Molecular Biology of the Gene

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Molecular Biology of the Gene

• Chapter 10

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• In 2002, 5 million people worldwide were newly
  infected with HIV

• Because all organisms use the same genetic code,
  scientists can make a plant glow like a firefly

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• The loss of a single nucleotide from a
  1,000-nucleotide gene can completely destroy the
  genes function

• Many viruses have genes that are not made of DNA

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BIOLOGY AND SOCIETY SABOTAGING HIV

• AIDS is one of the most challenging health
  problems facing the world today

• Infection by HIV can cause AIDS

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• The drug AZT (3-azido-3deoxythymidine or
  Azidothymidine/Zidovudine) is effective at
  preventing the spread of HIV

Thymine (T)
Part of a T nucleotide
AZT
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THE STRUCTURE AND REPLICATION OF DNA

• DNA

• Was known as a chemical in cells by the end of
  the nineteenth century
• Has the capacity to store genetic information
• Can be copied and passed from generation to
  generation

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DNA and RNA Polymers of Nucleotides

• DNA and RNA are nucleic acids

• They consist of chemical units called nucleotides
• The nucleotides are joined by a sugar-phosphate
  backbone

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Phosphate group
Nitrogenous base
Sugar
Nitrogenous base (A,G,C, or T)
Nucleotide
Thymine (T)
Phosphategroup
Sugar (deoxyribose)
DNA nucleotide
Polynucleotide
Sugar-phosphate backbone
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• The four nucleotides found in DNA

• Differ in their nitrogenous bases
• Are thymine (T), cytosine (C), adenine (A), and
  guanine (G)
• RNA has uracil (U) in place of thymine

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Watson and Cricks Discovery of the Double Helix

• James Watson and Francis Crick determined that
  DNA is a double helix

James Watson and Francis Crick
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James D. Watson and Francis CrickDNA
characteristics

• Ability to replicate itself
• Encode information
• Control the destiny and function of each cell
• Structure should change as a result of mutation
  it should be flexible to minor changes

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• Watson and Crick used X-ray crystallography data
  to reveal the basic shape of DNA

• Rosalind Franklin collected the X-ray
  crystallography data

(b) Rosalind Franklin
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• The model of DNA is like a rope ladder twisted
  into a spiral

Twist
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• Detailed representations of DNA

• Notice that the bases pair in a complementary
  fashion

(a)
(b)
(c)
Hydrogen bond
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DNA Replication

• When a cell or organism reproduces, a complete
  set of genetic instructions must pass from one
  generation to the next

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• Watson and Cricks model for DNA suggested that
  DNA replicated by a template mechanism

Parental (old) DNA molecule
Daughter (new) strand
Daughter DNA molecule (double helices)
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Watson-Crick DNA replication depends on specific
base pairing
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Semiconservative replication
H-bonds break, parent strands separate
Enzymes use each parent strand as template
to assemble new strands
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• DNA can be damaged by ultraviolet light

• The enzymes and proteins involved in replication
  can repair the damage

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DNA replication

• DNA replication requires more than a dozen
  enzymes and proteins to work together
• Rate of nucleotide addition
• 50 per second in mammals
• 500 per second in bacteria

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DNA replication begins at specific sites
Origin of replication
Parental strand
Daughter strand
Bubble
Two daughter DNA molecules
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Origin of replication
Origin of replication

• DNA replication

• Begins at specific sites on a double helix
• Proceeds in both directions

Origin of replication
Parental strand
Daughter strand
Bubble
Two daughter DNA molecules
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3 end
5 end
Each strand of the double helix is oriented in
the opposite direction
3 end
5 end
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3?

• Helicase
• Unwinds DNA
• double helix

DNA polymerasemolecule
5?
daughter strand synthesized continuously
Parental DNA
5?
3?

• DNA polymerases
• link nucleotides in
• the new DNA strand
• adds nucleotides only
• in the 5 ? 3
• direction

Helicase
daughter strand synthesized in pieces
3?
5?
3?
5?
DNA ligase - links up the short segments (or
Osazaki fragments) of newly synthesized DNA
5?
3?
3?
DNA ligase
5?
Overall direction of replication
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DNA replication is very accurate one in 10,000
nucleotides are incorrectly paired DNA
polymerases also proofread the newly synthesized
DNA DNA polymerases and DNA ligase repair DNA
damaged by harmful radiations (UV or X-rays) or
toxic chemicals in the environment After
proofreading and repair, one in a billion
nucleotides are incorrectly paired
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The flow of genetic information is from DNA to
RNA to PROTEIN

• DNA functions as the inherited directions for a
  cell or organism

• How are these directions carried out?

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How an organisms DNA genotype produces its
phenotype

• The information of an organisms genotype is
  carried in its sequence of bases
• The genotype is expressed as proteins, which
  provide the molecular basis of phenotypic traits
• DNA does not directly synthesize proteins, but
  passes on specific information for protein
  synthesis via the RNA to the cytoplasm

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A specific gene specifies a polypeptideDNA is
transcribed into RNA, which is translated into
the polypeptide
DNA
Nucleus
TRANSCRIPTION
Nuclear membrane
mRNA
tRNA Ribosomes (rRNA)
TRANSLATION
Cytoplasm
Protein
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DNA are a small number of large immobile
molecules that change little during their
lifetime RNA are small, highly mobile and
short lived
DNA
TRANSCRIPTION
mRNA
TRANSLATION
tRNA Ribosomes (rRNA)
Protein
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• The one geneone protein hypothesis states that
  the function of an individual gene is to dictate
  the production of a specific protein
• One gene one polypeptide hypothesis

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From nucleotide sequence to amino acid sequence
an overview

• The information, or language, in DNA is
  ultimately translated into the language of
  polypeptides

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Gene 1
Gene 3
DNA molecule
Gene 2
DNA strand
TRANSCRIPTION
mRNA
Codon
TRANSLATION
Polypeptide
Amino acid
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An exercise in translating the genetic code
_____________________ T A C T T C A A A A T C A T
G A A G T T T T A G
transcribed strand
DNA helix
What is the sequence of bases in the mRNA?
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An exercise in translating the genetic code
Transcribed strand
DNA
Transcription
mRNA
Stopcodon
Startcodon
Translation
Polypeptide
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Genetic information written in codons is
translated in amino acid sequences

• Four different nucleotides code for twenty amino
  acids
• The words of the DNA language are triplets of
  bases called codons
• The DNA code is
• - universal (applies to all species, almost)
• - degenerate (redundant)
• - unambiguous
• - non-overlapping

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The genetic code
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Cracking the genetic code
Marshal Nirenberg and Heinrich Matthaei
(1961) Artificial mRNA poly U ? poly
phenylalanine poly A ? poly lysine poly
C ? poly proline etc. 61 of 64 triplets code
for amino acids AUG is the start codon and codes
for methionine UAA, UAG and UGA are stop codons
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• The genetic code is shared by all organisms

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Decoding the DNAPart I TRANSCRIPTION
DNA
Nucleus
TRANSCRIPTION
Nuclear membrane
mRNA
TRANSLATION
tRNA Ribosomes (rRNA)
Cytoplasm
Protein
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Transcription From DNA to RNA

• In transcription

• Genetic information is transferred from DNA to
  RNA
• An mRNA (messenger RNA) molecule is transcribed
  from a DNA template

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Initiation of Transcription

• The start transcribing signal is a nucleotide
  sequence called a promoter

• The first phase of transcription is initiation
• RNA polymerase attaches to the promoter
• RNA synthesis begins

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RNA Elongation

• The second phase of transcription is elongation

• The RNA grows longer

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Termination of Transcription

• The third phase of transcription is termination

• RNA polymerase reaches a sequence of DNA bases
  called a terminator

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• Transcription of an entire gene

RNA polymerase
DNA of gene
Promoter DNA
Terminator DNA
Initiation
mRNA
Elongation
Termination
Growing mRNA
Completed mRNA
RNA polymerase
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A close-up view of transcription
RNA nucleotides
RNA polymerase
Newly made mRNA
Direction of transcription
Template strand of DNA
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Overview of transcription

• Initiation
• - RNA polymerase binds to promoter sequence
• Elongation
• - mRNA synthesis continues, RNA peels off
  from DNA
• Termination
• - RNA polymerase reaches termination
  sequence
• - RNA polymerase detaches from DNA strand
  and mRNA strand

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The Processing of Eukaryotic RNA

• The eukaryotic cell processes the mRNA after
  transcription

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Intron
Exon

• RNA processing includes

Exon
Intron
Exon
DNA
Transcription Addition of cap and tail
Cap
RNA transcript with cap and tail

• Adding a cap (G) and tail (poly A)
• Removing introns
• Splicing exons together

Tail
Introns removed
Exons spliced together
mRNA
Coding sequence
Nucleus
Cytoplasm
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Decoding the DNAPart II TRANSLATION
DNA
Nucleus
TRANSCRIPTION
Nuclear membrane
mRNA
TRANSLATION
tRNA Ribosomes (rRNA)
Cytoplasm
Protein
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Key players in translation

• - Messenger RNA
• - Transfer RNAs
• - Ribosomes
• (amino acids, ATP, specific enzymes and protein
  factors)

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Messenger RNA (mRNA)

• mRNA

• Is the first ingredient for translation

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Transfer RNA (tRNA)

• tRNA

Amino acid attachment site

• Acts as a molecular interpreter
• Carries amino acids
• Matches amino acids with codons in mRNA using
  anticodons

Hydrogen bond
RNA polynucleotide chain
Anticodon
Anticodon
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Structure of transfer RNA
Amino acid attachment site
5
3
CCA
Hydrogen bond
RNA polynucleotide chain
Anticodon
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Transfer RNAs
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Ribosomes

• Ribosomes

tRNA binding sites

• Are organelles that actually make polypeptides
• Are made up of two protein subunits
• Contain ribosomal RNA (rRNA)

A site
P site
Large subunit
mRNA binding site
A
P
Small subunit
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• A fully assembled ribosome holds tRNA and mRNA
  for use in translation

Next amino acid to be added to polypeptide
Growing polypeptide
tRNA
mRNA
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Translation The Process

• Translation is divided into three phases

• Initiation
• Elongation
• Termination

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Initiation

• The first phase brings together

• The mRNA
• The first amino acid with its attached tRNA
• The two subunits of the ribosome

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• An mRNA molecule has a cap and tail that help it
  bind to the ribosome

Start of genetic message
End
G cap
Poly A tail
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Met
Initiator tRNA

• The process of initiation

mRNA
Start codon
1
Small ribosomal subunit
Large ribosomal subunit
A site
P site
Initiation
2
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Elongation

• Step 1, codon recognition

• The anticodon of an incoming tRNA pairs with the
  mRNA codon

• Step 2, peptide bond formation

• The ribosome catalyzes bond formation between
  amino acids

• Step 3, translocation

• A tRNA leaves the P site of the ribosome
• The ribosome moves down the mRNA

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• The process of elongation

Amino acid
Polypeptide
P site
Anticodon
mRNA
A site
Codons
1
Codon recognition
Elongation
2
Peptide bond formation
New peptide bond
mRNA movement
3
Translocation
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Termination

• Elongation continues until the ribosome reaches a
  stop codon

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Overview of translation

• Initiation
• - mRNA attaches to small subunit of ribosome
• - initiator tRNA carrying methionine binds to
  start codon
• - large subunit of ribosome binds to small
  subunit such that the initiator tRNA fits to
  the P site
• Elongation
• - codon recognition by specific tRNA that
  enters the A site
• - peptide bond formation between amino acids at
  P and A site
• - translocation ribosome moves along mRNA,
  tRNA with attached polypeptide moves from A
  to P site
• Termination
• - stop codon reaches A site
• - release factor binds to A site
• - polypeptide detaches from ribosome

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Review DNA? RNA? Protein

• The flow of genetic information in a cell

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RNA Polymerase
1
1
Transcription
Nucleus
RNA transcript
DNA
2
RNA processing
Intron
Amino acid
CAP
Tail
mRNA
Intron
Enzyme
tRNA
3
Amino acid attachment
Ribosomal subunits
4
4
Initiation of translation
Stop codon
Anticodon
Codon
6
Termination
5
Elongation
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• In eukaryotic cells

• Transcription occurs in the nucleus
• Translation occurs in the cytoplasm

• Transcription and translation

• Are the processes whereby genes control the
  structures and activities of cells

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Mutations

• A mutation

• Is any change in the nucleotide sequence of DNA

Normal hemoglobin DNA
Mutant hemoglobin DNA
mRNA
mRNA
Sickle-cell hemoglobin
Normal hemoglobin
Glu
Val
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Mutagens

• Mutations may result from

• Errors in DNA replication
• Physical or chemical agents called mutagens

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Types of Mutations

• Mutations within a gene

• Can be divided into two general categories
• Can result in changes in the amino acids in
  proteins

mRNA
Protein
Met
Lys
Phe
Gly
Ala
(a) Base substitution
Met
Lys
Phe
Ser
Ala
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• Insertions and deletions

• Can have disastrous effects
• Change the reading frame of the genetic message

mRNA
Protein
Met
Lys
Phe
Gly
Ala
(b) Nucleotide deletion
Met
Lys
Leu
Ala
His
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• SILENT MUTATION when substitution of one base
• for another leads to no change in the amino acids
• Mutation at position 12 in DNA C ? A

DNA template strand
GGC
3------TAC ACC GAG GGA CTA ATT------5
Transcription
CCG
5------AUG UGG CUC CCU GAU UAA------3
mRNA
Translation
Met Trp Leu Pro Asp Stop
Peptide
Result no change in amino acid sequence
CCG codes for Proline, as does CCU, CCA and CCC
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The genetic code is degenerate
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2. MISSENSE MUTATION a base change that results
in the substitution of one amino acid for another
in the protein Mutation at position 14 in the
DNA T?A
CTA
DNA template strand
3------TAC ACC GAG GGC CAA ATT------5
Transcription
GAU
5------AUG UGG CUC CCG GUU UAA------3
mRNA
Translation
Met Trp Leu Pro Val Stop
Peptide
Result Amino acid change at position 5 Asp ? Val
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Sickle Cell Anemia
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3. NONSENSE MUTATION a base substitution that
causes a chain terminator (stop) codon to form in
the mRNA Mutation at position 5 in DNA C?T
DNA template strand
ACC
3------TAC ATC GAG GGC CTA ATT------5
Transcription
UGG
5------AUG UAG CUC CCG GAU UAA------3
mRNA
Translation
Met Stop
Peptide
Result Only one amino acid translated no
protein made
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4. FRAMESHIFT MUTATION single base insertions
or deletions into the DNA that shifts the reading
frame of the genetic code Mutation by insertion
of T between bases 6 and 7 in DNA
DNA template strand
3------TAC ACC GAG GGC CTA ATT------5
T
DNA template strand
3------TAC ACC GAG GGC CTA ATT------5
Transcription
5------AUG UGG ACU CCC GGA UUA A------3
mRNA
Translation
Met Trp Thr Pro Gly Leu ---
Peptide
Result All amino acids changed beyond insertion
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Neutral mutations occur in sequences of DNA in
the non-coding regions that have no apparent
function Mutation is the only way new alleles
are produced. Thus, they play a key role in the
evolution of life forms
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• Although mutations are often harmful

• They are the source of the rich diversity of
  genes in the living world
• They contribute to the process of evolution by
  natural selection

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VIRUSES GENES IN PACKAGES

• Viruses sit on the fence between life and non-life

• They exhibit some but not all characteristics of
  living organisms

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Bacteriophages

• Bacteriophages, or phages

• Attack bacteria

Head
Tail
Tail fiber
DNA of virus
Bacterial cell
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• Phages have two reproductive cycles

Bacterial chromosome (DNA)
Phage DNA
4
Cell lyses, releasing phages
1
Many cell divisions
7
Occasionally a prophage may leave the
bacterial chromosome
Lysogenic cycle
Lytic cycle
2
Phage DNA circularizes
6
Lysogenic bacterium reproduces normally, replicati
ng the prophage at each cell division
Prophage
3
5
New phage DNA and proteins are sythesized
Phage DNA inserts into the bacterial chromosome
by recombination
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Plant Viruses

• Viruses that infect plants

Protein
RNA

• Can stunt growth and diminish plant yields
• Can spread throughout the entire plant

• Genetic engineering methods

• Have been used to create virus-resistant plants

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Animal Viruses

• Molecular genetics helps us understand viruses

• Virus studies help establish molecular genetics

Membranous envelope
RNA
Protein coat
Protein spike
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Protein spike
VIRUS

• The reproductive cycle of an enveloped virus

Protein coat
Viral RNA (genome)
Envelope
1
Entry
Plasma membrane of host cell
2
Uncoating
RNA synthesis by viral enzyme
3
4
5
Protein synthesis
RNA synthesis (other strand)
mRNA
Template
New viral genome
6
Assembly
New viral proteins
Exit
7
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HIV, the AIDS Virus

• HIV is a retrovirus

Envelope

• A retrovirus is an RNA virus that reproduces by
  means of a DNA molecule
• It copies its RNA to DNA using reverse
  transcriptase

Protein
Protein coat
RNA (two identical strands)
Reverse transcriptase
HIV
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• How HIV reproduces inside a cell

Reverse transcriptase
Viral RNA
Cytoplasm
1
Nucleus
Chromosomal DNA
DNA strand
2
3
Provirus DNA
Double-stranded DNA
4
5
Viral RNA and proteins
6
The behavior of HIV nucleic acid in an infected
cell
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• AIDS is

• Acquired immune deficiency syndrome
• The disease caused by HIV infection
• Treated with the drug AZT

HIV infecting a white blood cell
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EVOLUTION CONNECTIONEMERGING VIRUSES

• Many new viruses have emerged in recent years

• HIV
• Ebola
• Hantavirus

(a) Ebola virus
(b) Hantavirus
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• How do new viruses arise?

• Mutation of existing viruses
• Spread to new host species

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SUMMARY OF KEY CONCEPTS

• DNA and RNA Polymers of Nucleotides

Nitrogenous base
Phosphate group
Sugar
DNA
Nucleotide
Polynucleotide
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• DNA Replication

Parental DNA molecule
Identical daughter DNA molecules
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• Translation The Players

Amino acid
Large ribosomal subunit
tRNA
Anticodon
mRNA
Codons
Small ribosomal subunit
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