biology sylvia s. mader michael windelspecht chapter 12 molecular biology of the gene lecture...

BiologySylvia S. Mader

Michael Windelspecht

Chapter 12Molecular Biology

of the GeneLecture Outline

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

See separate FlexArt PowerPoint slides for all figures and tables pre-inserted into

PowerPoint without notes.

1

12.1 The Genetic Material

• Frederick Griffith investigated virulence of Streptococcus pneumoniae Concluded that virulence could be passed

from a dead strain to a nonvirulent living strain Transformation

• Further research by Avery et al. Discovered that DNA is the transforming

substance DNA from dead cells was being incorporated

into the genome of living cells2

The Genetic Material

• 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.

3

Griffith’s Transformation Experiment

4


capsule

Injected live S strain hascapsule and

causes mice to die.

a. b.

Injected live R strain hasno 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 liveR strain causes

mice to die. Live S strain iswithdrawn from

dead mice.


• Transformation of organisms today: Result is the so-called genetically modified

organisms (GMOs) • Invaluable tool in modern biotechnology today• Commercial products that are currently much used • Green fluorescent protein (GFP) can be 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

5

Transformation of Organisms

6


• DNA contains: Two Nucleotides with purine bases

• Adenine (A)• Guanine (G)

Two Nucleotides with pyrimidine bases• Thymine (T)• Cytosine (C)

7

The Genetic Material• Chargaff’s Rules:

The amounts of A, T, G, and C in DNA:• Are constant among members of the same species• Vary from species to species

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

All this suggests that DNA uses complementary base pairing to store genetic information

Each human chromosome contains, on average, about 140 million base pairs

The number of possible nucleotide sequences is 4140,000,000

8

Nucleotide Composition of DNA

9


O

N

N

CH

CH

C

C

NH2

cytosine(C)

3C C2

C1

OHO P O

O

H

HH

HH

OH

CH3

O

HN

N

C

CH

C

C

OHO P O

O

H

HH

HH

OH

HN

N

N

CCH

O

C

CC

NH2N

C2

C2

C1

C1

OHO P O

O

guanine(G)

phosphate

H

HH

HH

OH

N

N

N

HCCH

NH2

C

CC

N

4

3C

2

C1

5 O

O

O

O

O

O

H

HH

HH

OH

c. Chargaff’s data

DNA Composition in Various 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-containingbase

sugar = deoxyribose

thymine(T)

adenine(A)

HO P O CH2

5CH2

5CH2

5CH2

C

4C

4C

4C

C

3C

3C

O


• X-Ray diffraction: 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 an

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.

10

X-Ray Diffraction of DNA

11


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

X-ray beam

b.c.

Rosalind Franklin

diffraction pattern

CrystallineDNA

diffractedX-rays

a.


• The Watson and Crick Model (1953)

Double helix model is similar to a twisted ladder

• Sugar-phosphate backbones make up the sides

• Hydrogen-bonded bases make up the rungs

Complementary base pairing ensures that a purine is always bonded to a pyrimidine (A with T, G with C)

Received a Nobel Prize in 1962

12

Watson and Crick Model of DNA

13


P

P

P

P

c.

P

S SCG

PP

S

SAT

G

C

C

G

T

T

A

A

C

G

P

0.34 nm3.4 nm

sugar-phosphatebackbone

a.

b.

d.

5′ end 3′ end

3′ end 5′ endcomplementarybase pairing

hydrogen bondssugar

2 nm

a: © Photodisk Red/Getty RF; d: © A. Barrington Brown/Photo Researchers

12.2 Replication of DNA

• DNA replication is the process of copying a DNA molecule.

• Semiconservative replication - each strand of the original double helix (parental molecule) serves as a template (mold or model) for a new strand in a daughter molecule.

14

Semiconservative Replication

15


oldstrand

newstrand

A

A

A

A

A

A

A

A

AA

A

A

AA

A

A

A

A

A

A

T

T

T

T

T

TT

T

T

T

TT

T

G

G

G

G

GG

GG

G

G

G

G

GG

G

G

C

C

C

CC

C

C

C

C

C C

C

C

A

T

TG

C

A

T

T

GC

C

region of parentalDNA double helix

region ofreplication:new nucleotidesare pairingwith those ofparental strands

region ofcompletedreplication

oldstrand

newstrand

daughter DNA double helix

daughter DNA double helix

DNA polymeraseenzyme

5′ 3′

3′

5′

16

Replication of DNA

• Replication requires the following steps:

Unwinding, or separation of the two strands of the parental DNA molecule

Complementary base pairing between a new nucleotide and a nucleotide on the template strand

Joining of nucleotides to form the new strand

• Each daughter DNA molecule contains one old strand and one new strand

Replication of DNA

• Prokaryotic Replication Bacteria have a single circular loop of DNA

Replication moves around the circular DNA molecule in both directions

Produces two identical circles

The process begins at the origin of replication

Replication takes about 40 minutes, but the cell divides every 20 minutes

• A new round of replication can begin before the previous round is completed

17

Replication of DNA• Eukaryotic Replication

DNA replication begins at numerous points along each 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 a new strand paired with each old strand

• Requires enzyme DNA polymerase

18

Replication of DNA• Eukaryotic Replication

Replication bubbles spread bidirectionally until they meet

The complementary nucleotides are joined 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.

19

Replication of DNA

• Accuracy of Replication

DNA polymerase is very accurate, yet makes a mistake about once per 100,000 base pairs.

• Capable of identifying and correcting errors

20

12.3 The Genetic Code of Life

• Genes Specify Enzymes Beadle and Tatum:

• Experiments on the fungus Neurospora crassa

• Proposed that each gene specifies the synthesis of one enzyme

• One-gene-one-enzyme hypothesis

21

The Genetic Code of Life• The mechanism of gene expression

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

Genetic information is expressed into structure and function through protein synthesis

• The expression of genetic information into structure and function: DNA in a gene determines the sequence of

nucleotides in an RNA molecule RNA controls the primary structure of a protein

22

The Central Dogma of Molecular Biology

23

codon 1 codon 2

nontemplate strand

template strand

codon 3

polypeptide N N NC C C C C C

R1 R2 R3

Serine Aspartate Proline

O O O

C

G

G

CA

G

CC

G

C

GTT

AG

C G

C

G CA CC CAG C

transcriptionin nucleus

translationat ribosome

mRN A

35

35

53


DNA

The Genetic Code of Life

• RNA is a polymer of RNA nucleotides RNA nucleotides contain the sugar ribose instead of

deoxyribose RNA nucleotides are of four types: uracil (U),

adenine (A), cytosine (C), and guanine(G) Uracil (U) replaces thymine (T) of DNA

• Types of RNA• Messenger (mRNA) - Takes a message from DNA in the

nucleus to ribosomes in the cytoplasm• Ribosomal (rRNA) - Makes up ribosomes, which read the

message in mRNA• Transfer (tRNA) - Transfers the appropriate amino acid to

the ribosomes for protein synthesis

24

Structure of RNA

25


G

U

A

C

S

S

S

S

P

P

P

P

base isuracil insteadof thymine

one nucleotideribose3′ end

5′ end

RNA Structure Compared to DNA structure

26

The Genetic Code of Life• The unit of the genetic 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”

27

Messenger RNA Codons

28

U C A G

U

C

A

G

U

C

A

G

U

C

A

G

U

C

A

G

U

C

A

G

Second Base ThirdBase

FirstBase

CGAarginine

CGGarginine

AGUserine

AGCserine

AGAarginine

AGGarginine

GGUglycine

GGCglycine

GGAglycine

GGGglycine

UGGtryptophan

CGUarginine

CGCarginine

UGAstop

AUG (start)methionine

CAChistidine

CAAglutamine

CAGglutamine

AAUasparagine

AACasparagine

AAAlysine

AAGlysine

GAUaspartate

GACaspartate

GAAglutamate

GAGglutamate

UUUphenylalanine

CUUleucine

CUCleucine

CUAleucine

CUGleucine

AUUisoleucine

AUCisoleucine

AUAisoleucine

GUUvaline

GUCvaline

GUAvalineGUGvaline

UCAserine

CCUproline

CCCproline

CCAproline

CCGproline

ACUthreonine

ACCthreonine

ACAthreonine

ACGthreonine

GCUalanine

GCCalanine

GCAalanineGCG

alanine

CAUhistidine

UAAstop

UCUserine

UAUtyrosine

UGUcysteine

UUCphenylalanine

UCCserine

UACtyrosine

UGUcysteine

UUAleucine

UCGserine

UAGstop

UUGleucine


The Genetic Code of Life

• 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

29

12.4 First Step: Transcription• Transcription

A segment of DNA serves as a template for the production of an RNA molecule

The gene unzips and exposes unpaired bases Serves as template for mRNA formation Loose RNA nucleotides bind to exposed DNA

bases using the C=G and A=U rule When entire gene is transcribed into mRNA, the

result is a pre-mRNA transcript of the gene The base sequence in the pre-mRNA is

complementary to the base sequence in DNA

30

First Step: Transcription • A single chromosome 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

31

Transcription

32


T

TG

C

A

T

T

G

C

A

terminator

gene strand

promoter

templatestrand

direction ofpolymerasemovement

RNA polymerase

DNA template strand

mRNAtranscript

to RNA processing

3′

3′5′

5′

5′

3′

RNA Polymerase

33© Oscar L. Miller/Photo Researchers, Inc.


a. 200m

b.

spliceosome

DNA

RNApolymerase

RNAtranscripts

First Step: Transcription

• Pre-mRNA is modified before leaving the eukaryotic nucleus. Modifications to the ends of the primary

transcript:• Cap on the 5′ end

– The cap is a modified guanine (G) nucleotide

– Helps a ribosome determine where to attach when translation begins

• Poly-A tail of 150-200 adenines on the 3′ end– Facilitates the transport of mRNA out of the nucleus

– Inhibits degradation of mRNA by hydrolytic enzymes. 34

First Step: Transcription• 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 a mature mRNA transcript35

First Step: Transcription

• 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

36

Messenger RNA Processing in Eukaryotes

37

exon

intron intron

exon exonDNA

transcription

exon

intron intron

exon exon

5 3

exon

intron intron

exon exon

exon

intron RNA

exon

pre-mRNAsplicing

exon

cap poly-A tail

spliceosome

nucleus

mRNA

cytoplasm

nuclear porein nuclear envelope

cap poly-A tail

cap poly-A tail

3

3

5

5

pre-mRNA

3 5


First Step: Transcription• Functions of introns:

As organismal complexity increases;• The number of protein-coding genes does not keep pace• But the proportion of the genome that is introns increases

Possible functions of introns:• Exons might combine in various combinations

– Would allow different mRNAs to result from one segment of DNA• Introns might regulate gene expression• Introns may encourage crossing-over during meiosis

Exciting new picture of the genome is emerging

38

12.5 Second Step: Translation

• Translation The sequence of codons in the mRNA at a ribosome directs the sequence of

amino acids into a polypeptide A nucleic acid sequence is translated into a protein sequence

• 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

types of tRNA• Translation

An mRNA transcript associates with the rRNA of a ribosome in the cytoplasm or a ribosome associated with the rough endoplasmic reticulum

The ribosome “reads” the information in the transcript Ribosome directs various types of tRNA to bring in their specific amino acid

“fares” The tRNA specified is determined by the code being translated in the mRNA

transcript

39

Second Step: Translation

• tRNA molecules come in 64 different kinds• All are very similar except that

One end bears a specific triplet (of the 64 possible) called the anticodon

The other end binds with a specific amino acid type

tRNA synthetases attach the correct amino acid to the correct tRNA molecule

• All tRNA molecules with a specific anticodon will always bind with the same amino acid

40

Structure of a tRNA Molecule

41


G

GA

A A

CCC CC CU U UU

3’

5’

hydrogenbonding

amino acid end

anticodon end

mRNA5’ 3’

aminoacid

leucine

anticodon

a. b.codon


• Ribosomes

Ribosomal RNA (rRNA):

• Produced from a DNA template in the nucleolus of a nucleus

• 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

42

Ribosome Structure and Function

43

tRNA bindingsites

outgoingtRNA

3

mRNA

incomingtRNA

polypeptide

5

small subunit

mRNA

large subunit

a. Structure of a ribosome b. Binding sites of ribosome

c. Function of ribosomes d. Polyribosome


Courtesy Alexander Rich

EP

A


• 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

44


• 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

45

Initiation

46

U A CA U G

U A CA U G

A small ribosomal subunitbinds to mRNA; an initiatortRNA pairs with the mRNAstart codon AUG. The large ribosomal subunit

completes the ribosome.Initiator tRNA occupies theP site. The A site is readyfor the next tRNA.

Met

amino acid methionine

initiator tRNA

mRNA

3

5

small ribosomal subunit

large ribosomal subunit

P site A siteE site

35

Met

Initiation

start codon


Second Step: 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• The incoming tRNA joins the ribosome at its A site

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

47

Second Step: Elongation

• The second tRNA moves to P site (translocation)• The spent initiator tRNA moves to the E site and

exits• The 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 its A site

The dipeptide on the 2nd amino acid is connected to the amino acid of the 3rd tRNA by a peptide bond

48

Elongation

49

Elongation


UA

C AUG

C U G

G A C

U

A

C A

UG G A C

C U G

A tRNA–amino acidapproaches theribosome and bindsat the A site.

Two tRNAs can be at aribosome at one time;the anticodons arepaired to the codons.

anticodon

tRNApeptidebond

asp

35 5

Asp

1 2 4

UA

C A

UG G A C

C U G

UC

A

G A C

C U G

AUG

U G G

A C C

Peptide bond formationattaches the peptidechain to the newlyarrived amino acid.

The ribosome moves forward; the“empty” tRNA exits from the E site;the next amino acid–tRNA complexis approaching the ribosome.

35

Met

Val

Asp

Ala

Trp

Ser

3 35

Met

Val

Asp

Ala

Trp

Ser Thr

3

peptidebond

Met

Ala

Trp

Ser

Val

Met

Ala

Trp

Ser

Val


• Termination: Previous tRNA moves to the P site Spent tRNA moves to the 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

A protein called a release factor binds to the stop codon and cleaves the polypeptide from the last tRNA

The ribosome releases the mRNA and dissociates into subunits

The same mRNA may be read by another ribosome

50

Termination

51


The ribosome comes to a stopcodon on the mRNA. A releasefactor binds to the site.

The release factor hydrolyzes the bondbetween the last tRNA at the P site andthe polypeptide, releasing them. Theribosomal subunits dissociate.

release factorAla

Asp

GluUA

UA U G A

AG A

U G A

UC

U

Termination

stop codon

Asp

Ala

Trp

Trp

Val

Val

Glu

3′

5′

5′ 3′

Summary of Protein Synthesis in Eukaryotes

52

5

5

3mRNA

pre-mRNA

DNA

introns

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.

3. mRNA moves into cytoplasm and becomes associated with ribosomes.

4. tRNAs with anticodons carry amino acids to mRNA.

TRANSLATIONTRANSCRIPTION

nuclear pore

mRNA

large and smallribosomal subunits

aminoacids

anticodon

tRNA

peptide

codon

ribosome

5

3

3

C CG G

GCG

CG

C

CCC

GUA

CU A

UA

UA

UUA

UA

AU

CG

A

1. DNA in nucleus serves as a template for mRNA.

2. mRNA is processed before leaving the nucleus.

5. During initiation, anticodon-codon complementary base pairing begins as the ribosomal subunits come together at a start codon.

8. During termination, a ribosome reaches a stop codon; mRNA and ribosomal subunits disband.


12.6 Structure of the Eukaryotic Chromosome

• Each chromosome 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 The most abundant proteins in chromosomes Five primary types of histone molecules Responsible for packaging the DNA

• The DNA double helix is wound at intervals around a core of eight histone molecules (called a nucleosome)

• Nucleosomes are joined by “linker” DNA. 53

Structure of Eukaryotic Chromosomes

54a: © Ada L. Olins and Donald E. Olins/Biological Photo Service; b: Courtesy Dr. Jerome Rattner, Cell Biology and Anatomy, University of Calgary; c: Courtesy of Ulrich Laemmli and J.R. Paulson, Dept. of Molecular Biology, University of Geneva, Switzerland; d: © Peter Engelhardt/Department of Pathology and Virology, Haartman Institute/Centre of Excellence in Computational Complex Systems Research,

Biomedical Engineering and Computational Science, Faculty of Information and Natural Sciences, Helsinki University of Technology, Helsinki, Finland


1. Wrapping of DNA around histone proteins.

2. Formation of a three-dimensional zigzag structure via histone H1 and other DNA-binding proteins.

3. Loose coiling into radial loops .

4. Tight compaction of radial loops to form heterochromatin.

5. Metaphase chromosome forms with the help of a protein scaffold.

DNAdouble helix

nucleosome

euchromatin

c. Radial loop domains

d. Heterochromatin

e. Metaphase chromosome

1,400 nm

700 nm

300 nm

30 nm

histone H1

histones

11 nm

2nm

a. Nucleosomes (“beads on a string”)

b. 30-nm fiber

biology sylvia s. mader michael windelspecht chapter 12 molecular biology of the gene lecture...

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