Chapter 12 Molecular Biology Of the Gene. One gene differs from another only by the sequence of the nucleotide bases in DNA How does base sequence determine

Chapter 12

Molecular Biology

Of the Gene

• One gene differs from another only by the sequence of the nucleotide bases in DNA

• How does base sequence determine the uniqueness of a species or of individual traits between members of a species?

• DNA specifies proteins which make unique structures that make up all the characteristics of an organism

• DNA’s sequence of nucleotides sequence of amino acids specific enzymes structures

• In 1900’s, scientists knew the genetic material:• Must store information about development,

structure, and metabolic activities of a cell• Must be stable, so it could be replicated in cell

division and passed on • Must be able to undergo rare changes called

mutations to provide variability required for evolution

• In the1920’s, Frederick Griffith was working on a vaccine against Streptococcus pneumoniae

• Notices some colonies were shiny and smooth• Other colonies had a rough appearance

• Smooth colony individuals had a capsule, but those of the rough colony did not

• S strain caused death when injected into mice• R strain did not• Heat-killed S strain mixed with living R strain

caused death in mice when injected and when they were isolated from the mice had capsules

Fig. 12.1

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

• Oswald Avery worked on whether genetic material was protein or DNA

• Subjected materials to proteinases and a capsule was still produced

• Subjected materials to DNase and it was not• DNA was shown to be the genetic material• DNA structure:• Contains four nitrogenous bases • -Two purines- adenine (A) and guanine (G) that

were double ringed• -Two pyrimidines- thymine (T) and cytosine (C)

that were single ringed

• Percentage of each type of nucleotide differs from species to species

• Within a species, DNA has a constancy of bases• % of A always equals % of T and % of G always

equally % of C. These relationships are called Chargaff’s rules

Fig. 12.3 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

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

5 CH2

5 CH2

5 CH2

C

4C

4C

4C

C

3C

3C

• Each human chromosome usually contains about 140 million base pairs

• Because any of the four nucleotides can be present at each position, the total possible nucleotide sequences is 4 to the 140th x 10 to the 6th or 4 to the 140,000,000th

• Rosalind Franklin’s work with x-ray diffraction determined that DNA was a double helix

Fig. 12.4

X-ray beam

b. c.

Rosalind Franklin

diffraction pattern

CrystallineDNA

diffractedX-raysa.

© Photo Researchers, Inc.; 12.4c: © Science Source/Photo Researchers, Inc.


• Watson and Crick constructed a model of DNA and received the 1962 Nobel prize

• Polymers of nucleotides in a double helix form• Sugar-phosphate back bones on outside and

paired bases inside• Two DNA strands are antiparallel, meaning that

the sugar-phosphate groups of each strand are oriented in opposite directions

• 5’ end of one strand is paired to the 3’ end of the other strand

• Complementary base pairing means a purine always bonds to a pyrimidine

• They have an antiparallel arrangement to insure that bases are oriented properly so can interact

• This is the only model having molecular width revealed by Franklin’s x-ray diffraction pattern


P

P

P

P

c.

b.

Complementarybase pairing

sugar-phosphatebackbone

3.4 nm

2 nm

0.34 nm

P

P

S S4

5 end3 end

11

23

2 35

4

5

CG

G

C

C

G

T

T

A

A

C

Ga.

d.d.

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

• Term DNA replication refers to process of copying a DNA molecule

• A template is a mold used to produce a shape complementary to itself

• During DNA replication, each DNA strand serves as a template for a new strand in a daughter molecule

• DNA replication is semiconservative replication because each daughter DNA double helix contains an old strand from parential DNA double helix and a new strand


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

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

G

G

GG

G

G

G

G

GG

G

G

C

C

C

CC

C

C

C

C

C C

C

C

oldstrand

newstrand

• Steps in replication:• 1) Unwinding of parental DNA is caused by the

breaking of weak hydrogen bonds between paired bases

• Enzyme called helicase necessary to unwind the molecule

• 2) Complementary base pairing occurs when new complementary nucleotides, always in the nucleus, are paired

• 3) Joining finishes replication by joining the complementary nucleotides to form new strands

• Each new daughter DNA molecule contains an old strand and a new strand

• Steps 2 and 3 are done with the enzyme complex called DNA polymerase

• DNA replication must occur before a cell can divide

• Cancer is characterized by rapid cell division• Sometimes treated with chemotherapeutic drugs

that are analogs to one of four nucleotides• Analogs have similar but not identical structure• They cause replication to stop and cells to die• Bacteria have a single circular loop of DNA that

must be replicated before the cell divides• Process begins at origin of replication site

• The strands are separated, unwound, and DNA polymerase binds to each side and begins copying

• The two DNA polymerases meet at a termination region, then the chromosomes separate

• Bacteria cells require about 40 minutes to replicate, but bacterial cells can divide every 20 minutes

• So replication can begin even before previous round is complete

• In eukaryotes, DNA replication begins at numerous origins of replication along the length of the chromosome

• Replication fork is the V shape of strands formed when replication bubbles spread bidirectionly until they meet

• Chromosomes are long and linear and replicate at about 500-5,000 base pairs per minute

• Because there are many individual origins of replication, the diploid DNA in humans of over 6 billion base pairs takes some hours

• In linear chromosomes, DNA polymerase cannot replicate to the ends

• Ends of chromosomes composed of telomeres which are short DNA sequences that are repeated over and over

• Telomeres are added back by the enzyme telomerase

• Eventually telomeres are lost and the cell cannot replicate

• In stem cells, this process preserves the ends of chromosomes and prevents the loss of DNA after successive rounds of replication

• DNA polymerase is very accurate and makes a mistake approximately once per 100,000 base pairs, but has proof reading capability so overall error rate is one in 100 million base pairs


replication fork replication bubble

parental strand

daughter strand

new DNAduplexes

a. Replication in prokaryotes

b. Replication in eukaryotes

replication isoccurring in

two directions

replication iscomplete

origin

• RNA is a polymer composed of nucleotides• Contains sugar ribose and bases adenine (A)

cytosine, (C), guanine (G),and uracil (U) which replaces the thymine in DNA

• Single stranded and does not form a helix• RNA comes in three major classes:• Messenger RNA (mRNA) takes message from

DNA in nucleus to ribosomes in cytoplasm• Transfer RNA (tRNA) transfers amino acids to

the ribosomes• Ribosomal RNA (rRNA) along with ribosomal

proteins, make up the ribosomes where polypeptides are synthesized

• Two major steps in synthesizing a protein from information in the DNA

• Transcription where one of the DNA strands acts as a template to make messenger RNA

• Translation where the messenger RNA directs the sequence of amino acids into a polypeptide

• Sequence of nucleotides in DNA to mRNA specify the order of amino acids in polypeptide

• Genetic code:• Codon is a triplet code where three nucleotides

code for one of the twenty amino acids• Code is degenerate meaning most amino acids

have more than one codon

• Code is unambiguous meaning each triplet codon has only one meaning

• Code has one start signal (AUG) and three stop signals (UAA) (UGA) and (UAG)

• Code is universal

Fig. 12.10

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

GUAvaline

GUGvaline

UCAserine

CCUproline

CCCproline

CCAproline

CCGproline

ACUthreonine

ACCthreonine

ACAthreonine

ACGthreonine

GCUalanine

GCCalanine

GCAalanine

GCGalanine

CAUhistidine

UAAstop

UCUserine

UAUtyrosine

UGUcysteine

UUCphenylalanine

UCCserine

UACtyrosine

UGUcysteine

UUAleucine

UCGserine

UAGstop

UUGleucine


• Messenger RNA has a sequence of bases complementary to a portion of one DNA strand

• When a gene is transcribed, a segment of DNA helix unwinds and unzips

• Rna nucleotides pair with complementary DNA nucleotides; this is known as the template strand

• RNA polymerase joins the nucleotides together in the 5’ 3’ direction or adds a nucleotide to the 3’ end of polymer under construction

• Transcription begins when RNA polymerase attaches to promoter in DNA

• Promoter defines the start of transcription, the direction of transcription, and the strand to be transcribed

• Initiation of transcription is the binding of RNA polymerase to promoter

• Elongation of mRNA continues until RNA polymerase comes to DNA stop codon sequence

• Causes release of mRNA now called mRNA transcript

• RNA transcript is called pre-mRNA and is modified before leaving the nucleus

• Receives a cap at 5’ end and a tail at 3’ end

• Cap is modified guanine (G) nucleotide which tells ribosome where to attach when translation begins

• Tail consists of 150-200 adenine (A) nucleotides• This poly-A tail helps transport of mRNA out of

nucleus and inhibits degration of mRNA by hydrolytic enzymes

• Pre-mRNA is composed of exons and introns• Exons are segments that will be expressed• Introns are segments in between exons that will

not be expressed• During pre-mRNA splicing, introns are removed

• In prokaryotes, introns splice themselves out• In eukaryotes, DNA splicing is done by

spliceosomes which contain small nuclear RNA (snRNA)

• Spliceosomes use ribozymes whose catalytic activity works like enzymes that are made of protein

• Presence of introns allows a cell to pick and chose which exons will go into a particular mRNA

• What is an exon in one mRNA could be an intron in another mRNA

• Called alternate mRNA splicing

• Some introns give rise to micro RNA (miRNA) which are involved in regulating the translation of mRNA

• Introns may also encourage crossing-over during meiosis, and permit exon shuffling which can play a role in evolution of new genes

Fig. 12.13

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


• Translation takes place in the cytoplasm of eukaryotic cells

• Transfer RNA (tRNA) molecules transfer amino acids to the ribosomes

• tRNA is a single stranded nucleic acid that doubles back on itself to create regions where complementary bases hydrogen bond to one another

• There is at least one tRNA molecule for each of the 20 amino acids

• Amino acids bind to 3’ end of tRNA• The opposite end contains an anticodon that is

complementary to a specific mRNA codon

Fig. 12.14

G

GA

A A

CCC CC CU U UU

Hydrogenbonding

codon

anticodon

mRNA35

aminoacid

leucine

3

5

anticodon end

amino acid end

b.a.


• In eukaryotes, ribosomal RNA (rRNA) is produced from a DNA template in the nucleolus of a nucleus

• Packaged with proteins into two ribosomal subunits one larger than the other

• They move into the cytoplasm where they combine when translation begins

• May remain in cytoplasm or attach to endoplasmic reticulum

• Ribosomes have a binding site for mRNA and three binding sites for tRNA

• When ribosome moves down a mRNA molecule, the polypeptide increases by one AA at a time

• Several ribosomes are often attached to and translating the same mRNA

• The entire complex is called a polyribosome

Fig. 12.15

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

• Translation requires three steps:• 1) Initiation brings all the translation components

together• Initiation factors (proteins) assemble small

ribosome subunit, mRNA, initiator tRNA, and large ribosomal subunit

• 2) Elongation where polypeptide increases in length one amino acid at a time

• Requires elongation factors (proteins) for binding tRNA anticodons to mRNA codons

• 3) Termination of polypeptide occurs at a stop codon

• Requires protein called release factor which binds to stop codon and cleaves the polypeptide from the last tRNA

Fig. 12.17

UA

C AUG

C U G

G A C

UA

C AUG G A C

C U G

Elongation

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 AUG 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

Fig. 12.18


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.

3

release factorAla

Trp

Val

Asp

Glu

3

5

UA

UA U G A

AG A

U G A

UC

U

Termination

stop codon

• Gene has been expressed once its product is made and is operating in the cell

• Eukaryotic chromosome contains a single double helix DNA molecule, but is composed of more than 50% protein

• Histones are the large majority and play a primary structural role

• A human cell contains at least 2 meters of DNA

Documents

Chapter 12 Molecular Biology Of the Gene. One gene differs from another only by the sequence of the nucleotide bases in DNA How does base sequence determine