Transcript 02. Molecular basis of heredity. Realization of hereditary information

Theme: Molecular basis
of heredity.
Realization of hereditary
information
Lecturer: ass. prof. Tetyana Bihunyak
The questions of the lecture:
1. Molecular biology as science
2. The chemistry of nucleic acids
2.1. Deoxyribonucleic acid (DNA)
2.2. Ribonucleic acid (RNA)
3. DNA Replication
4. Genetic code
5. Gene Expression
6. Functions of Proteins in the organism
Molecular biology is the study of biology at
a molecular level.
Molecular biology concerns itself with
understanding the interactions between
the various systems of a cell, including the
interactions between DNA, RNA and
protein biosynthesis and learning how
these interactions are regulated.
Nucleus contains genetic materials encoded in
DNA of chromosomes
Only nucleus directs protein synthesis in the
cytoplasm via ribosomal RNA (rRNA),
messenger RNA (mRNA) and transport RNA
(tRNA), which are synthesized in the nucleus
Nucleic acids
• The nucleic acids are polymers of
smaller units called nucleotides.
• There are 2 types of nucleic acids:
DNA (deoxyribonucleic acid)
RNA (ribonucleic acid).
Structure of nucleotide
1) five-carbon sugar
(deoxyribose C5H10O4
in DNA and ribose
C5H10O5 in RNA);
2) a phosphate group
(PO4);
3) one of five types
nitrogen-containing
compounds called
nitrogenous bases.
Nucleic acids
The nitrogenous bases are:
•
Purines, which are
larger – Adenine (A),
Guanine (G);
•
Pyramidines, which
are smaller – Thymine
(T), Cytosine (C), Uracil
(U).
Chromosome
DNA located in
nucleus, packaged into
chromosomes
DNA
Nucleus
Nucleotides
DNA Basics
Organelles
(mitochondria,
chloroplasts) have
their own
chromosomes (DNA)
DNA Basics
• DNA is a long, double-stranded,
linear molecule composed of
multiple nucleotide sequences.
• DNA contains Adenine,
Guanine, Cytosine, Thymine.
Nitrogenous bases
Purines
Thymine (T)
Cytosine (C)
Pyrimidines
DNA Basics
The DNA double helix
consists of two
complementary DNA
strands held together by
hydrogen bonds between
the base pairs A-T and G-C
A-T base pair
Hydrogen bonding of the bases
G-C base pair
Chargaff’s rule: The content of A equals the content of T,
and the content of G equals the content of C
in double-stranded DNA from any species
Chargaff's rules said that A = T and G = C. The model
shows that A is hydrogen bonded to T and G is hydrogen
bonded to C.
This so-called complementary base pairing means that a
purine is always bonded to a pyrimidine.
Only in this way will the molecule have the width (2 nm)
dictated by its X-ray diffraction pattern, since 2
pyrimidines together are too narrow and 2 purines
together are too wide.
DNA Basics
In the formation of a
nucleic acid chain the
phosphate group of the
nucleotide binds to the
hydroxyl group of
another, forming what
is called a
phosphodiester bond,
which is very strong.
Crick Francis
Watson James
The double helix of DNA was discovered
in 1953 by Crick F. and Watson J. Nobel
prize in 1962
Structure of DNA
Watson and Crick model shows that DNA
is a double helix with sugar-phosphate
backbones on the outside and paired bases
on the inside. This arrangement first the
mathematical measurements provided by
the X-ray diffraction data for the spacing
between the base pairs (0.34 nm) and for a
complete turn of the double helix (3.4 nm)
The main biological DNA functions:
•
•
•
DNA stores hereditary
information about primary
protein structure.
The order of the bases
specifies the order of amino
acids in polypeptides.
DNA-replication –
maintaining genetic
information.
DNA Replication
• The Watson and Crick model suggests
that DNA can be replicated by means
of complementary base pairing.
• During replication, each old DNA
strand of the parent molecule serves as
a template for a new strand in a
daughter molecule.
• A template is most often a mold used to
produce a shape complementary to
itself
The human cell cycle
Rapid growth and
preparation for
DNA synthesis
S
phase
G0
Quiescent cells
G1
S phase
is the synthetic
phase, resulting in
duplication of the
chromosomes: one
replicated
chromosome
consisting of two
chromatids
phase
G2
M
phase
Mitosis
phase
Growth and
preparation for
cell division
Steps of replication:
Helicase
1. Unwinding
• The old strands that make
up the parent DNA
molecule are unwound and
"unzipped" (i.e., the weak
hydrogen bonds between the
paired bases are broken)
• There is a special enzyme
called helicase that unwinds
the molecule
DNA replication
• DNA helicase (enzyme)
unwinds the DNA. The
junction between the
unwound part and the open
part is called a replication
fork.
• DNA polymerase adds the
complementary nucleotides
and binds the sugars and
phosphates. DNA
polymerase travels from the
3' to the 5' end.
Steps of replication:
2. Complementary
base pairing
New complementary
nucleotides,
always present in the
nucleus,
are positioned by the
process
of complementary base
pairing.
DNA replication
• DNA polymerase adds
complementary nucleotides on
the other side of the ladder.
Traveling in the opposite
direction.
• One side is the leading strand it follows the helicase as it
unwinds.
• The other side is the lagging
strand - its moving away from
the helicase
DNA replication
• Problem: it reaches the
replication fork, but the
helicase is moving in the
opposite direction. It
stops, and another
polymerase binds farther
down the chain.
• This process creates
several fragments, called
Okazaki Fragments,
that are bound together
by DNA ligase.
Steps of replication:
3. Joining
•
The complementary nucleotides
become joined together to form
new strands.
•
Each daughter DNA molecule
contains an old strand and a
new strand.
•
Steps 2 and 3 are carried out by
the enzyme DNA polymerase.
DNA replication
• During replication, there are many points along the
DNA that are synthesized at the same time
(multiple replication forks).
• It would take forever to go from one end to the
other, it is more efficient to open up several points
at one time.
A model for DNA replication
DNA replication is termed semiconservative
replication because one of the old strands is
conserved, or present, in each daughter double
helix. Semiconservative replication was
experimentally confirmed by Matthew Meselson
and Franklin Stahl in 1958.
Accuracy of Replication
•
The mismatched nucleotide causes a
pause in replication, and during this time,
the mismatched nucleotide is excised from
the daughter strand.
•
The errors that slip through nucleotide
selection and proofreading cause a gene
mutation to occur.
•
Actually it is of benefit for mutations to occur
occasionally because variation is the raw
material for the evolutionary process.
Rate of Gene Mutations
•
Per cell cycle, gene mutations don't occur very often
•
There are several mechanisms that protect against the
occurrence of mutations.
•
The bases are on the interior of the DNA molecule, and
the supercoiling of the molecule in eukaryotes also
lends stability.
•
During replication, DNA polymerases proofread the new
strand against the old strand and detect any mismatched
pairs, which are then replaced with the correct
nucleotides. In the end, there is usually only one
mistake for every one billion nucleotide pairs
replicated.
deamination
Excision repair
ATGCUGCATTGA
TACGGCGTAACT
uracil DNA glycosylase
ATGC GCATTGA
TACGGCGTAACT
repair nucleases
AT
GCATTGA
TACGGCGTAACT
DNA polymerase b
ATGCCGCATTGA
TACGGCGTAACT
DNA ligase
thymine dimer
ATGCUGCATTGATAG
TACGGCGTAACTATC
excinuclease
AT (~30 nucleotides) AG
TACGGCGTAACTATC
DNA polymerase b
ATGCCGCATTGATAG
TACGGCGTAACTATC
DNA ligase
ATGCCGCATTGA
TACGGCGTAACT
ATGCCGCATTGATAG
TACGGCGTAACTATC
Base excision repair
Nucleotide excision repair
Correlation between DNA repair activity in fibroblast cells
from various mammalian species and the life span of the
organism
100
human
elephant
Life span
cow
10
hamster
rat
mouse
shrew
1
DNA repair activity
Point Mutations
•
•
•
•
Point mutations involve a change in a single nucleotide and therefore a
change in a specific codon.
When one base is substituted for another, the results can be variable. For
example, if UAC is changed to UAU, there is no noticeable effect, because
both of these codons code for tyrosine. This is called a silent mutation.
If UAC is changed to UAG, however, the result could very well be a drastic
one because UAG is a stop codon. If this substitution occurs early in the gene,
the resulting protein may be too short and may be unable to function. Such an
effect is called a nonsense mutation. Finally, if UAC is changed to CAC, then
histidine is incorporated into the protein instead of tyrosine. This is a missense
mutation.
A change in one amino acid may not have an effect if the change occurs in a
noncritical area or if the 2 amino acids have the same chemical properties. In
this instance, the polarities of tyrosine and histidine differ; this substitution
most likely will have a deleterious effect on the functioning of the protein.
Recall that the occurrence of valine instead of glutamate in the beta (B) chain
of hemoglobin results in a sickle-cell disease.
Defects in DNA repair or replication
All are associated with a high frequency of chromosome
and gene (base pair) mutations; most are also associated with a
predisposition to cancer, particularly leukemias
• Xeroderma pigmentosum
• caused by mutations in genes involved in nucleotide excision repair
• associated with a >1000-fold increase of sunlight-induced
skin cancer and with other types of cancer such as melanoma
• Ataxia telangiectasia
• caused by gene that detects DNA damage
• increased risk of X-ray
• associated with increased breast cancer in carriers
• Fanconi anemia
• caused by a gene involved in DNA repair
• increased risk of X-ray and sensitivity to sunlight
• Bloom syndrome
• caused by mutations in a DNA helicase gene
• increased risk of X-ray
• sensitivity to sunlight
• Cockayne syndrome
• caused by a defect in transcription-linked DNA repair
• sensitivity to sunlight
• Werner’s syndrome
• caused by mutations in a DNA helicase gene
• premature aging
DNA and RNA differ
RNA is single-stranded
(but it can fold back upon
itself to form secondary
structure, e.g. tRNA)
In RNA, the sugar
molecule is ribose rather
than deoxyribose
In RNA, the fourth base is
uracil rather than thymine.
DNA
RNA
H
3
U
OH
2
OH
OH
OH
1
The major bases found in DNA and RNA
DNA
Adenine
Cytosine
Guanine
Thymine
thymine-adenine base pair
RNA
Adenine
Cytosine
Guanine
Uracil (U)
uracil-adenine base pair
RNA Basics
• Messenger RNA carries the genetic code to the
cytoplasm to direct protein synthesis.
• 1. This single-stranded molecule (hundreds to
thousands of nucleotides).
• 2. mRNA contains codons that are complementary
to the DNA codons from which it was transcribed
• Ribosomal RNA associates with many different
proteins (including enzymes) to form ribosomes.
• 1. rRNA associates with mRNA and tRNA during
protein synthesis.
• 2. rRNA synthesis takes place in the nucleolus and
is catalyzed by RNA polymerase.
Transfer RNA - the adapter
Transfer RNA is folded into a cloverleaf shape and contains about 80
nucleotides.
• 1. Each tRNA combines with a specific amino acid that has been
activated by an enzyme.
• 2. One end of the tRNA molecule possesses an anticodon, a triplet
of nucleotides that recognizes the complementary codon in mRNA.
Types of RNA
Transcription: DNA-Directed RNA Synthesis
• Transcription has three phases:
Initiation
Elongation
Termination
• RNA is transcribed from a DNA template
after the bases of DNA are exposed by
unwinding of the double helix.
• In a given region of DNA, only one of the
two strands can act as a template for
transcription.
Figure 12.4 – Part 1
Transcription: DNA-Directed RNA Synthesis Elongation
• Nucleotides are added by complementary
base pairing with the template strand
• The substrates, ribonucleoside
triphosphates, are hydrolyzed as added,
releasing energy for RNA synthesis.
DNA
RNA
RNA
U
OH
U
OH
OH
OH
OH
OH
OH
OH
OH
OH
DNA Replication figure adapted for Transcription
Genetic code
All organisms use the same genetic code
Each set of three nucleotides codes for an
amino acid = “The Genetic Code”
The Genetic Code is universal
• All organisms use the same genetic code
• Each set of three nucleotides codes for an
amino acid = “The Genetic Code”
AUG = Met
The genetic code
–
–
–
–
–
–
consists of 64 triplet codons (A, G, C, U) 43 = 64
all codons are used in protein synthesis
• 20 amino acids
• 3 termination (stop) codons: UAA, UAG, UGA
• AUG (methionine) is the start codon (also used
internally)
multiple codons for a single amino acid =
degeneracy
Genetic code is unambiguous. Each triplet codon has
only one meaning
5 amino acids are specified by the first two
nucleotides only
3 additional amino acids (Arg, Leu, and Ser) are
specified by six different codons
Gene Expression
•
•
•
•
•
•
The process by which a gene produces a product, usually a
protein, is called gene expression.
DNA not only serves as a template for its own replication, it is also a
template for RNA formation.
Gene Expression in prokaryotes:
transcription, translation.
Gene Expression in eukaryotic cells:
transcription, processing, translation.
Transcription
The
DNA
process by which a mRNA copy is made of a portion of
• It is the first step required for
gene expression.
• During transcription, a mRNA
molecule is formed that has a
sequence of bases
complementary to a portion of
one DNA strand;
• A, T, G, or С is present in the
DNA template,
• U, A, C, or G is incorporated
into the mRNA molecule
Transcription
• Transcription begins at a region of DNA called a
promoter.
• A promoter is a special sequence of DNA bases
where RNA polymerase attaches and the
transcribing process begins. A promoter is at the
start end of the gene to be transcribed.
• Elongation of the mRNA molecule occurs as long as
transcription proceeds. Finally, RNA polymerase
comes to a terminator sequence at the other end of
the gene being transcribed.
• The terminator causes RNA polymerase to stop
transcribing the DNA and to release the mRNA
molecule, now called a RNA transcript
Transcription: DNA-Directed RNA
Synthesis - Termination
• Termination: Special DNA sequences
and protein helpers terminate
transcription.
• The transcript is released from the DNA.
• This Primary Transcript is called the “premRNA”
• The pre-mRNA is processed to generate
the mature mRNA
RNA Processing
• introns are noncoding portions of the original mRNA
tape; they do not contain information for the
sequencing of amino acids in a protein
• exons: portions of the mRNA transcript that code for
amino acids
• special molecular splicing complexes cut out sections
of introns. Then the remaining portions of the mRNA
(exons) tape are spliced together
Translation
During translation, the sequence
of codons in mRNA directs the
sequence of amino acids in a
protein.
Two other types of RNA are needed for
protein synthesis.
• rRNA is contained in the ribosomes,
where the codons of mRNA are read
•
tRNA carries amino acids to the
ribosomes so that protein synthesis
сan occur.
Ribosomes are the protein synthesis machines,
and use RNA as the template for translation
Codon-anticodon interactions
• codon-anticodon base-pairing is antiparallel
• the third position in the codon is frequently degenerate
• one tRNA can interact with more than one codon (therefore 50
tRNAs)
• wobble rules
3’
5’ tRNAmet
C with G
A with U
G with C
U with A
UAC
AUG
5’
mRNA
3’
3’
5’
• one tRNAleu can read two
of the leucine codons
5’
mRNA
GAU
CUA
G
tRNAleu
wobble base
3’
PROTEIN SYNTESIS ON RIBOSOMES
The Central Dogma
DNA Replication
Transcription
Translation
The Flow of Information: DNA  RNA  protein
Regulation of Gene expression in prokaryotes
lac operon in E. Coli
•Function - to produce enzymes which break down lactose (milk sugar).
When lactose (inducer)is present, they turn on and produce enzymes (5)
•Two components - repressor genes (1) and functional strutural genes
(4)
•Promoter (P) - aids in RNA polymerase binding
•Operator (O) - "on/off" switch (3) - binding site for the repressor
protein
•Repressor (lacI) gene - produces repressor protein w/ two binding sites, one for
the operator and one for lactose. The repressor protein is under allosteric control when not bound to lactose, the repressor protein can bind to the operator
Regulation of Gene expression in prokaryotes
lac operon in E. Coli
Operation if lactose is present:
When lactose is present, an isomer of lactose,
allolactose, will also be present in small
amounts. Allolactose binds to the allosteric site and
changes the conformation of the repressor protein so
that it is no longer capable of binding to the operator
Operation if lactose is not present:
the repressor gene produces repressor,
which binds to the operator. This blocks
the action of RNA polymerase, thereby
preventing transcription
Proteins are polymers of amino acids
•
There are 20 different
amino acids in cells that
differ only by R groups
•
All amino acids contain 2
important functional
groups: an amino, - NH2,
group and a carboxyl (acid)
–СООН, group, both of
which ionize at normal
body pH
Functions of Proteins in the organism
•All enzymes are proteins.
• Storing amino acids as nutrients and
as building blocks for the growing
organism.
•Transport function (proteins transport
fatty acids, bilirubin, ions, hormones,
some drugs etc.).
•Proteins are essential elements in
contractile and motile systems (actin,
myosin).
• Protective or defensive function
(fibrinogen, antibodies).
• Some hormones are proteins (insulin,
somatotropin).
•Structural function (collagen, elastin).
Types of proteins
• Enzymes -Quicken chemical reactions (surcease
•
•
•
•
•
•
•
•
brocks sugar to glucose and fructose)
Hormones - chemical messengers (growth hormone)
Transport –move other molecules (hemoglobin)
Contractive –movement (myosin and actin -allow
muscles to contract)
Protective - healing, defense against invader
(fibrinogen: stops bleeding antibodies: kill bacterial
invaders)
Structural –mechanical support (keratin: hair
collagen: cartilage)
Storage - stores nutrients (ovalbumin: egg white:
used as nutrient for embryos)
Toxins - defense, predations (bacterial diphteria
toxin)
Communication – cell signaling (glicoprotein:
receptors on cell surface)
TEST QUESTIONS
1. Which one of the following nucleotides is not present in DNA?
A. Thymine. B. Adenine. C. Uracil. D. Cytosine. E. Guanine.
2. DNA is duplicated in the cell cycle during the:
A. G1 phase. B. S-phase. C. G2-phase. D. Prophase. E. Metaphase.
3. If one strand of DNA has the base sequence ATCGTA, what will the
complementary strand of mRNA have?
A. TAGCAT
B. UAGCAU
C. CAGTCT
D. ATCGTA
E. All of these.
4. Which of the following statements concerning transcription is false:
A. A gene is transcribed into DNA on RNA molecule by RNA
polymerase.
B. Both exons and introns are transcribed.
C. DNA molecule unwinds at the end of transcription
D. The codon always represents a single amino acid.
E. The chain terminator on the DNA molecule stops the transcription
process.
“You can take a horse to the
water, but you cannot make
him drink”
(English proverb)
Thank you for attention !