecules have two binding sites

One associates with the mRNA transcript

The other associates with a specific amino acid

Each of the 20 amino acids in proteins associates with one or more of 64 species of tRNA

Translation

An mRNA transcript migrates to rough endoplasmic reticulum

Associates with the rRNA of a ribosome

The ribosome “reads” the information in the transcript

Ribosome directs various species of tRNA to bring in their specific amino acid “fares”

tRNA specified is determined by the code being translated in the mRNA transcript

61

tRNA

tRNA molecules come in 64 different kinds

All very similar except that

One end bears a specific triplet (of the 64

possible) called the anticodon

Other end binds with a specific amino acid type

tRNA synthetases attach correct amino acid to the

correct tRNA molecule

All tRNA molecules with a specific anticodon

will always bind with the same amino acid

Animation













63

Structure of tRNA


amino

acid

leucine

3

5

Hydrogen

bonding

anticodon

mRNA

5'

codon

3'

b.

anticodon end

amino acid end

64

Ribosomes

Ribosomal RNA (rRNA):

Produced from a DNA template in the nucleolus

Combined with proteins into large and small ribosomal

subunits

A completed ribosome has three binding sites to

facilitate pairing between tRNA and mRNA

The E (for exit) site

The P (for peptide) site, and

The A (for amino acid) site

65

Ribosomal Structure and Function


Courtesy Alexander Rich

large subunit

small subunit

a. Structure of a ribosome

tRNA binding

sites

outgoing

tRNA

3 5

mRNA

b. Binding sites of ribosome

polypeptide incoming

tRNA

incoming

tRNA

c. Function of ribosomes d. Polyribosome

66

Steps in Translation: Initiation

Components necessary for initiation are: Small ribosomal subunit mRNA transcript Initiator tRNA, and Large ribosomal subunit Initiation factors (special proteins that bring the above

together)

Initiator tRNA: Always has the UAC anticodon Always carries the amino acid methionine Capable of binding to the P site

67


Small ribosomal subunit attaches to mRNA

transcript

Beginning of transcript always has the START

codon (AUG)

Initiator tRNA (UAC) attaches to P site

Large ribosomal subunit joins the small

subunit

Animation













69



A small ribosomal subunit

binds to mRNA; an initiator

tRNA pairs with the mRNA

start codon AUG. The large ribosomal subunit

completes the ribosome.

Initiator tRNA occupies the

P site. The A site is ready

for the next tRNA.

Initiation

Met

amino acid methionine

initiator tRNA

mRNA

small ribosomal subunit

3'

5'

P site A site E site

Met

large ribosomal subunit

U A C

A U G

start codon 5' 3'

70

Steps in Translation: Elongation

“Elongation” refers to the growth in length of the polypeptide

RNA molecules bring their amino acid fares to the ribosome Ribosome reads a codon in the mRNA

Allows only one type of tRNA to bring its amino acid

Must have the anticodon complementary to the mRNA codon being read

Joins the ribosome at it’s A site

Methionine of initiator is connected to amino acid of 2nd tRNA by peptide bond

71


Second tRNA moves to P site (translocation)

Spent initiator moves to E site and exits

Ribosome reads the next codon in the mRNA

Allows only one type of tRNA to bring its amino acid

Must have the anticodon complementary to the mRNA codon

being read

Joins the ribosome at it’s A site

Dipeptide on 2nd amino acid is connected to amino acid

of 3nd tRNA by peptide bond

Animation













73



A tRNA–amino acid

approaches the

ribosome and binds

at the A site.

Two tRNAs can be at a

ribosome at one time;

the anticodons are

paired to the codons.

Peptide bond formation

attaches the peptide

chain to the newly

arrived amino acid.

The ribosome moves forward; the

“empty” tRNA exits from the E site;

the next amino acid–tRNA complex

is approaching the ribosome.

1 2 3 4

Elongation

peptide

bond

Met

Ala

Trp

Ser

Val

U

A

C A

U G G A C

3 3

C G

anticodon

tRNA

asp

U

Met

Ala

Trp

Ser

Val

U

A

C A

U G G A C

C U G

Asp

6

U

A

C A

U G G A C

C U G

Met

Val

Asp

Ala

Trp

Ser

peptide

bond

6 3

G A C

C U G

A U G A C C

Met

Val

Asp

Ala

Trp

Ser Thr

6 3

74

Steps in Translation: Termination

Previous tRNA moves to P site

Spent tRNA moves to E site and exits

Ribosome reads the STOP codon at the end of the mRNA UAA, UAG, or UGA

Does not code for an amino acid

Polypeptide is released from last tRNA by release factor

Ribosome releases mRNA and dissociates into subunits

mRNA read by another ribosome

Animation













76

Steps in Translation: Termination


Termination

The release factor hydrolyzes the bond

between the last tRNA at the P site and

the polypeptide, releasing them. The

ribosomal subunits dissociate.

3

5

The ribosome comes to a stop

codon on the mRNA. A release

factor binds to the site.

U A

U A U G A

stop codon 5' 3'

Asp

Ala

Trp

Val Glu

release factor

Animation

77













78

Summary of Gene Expression (Eukaryotes)


TRANSCRIPTION

1. DNA in nucleus serves

as a template for mRNA.

2. mRNA is processed

before leaving the nucleus.

mRNA

pre-mRNA

DNA

introns

3. mRNA moves into

cytoplasm and

becomes associated

with ribosomes.

TRANSLATION

mRNA large and small

ribosomal subunits

5

3'

nuclear pore

4. tRNAs with

anticodons carry

amino acids

to mRNA.

5

peptide

codon

ribosome

3 U A

A U

C G

5 C C G G

G C G

C G

C G

U A

U A

U A

U A

6. During elongation, polypeptide

synthesis takes place one

amino acid at a time. 7. Ribosome attaches to rough

ER. Polypeptide enters lumen,

where it folds and is

modified.

8. During termination, a

ribosome reaches a stop

codon; mRNA and

ribosomal subunits

disband.

5. During initiation, anticodon-codon

complementary base pairing begins

as the ribosomal subunits come

together at a start codon.

amino

acids

anticodon

tRNA

3'

Animation













80


Contains a single linear DNA molecule, but is composed of more than 50% protein.

Some of these proteins are concerned with DNA and RNA synthesis,

Histones, play primarily a structural role

Five primary types of histone molecules

Responsible for packaging the DNA DNA double helix is wound at intervals around a

core of eight histone molecules (called nucleosome)

Nucleosomes are joined by “linker” DNA.

81



a. Nucleosomes (“beads on a string”)

b. 30-nm fiber

c. Radial loop domains

d. Heterochromatin

e. Metaphase chromosome

1. Wrapping of DNA

around histone proteins.

4. Tight compaction of radial

loops to form heterochromatin.

3. Loose coiling into radial

loops.

2. Formation of a three-dimensional

zigzag structure via histone H1 and

other DNA-binding proteins.

5. Metaphase chromosome forms

with the help of a protein

scaffold.

2 nm

1 nm

300 nm

1,400 nm

700 nm

30 nm

DNA

double helix

histones

histone H1

nucleosome

euchromatin

Animation













83

Review

Genetic Material

Transformation

DNA Structure

Watson and Crick

DNA Replication

Prokaryotic versus Eukaryotic

Replication Errors

Transcription

Translation


Sylv

ia S

. Ma

der

Copyright © The McGraw Hill Companies Inc. Permission required for reproduction or display

PowerPoint® Lecture Slides are prepared by Dr. Isaac Barjis, Biology Instructor

BIOLOGY 10th Edition

Molecular Biology of the Gene

Chapter 12: pp. 211 - 232

84


b.

3.4 nm

2 nm

0.34 nm

G C

A

T

T A

P

P

P

P

C G

G

C

Sugar hydrogen bonds

sugar-phosphate

backbone

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BIOLOGY Chapter 12: 10th Edition Molecular Biology of the Gene · Green fluorescent protein (GFP) used as a marker A jellyfish gene codes for GFP The jellyfish gene is isolated and