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Molecular Genetics
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Introduction
In this unit you will learn:
 how living cells store, transmit, & express
genetic information
 how the external environment affects the
nature and expression of genetic material
within cells, and
 how new and genetic technologies are
transforming science & society.
Blake, Lessa, et al. (2002). Biology 12. Toronto: McGraw-Hill Ryerson Limited.
History
Gregor Mendel
 In 1865, an Austrian monk, named Gregor
Mendel found that individual traits are
determined are determined by discrete
“factors” (later determined to be genes)
contributed by both parents.
 Further, he stated that for any visible trait
there exists two factors, but one (the
recessive factor) may not be expressed.
History, continued …
Friedrich Miescher
 In 1869, a Swiss physician & scientist,
Friedrich Miescher isolated a phosphoruscontaining material from the nuclei of cells
found in pus from discarded surgical
bandages, and called in “nuclein”.
 Later, he found the nuclein was made up of
an acidic portion (nucleic acid) & an
alkaline portion (a protein).
History, continued …
Phoebus Levene
 During the 1920s, Levene, a biochemist,
isolated two types of nucleic acids
(distinguished by the different sugars
involved in their composition):
• a five-carbon sugar ribose (he called “ribose
nucleic acid” or RNA) &
• a previously unknown sugar, similar to ribose but
lacking one oxygen molecule (he later called
“deoxyribose nucleic acid” or DNA).
O
History, continued …
O
H
CH3
H
N
O
CH3
N
N
O
O
N
O
O
O
O
O
P
O
O
O
-
P
O
-
 Levene went on to show that nucleic acids
are made up of long chains of individual
units (he called nucleotides).
 He found that both DNA & RNA contained:
• four nitrogenous bases:
• adenine (A), guanine (G), cytosine (C), &
thymine (T) (or uracil (U) in RNA);
• a five-carbon sugar (deoxyribose in DNA & ribose
in RNA); and
• a phosphate group.
History, continued …
 However, Levene incorrectly concluded:
• that nucleic acids contained equal amounts of
each one of these nucleotides, and
• that the structure of DNA & RNA where made of
these nucleic acids in a repeating pattern or
sequence.
 This error meant that most scientists
concluded that DNA could not be the
material of heredity because of its lack of
complexity.
History, continued …
Fred Griffith
 In 1928, Griffith, a British researcher, did a
series of experiments on mice infected
with Streptococcus bacteria.
• He had 2 strains of Streptococcus.
• The S strain was virulent: when injected into
mice, they developed pneumonia and died.
• The R strain was avirulent: it did not kill the
mice upon injection.
History, continued …
• When S cells were killed by heat, injecting them
had no effect on the mice.
• Heat killed R cells also had no effect.
• The surprising result:
• When live R cells were mixed with heat-killed
S cells and injected, the mice developed an
infection and died.
• When bacteria were isolated from the dead
mice, they were found to be S type.
• Conclusion: something from the dead S cells had
“transformed” the live R cells into S.
History, continued …
Oswald Avery, Colin MacLeod, & Maclyn
McCarty
 In 1943-44, following Griffith’s experiments,
this team demonstrated that the
transforming material was DNA.
 However, many scientists, who had
accepted Levene’s erroneous theory,
refused to believe DNA played a key role in
heredity.
History, continued …
 During this time period, the evidence for
the importance of the role of DNA in
heredity accumulated.
 A key discovery was also made, that the
quantity of DNA in somatic cells is both
constant and double the quantity in
gametes.
• However, you would expect reproductive cells to
have half as much hereditary material as the
other cells of the body, since two gametes come
together to form a zygote with a full complement
of hereditary material.
History, continued …
Erwin Chargaff
 In the late 1940s, Chargaff overturned one
of Levene’s main conclusions, that the
nucleotides were present in equal
quantities.
 Chargaff, discovered that although the
nucleotide composition varied between
species it was same with species, despite
changes in age, physical state, or its
environment.
History, continued …
 Most importantly, Chargaff discovered that,
in any sample of DNA, the amount of
adenine present is always equal to the
amount of thymine, and the amount of
cytosine is always equal to the amount of
guanine.
 This constant relationship is known as:
Chargaff’s Rule.
History, continued …
 Alfred Hershey & Martha Chase
 In 1952, Hershey & Chase, in a series of
experiment, again demonstrate the
essential role of DNA in the transmission of
genetic information.
 They determined that DNA was the
physical carrier of heredity.
History, continued …
Rosalind Franklin & Maurice Wilkins
 In the early 1950s, the X-ray
crystallography data (diffraction pattern of
X-rays through crystallized DNA) provided
by Maurice Wilkins and Rosalind Franklin
provided the first clues to the molecular
structure of DNA
History, continued …
James Watson & Francis Crick
 In 1953, Watson & Crick published a model
of DNA structure in Nature magazine.
 They had determined that the DNA
molecule was shaped like a twisted ladder.
• More precisely, the DNA molecule consists of
two anti-parallel chains twisted into a helix.
• The nitrogenous bases are paired in the centre of
the molecule, and the phosphate-sugar
backbones are on the outside.
DNA – The Double Helix
 DNA is a double helix, with bases to the
centre (like rungs on a ladder) and with
sugar groups alternating with phosphate
groups along the sides of the helix (like the
sides of a twisted ladder).
 The strands are complementary, A pairs
with T and C pairs with G, and the pairs
held together by hydrogen bonds.
Nucleotides
 Each nucleotide of DNA or RNA has 3 parts:
a nitrogenous base, a sugar, and a
phosphate group.
 The sugar (ribose in RNA & deoxyribose in
DNA) has 5 carbons, numbered 1’ through
5’.
• the nitrogenous base is attached to the 1’ carbon
• the 2’ carbon has a free hydroxyl group (-OH) in
the case of RNA, but a hydrogen group (-H) in the
case of DNA, the lack of the oxygen atom makes
DNA far less reactive than RNA
• the 3’ carbon has an -OH group on it that links to
the phosphate group on the next base, thus the
“end” of the DNA molecule is a free 3’ OH group
• the 5’ carbon is attached to the phosphate group
Directionality
 The 5’ carbon of the pentose (5-carbon)
sugar of one nucleotide is connected to the
3’ hydroxyl (-OH) of the next.
 All the phosphate bridges have the same
orientation, thus each strand has a specific
directionality.
 Therefore any strand or severed fragment
of DNA had a 5’ & 3’ end, by convention the
strand is always read in the 5’ to 3’
direction.
Complementary Base Pairs
 There are 4 possible DNA bases: adenine
(A), guanine (G), cytosine (C), and thymine
(T).
• In RNA, thymine is replaced by uracil (U).
 Each strand of DNA pairs with a
complementary DNA strand (A with T & G
with C).
• Thus, the information on one DNA strand easily
allows the other strand to be deduced.
• The amount of A in DNA always equals the
amount of T, and the amount of G always equals
the amount of C.
Purines & Pyrimidines
 Adenine and guanine are purines: they
consist of two linked rings of mixed
nitrogen and carbon atoms.
 Thymine and cytosine are pyrimidines,
which consist of a single ring. In RNA,
thymine is replaced by uracil (U), which
looks like thymine except for a single
methyl group.
RNA
Ribose Nucleic Acid
 a five-carbon sugar (deoxyribose for DNA)
 the nucleotide thymine is not found in RNA,
in its place is the nucleotide uracil
 RNA is single stranded, although
sometimes is can fold back on itself to
produce regions of complementary base
pairs
Organization of Genetic Material
 How does this genetic material get stored
as it is hundreds of times longer than the
organism or cell itself?
• It has to be compact so it does not interfere with
each other or other cellular processes.
• It must be protected from enzymes that are
designed to break down this material into its
component nucleotides.
 Prokaryotes & eukaryotes have distinct
ways of arranging this genetic material to
meet both these requirements.
Genetic Material in Prokaryotes
 Most have a single, double-stranded DNA
molecule.
 They have no nucleus, the genetic material
is packed into a specific region (the
nucleoid or nuclear zone) of the cell.
 The ends bind together, then is further
twisted upon itself into a series of small
loops (said to be supercoiled) held in place
by proteins.
Plasmids
 Prokaryotes often have one or more small,
circular double-stranded DNA molecules
floating free in the cytoplasm in addition to
the DNA found in the nucleoid.
 Although not physically part of the nucleoid
DNA, plasmids can:
• contribute to cell metabolism and to the
hereditary mechanism;
• be copied and transmitted between cells; and
• be incorporated into the nucleoid DNA and
reproduced during cell division.
Genetic Material in Eukaryotes
 Supercoiling is not effective in eukaryotes
as the simplest DNA of an eukaryotic cell is
ten times that for a prokaryote.
 In plants and animals this DNA is organized
into chromosomes.
• There are multiple chromosomes.
• Each chromosome contains one linear doublestranded DNA molecule together with the protein
histone.
• These components are organized into long fibres
called chromatin (the “nuclein” observed by
Miescher).
• The chromatin fibres can form loops attached to
a protein scaffold.
DNA Replication
 When a cell or organism reproduces, a
complete set of genetic instructions must
pass from one generation to the next.
 DNA functions as the inherited directions
for a cell or organism.
 An organism’s genotype, its genetic
makeup is the sequence of nucleotide
bases in DNA.
 Watson and Crick recognized that the
double stranded DNA molecule could
replicate by unwinding, then synthesizing a
new strand for each of the old stands.
Semi-conservative Replication
 This mode of replication is called “semiconservative”. It means that after one
DNA molecule has replicated to become 2
DNA molecules, each new molecule
consists of one old strand (from the
original molecule) and one new strand.
• The information from each old strand can be
used to create the new strands, since A always
pairs with T, and G always pairs with C.
• DNA replication starts at specific locations
“origins of replication”, and proceeds in both
directions.
DNA Replication Process
 There are three phases:
 Initiation:
• a portion of the double helix is unwound to
expose the bases for new base pairing
 Elongation:
• two new strands are assembled using the
parental DNA as a template
 Termination:
• the replication process is completed and the new
DNA molecule reform into helices
Initiation
 The DNA is unwound and opened by a
group of enzymes (always at a certain set
of nucleotides – the origin of replication),
this section is called the replication
bubble.
 After the bubble has opened the enzyme,
DNA polymerase insert themselves into the
space between the two strands.
 The point at which the helix is unwound
and new strands develop are called the
replication forks, there is one at each end.
Initiation, continued …
 New nucleotides are placed in the fork and
link to the corresponding parental
nucleotide already there (A with T, C with
G).
 The enzyme helicases cleave and unravel
short segments of DNA just ahead of the
replicating fork.
 The entire length of the DNA molecule is
replicated as the bubbles meet.
Elongation
 DNA polymerase can only attach new
nucleotides to the free 3’ hydroxyl end of
pre-existing chain of nucleotides.
 Thus DNA can only be synthesized from 5’
to 3’.
 Thus a primer or initial chain (a small
strand of RNA) of nucleotides are needed
as a starting point.
• The primer is created through the action of the
enzyme primase.
Elongation, continued …
 As DNA synthesis is from 5’ to 3’, it
becomes a problem, because both strands
must be synthesized at the replication fork,
and one strand will necessarily be
synthesized in the opposite direction from
the movement of the replication fork.
 In reality, one strand is synthesized
continuously, in the same direction that the
replication form is moving, called the
leading strand.
 The other strand is synthesized in short,
discontinuous pieces, that are then
attached together to form the final DNA
strand this is the lagging strand.
Elongation, continued …
 Each fragment of the lagging strand is
called an Okazaki fragment, and they are
synthesized in the opposite direction that
the replication fork moves.
 The RNA primers are removed by DNA
polymerase during the synthesis of the
next Okazaki fragment.
 Joining of the Okazaki fragments is done
by the enzyme DNA ligase.
Termination
 Once the new strands are complete, the
molecules rewind automatically in order to
regain their stable helical structure.
 A problem is created once the RNA primer
is removed from the 5’ end of each
daughter strand, there is no adjacent
fragment for which new nucleotides can be
added to fill this gap, resulting in a slightly
shorter daughter chromosomes.
• This occurrence is not a problem in circular DNA,
but human cells loose about 100 base pairs from
each end of each chromosome with each
replication.
Termination, continued …
 This loss of genetic material could result in
critical code being eliminated, however
there are buffer zones of repetitive
nucleotide sequences, called the
telomeres.
• In humans the sequence is TTAGGG repeated
several thousand times.
 Their erosion does not affect cell function,
but protects against lost of important
genetic material.
• The erosion of the telomeres are related to the
death of the cell.
Termination, continued …
 Thus the extension of the telomeres is
linked to a longer lifespan for the cell.
 The enzyme telomerase is responsible for
the extension of the telomeres.
 Research has found that the activity of the
gene that codes for telomerase is directly
linked to the longevity in worms & fruit
flies, cancer cells (which divides beyond
normal lifespan of somatic cells) also
contains telomerase.
Proofreading & Correction
 Errors occur in DNA replication fairly
frequently: the wrong base gets inserted
due to the peculiarities of nucleotide
chemistry, however, DNA polymerase has a
built-in editing function that removes most
of the incorrect bases.
• DNA polymerase detects the absence of
hydrogen bonding (when a mismatch occurs),
then removes the incorrect base and inserts the
correct one using the parent strand as a
template.
 This complex process of replication is
known as the replication machine.
Genes
 A gene can be defined as a region of DNA that
controls a hereditary characteristic, it usually
corresponds to a sequence used in the production
of a specific protein or RNA.
 A gene carries biological information in a form that
must be copied and transmitted from each cell to all
its progeny.
 Genes can be as short as 1000 base pairs or as long
as several hundred thousand base pairs, it can even
be carried by more than one chromosome.
 The current estimate for the number of genes in
humans about 30,000 to 35,000.
Arrangement of the Genome
 The genome is all of the genetic
information or hereditary material
possessed by an organism; the entire
genetic complement of an organism.
• Genes are not regularly spaced along
chromosomes.
• There is no set relationship between the number
of genes on a chromosome and the total length
of the chromosome.
• The same is true for the number of genes in an
organism and the overall size of its genome.
• The genomes of organisms also contain varying
amounts of DNA that do not serve as genes or
regulatory sequences.
Exons & Introns
 Exon – The region of a gene that contains
the code for producing the gene's protein.
• Each exon codes for a specific portion of the
complete protein.
• In some species (including humans), a gene's
exons are separated by long regions of DNA
(called introns or sometimes “junk DNA”) that
have no apparent function*.
 Intron* – A non-coding sequence of DNA
that is initially copied into RNA but is cut
out of the final RNA transcript.
Introns
 Introns are NOT junk DNA but actually
support a variety of developmental &
regulatory functions.
 The existence of introns allow for some
genes to code for more than one
polypeptide by using different combinations
of exons & introns.
 Introns can also be the location of various
regulatory sequences that initiate or stop
gene activity.
Multi-gene Families
 Collection of identical or similar genes.
 Common origin from a single ancestral
gene during evolution.
 Probably arose by gene duplication events.
 Such genes may be clustered together on
the same chromosome or dispersed on
different chromosomes.
Transposons
 Transposons (or “jumping genes) are
segments of DNA that can move around to
different positions in the genome of a
single cell. In the process, they may:
• cause mutations; or
• increase (or decrease) the amount of DNA in the
genome.
Pseudogenes
 A pseudogene is a nucleotide sequence
that is part of the DNA of an organism that
are regarded as non-functional copies or
close relatives of genes.
 Pseudogenes are believed to have resulted
as mutation to an original functioning
coding sequence.
• One scenario for the evolution of a pseudogene is
that the environment of an organism changes
sufficiently such that the gene product is no
longer necessary for the survival of that
organism, thus there is no longer selection
pressure to maintain this sequence, and
organisms can continue to survive by
accumulating mutations in the sequence.
Repetitive Sequences
 Repetitive sequences are regions in the
DNA that contain short sequences repeated
thousands or even millions of times.
 It is suggested that in some cases they are
a result of mutations.
 While they have no coding function, they
can play an important role, for example:
• telomeres
The Triplet Hypothesis
What Crick knew:
 Proteins are made up of 20 amino acids.
 DNA only composed of 4 different nucleotides.
Therefore:
 if 1 nucleotide = 1 amino acid
 only 4 amino acids
 if 2 nucleotides = 1 amino acid
 42 or only 16 amino acids
 if 3 nucleotides = 1 amino acid
 43 or 64 possible combinations to code for amino
acids
Transfer of Genetic Information
How?
They knew the following:
1. DNA never leaves the nucleus of
eukaryotic cells
2. most of the structures & processes
involved in protein synthesis is found only
in the cytoplasm of the cell
3. RNA found in both the nucleus &
cytoplasm
The Central Dogma
 Proposed by Francis Crick in 1958 to
describe the flow of information in a cell.
 It is a two-step process by which the
genetic code is converted to proteins.
1. the strand of DNA serves as a template for the
construction of a complementary strand of RNA
2. RNA moves from the nucleus to the cytoplasm
3. in the cytoplasm, the RNA guides the synthesis of
the polypeptide chain or protein construction
The Two Steps
1st Step
 Transcription: the process of synthesis of the RNA
molecule from the DNA strand
2nd Step
 Translation: the code in the RNA molecule must be
converted to the amino acid sequence of a
polypeptide in order for a protein to be synthesized
DNA
protein transcription
RNA
translation
Why Two-step Process?
As it uses more energy.
Advantages are:
1. Only the required information is copied
and carried out of the nucleus, while the
DNA remains protected inside.


It takes more energy to transfer the entire
chromosome out of the nucleus in order to take
part in the process of protein synthesis.
Repeated transport of the DNA back & forth
between the nucleus & the cytoplasm would
also increase the risk of damage to the DNA.
Why Two-Steps?, continued …
2. A single strand of DNA can be used to
produce multiple strands of RNA to speed
up the process of protein synthesis.
3. Having more steps means there are
several more opportunities to regulate
gene expression, allowing for greater
sophistication in the regulatory functions.
The Genetic Code
Next, they needed to find this code.
 It was determined through the synthesis of
artificial RNA molecules (e.g. UUU), then
culturing this molecule in a medium that
contained the 20 amino acids + various
other substances required to catalyze the
formation of a protein.
 Note: The code is always presented in
terms of the RNA codon & in a 5’ to 3’
direction.
Characteristics of the Code
1. Continuity
2. Redundancy
3. Universality
Continuity
 long series of 3-letter codons
• no spaces
• no punctuation
• never overlaps
  knowing where to start transcription &
translation is essential
• in other words, each sequence of nucleotides
has a correct reading frame or grouping of
codons
• there is no process to reset the translation to the
correct reading frame if there is an insertion or
deletion of a nucleotide
Redundancy
 there are 64 possible combinations, but
only 20 amino acids, leaving potentially 44
stop signals
• however, this is not the case, cause otherwise
any random mutation would result in more than
twice as likely for the protein synthesis to stop,
which would result in a more severe mutation
rather than simply building the wrong amino acid
 further, redundancy is no random, it follows
a pattern, for example:
• proline has four codons – CC__ &
• arginine has six codons, 4 being – CG__
• this third position is referred to as the “wobble”
position
The “Wobble” Position
 in many cases, it can accommodate a
number of different nucleotides without
changing the resulting amino acid
• thus serving as a guard against harmful
mutations
 it also contributes to the efficiency of
protein synthesis
Universality
 the genetic code is the same in almost all
living organisms
• providing evidence that these organisms all
shared a common ancestor
• only exceptions known found in a few types of
unicellular eukaryotes and the mitochondria &
chloroplasts in eukaryotic cells
• the implications are that a gene taken from one
organism and inserted into another organism will
express the same polypeptide
• e.g. GM foods
Transcription: From DNA to RNA
4 Steps
1. Initiation
•
locates correct spot on the original DNA template to
begin transcription
2. Elongation
•
•
copies the correct number of nucleotides from the
DNA template to a RNA molecule, called the
messenger RNA or mRNA
this is the strand of RNA that carries information from
the DNA to the protein synthesis machinery
3. Termination
•
signals the correct place to stop the copying process
to make sure the mRNA molecule contains the
correct & complete set of instructions from the gene
4. Processing
•
the mRNA undergoes some final changes before it is
transported from the nucleus to the cytoplasm
Initiation of Transcription
 Initiation accomplished through promoter
sequences.
• promoter sequence usually rich in T & A
nucleotides
• for transcription to be initiated, both promoter
sequence must be present
 The stretch of DNA that includes the gene
is called the sense strand, the
complementary strand is called the antisense strand.
• however either strand can be the sense strand
• an mRNA molecule synthesized from the antisense strand will rarely code for a functional
protein
Initiation of Transcription, continued …
 RNA polymerase will only bind in one
direction.
• therefore transcription will only proceed in one
direction
• once bound, it will open up a section of the DNA
helix and begin the synthesis of the mRNA strand
 Note that only genes get transcribed.
 Thus transcription must begin precisely at
the correct nucleotide & direction on the
sense strand.
Elongation of mRNA
 Similar to DNA elongation.
 RNA polymerase works in a 5’ to 3’
direction.
 However, only utilizes one strand, therefore
no Okazaki fragments.
 RNA polymerase moves along the DNA
template opening it as it goes along the
helix which reforms as it passes.
• the RNA strand separates from the DNA
template
Elongation of mRNA, continued …
 Once one RNA polymerase passes, another
new RNA polymerase can bind to the DNA
template to begin transcribing another
mRNA molecule.
• Therefore many copies can be made at
the same time.
 But RNA polymerase has no proofreading.
• This is OK, as the mRNA does not
become part of the genetic material of
the organism.
• Also, the lack of a proofreading
mechanism speeds up the process of
protein synthesis.
Termination of Transcription
 RNA polymerase stops transcription once it
encounters a terminator sequence.
• at this point the mRNA strand separates
from RNA polymerase
• then the RNA polymerase can begin a
new transcription process
 The mRNA molecule is ready to begin the
process of protein synthesis.
• However, this process is slightly
different between prokaryotes and
eukaryotes.
Termination of Transcription, continued …
In Prokaryotes:
• protein synthesis takes place in the
cytoplasm of the cell
• the mRNA may even begin translation
before transcription is finished
In Eukaryotes:
• transcription & translation are physically
separated by the membrane around the
cell nucleus
Processing of mRNA
 In eukaryotes, the mRNA molecule that is
released after transcription is called
precursor mRNA or pre-mRNA.
 It undergoes several changes before being
exported out of the nucleus as mRNA.
• 5’ end is capped with a modified form of the G
nucleotide known as the 5’ cap
• at the 3’ end, an enzyme adds a long series of A
nucleotides referred to as a poly-A tail
• it serves to protect the mRNA from enzymes in
the cytoplasm that may break it down
• the greater the length of the poly-A tail, the
more stable the mRNA molecule
mRNA Splicing
 RNA polymerase does not distinguish
between introns & extrons.
• the introns need to be removed before
constructing the polypeptide
• this is done by the molecule called spliceosome
• composed of two compounds:
– Small nuclear RNA (of snRNA) + other
proteins
• it cleaves the pre-mRNA at both ends of each
intron and splices the remaining exons back
together
Complete mRNA Transcription Process
Source: http://io.uwinnipeg.ca/~simmons/cm1503/Image277.gif
Translation: From RNA to Protein
 The translation of messenger RNA (mRNA)
takes place on ribosomes,
• which include ribosomal RNA (rRNA),
• with the help of transfer RNA (tRNA),
• activating enzymes (amino-acyl tRNA
synthetase or aa-tRNA synthetase)
Transfer RNA (tRNA)
 tRNA are also derived from genes on the
DNA template.
 But they do not remain linear in structure,
complementary nucleotides on different
regions of the tRNA causes the molecule to
fold into a 3-lobed or “clover” shape.
Transfer RNA, continued …
 Two parts of the tRNA are important:
1. Lobe end
•
•
it is the anticodon, complementary to the
codon on the mRNA
by convention, the anticodon sequence is
written in a 3’ to 5’ direction
2. 3’ End
•
this is the attachment site for the amino
acid, specified by the mRNA codon
 The bound tRNA molecule is called aminoacyl tRNA or aa-tRNA.
Transfer RNA, continued …
 Remember, there are 64 possible
combinations coding for amino acids,
minus the 3 stop codes, still leaves 61
possible codes for amino acids.
 But only 30-45 different tRNA molecules
are found in each cell.
 Why?
• cause the anticodons on the tRNA can pair with
more than one codon
• remember the “wobble” position, thus saving the
cell energy by not having to produce all the
possible tRNA molecules
Activating Enzymes
amino-acyl tRNA synthetase
 they are genetic code-breakers or
chemical translators
 responsible for attaching the correct
amino acid to the tRNA molecule
 each activating enzyme has 2 binding
sites:
1. one for the anticodon
2. the other for the corresponding amino acid
Ribosomes
 It is responsible for combining mRNA & the
aa-tRNAs.
 It is a complex cluster of different kinds of
proteins with a third type if RNA.
• ribosomal RNA or rRNA
• it is a linear strand of RNA that always
stays bound to the proteins in the
ribosomal assembly
Ribosomes, continued …
 Each ribosome has 2 sub-units which sit
together:
• a small unit that contains 1 rRNA + 20 different
proteins (in prokaryotes)
• a large unit that contains 2 rRNA + 30 different
proteins (in prokaryotes)
•
eukaryotes tend to have more
 Each active ribosome has:
1. binding site for mRNA transcript
2. three binding sites for tRNA molecules
a. P site – holds 1 aa-tRNA & a growing chain of
amino acids
b. A site – holds the tRNA bringing the next amino
acid to be added to the chain
c. E site – releases the tRNA molecule back into the
cytoplasm
Translation – Initiation
 Initiated when mRNA molecule reached the
cytoplasm of the cell.
 The sequence of nucleotides at the 5’ end
of the mRNA molecule binds to a portion of
the rRNA strand.
 A special initiator (“starter”) tRNA
molecule also binds to the ribosomal-mRNA
complex, this carries the amino acid, at the
P site.
 The leader or starting sequence on the
mRNA established the starting point for
translation.
Translation – Elongation
 synthesis of polypeptide
 Three step process:
1. mRNA codon exposed in the A site forms a base
pair with the anticodon of the aa-tRNA
molecule.
2. enzymes + other molecules in the large sub-unit
catalyzes the formation of the peptide bond that
joins the last amino acid in the growing peptide
chain to the new amino acid
• at the same time, the polypeptide chain is
transferred from the tRNA in the P site to the
tRNA in the A site
Translation – Elongation, continued …
3. the mRNA and the tRNA complexes
move one codon distance in the 5’ to 3’
direction, this process is called
translocation
 Then the process is repeated.
Translation – Termination
 The elongation process continues until a
stop codon is reached on the mRNA.
 Instead of a tRNA, a release factor will
then bind with the stop codon.
 The release factor causes the polypeptide
to separate from the remaining tRNA
molecule and the ribosome assembly
separates into its elements, which can be
used again.
Polyribosome
 As soon as the first ribosome has moved
off the initiation sequence, another new
ribosome can move into place to begin the
synthesis of another polypeptide chain.
 This complex of a mRNA molecule bound to
multiple ribosomes is called a
polyribosome.
Transcription-Translation Summary
Source: http://fajerpc.magnet.fsu.edu/Education/2010/Lectures/26_DNA_Transcription_files/image018.jpg
Regulation of Gene Expression
Objectives:
 To examine how regulatory proteins act as
control mechanisms for genetic expression.
 To demonstrate how more than one form of
control can operate on a single gene at any
time.
 To discuss some of the reasons for the
differences in control mechanisms in
eukaryotes & prokaryotes.
Blake, Lessa, et al. (2002). Biology 12. Toronto: McGraw-Hill Ryerson Limited.
Gene Expression in Prokaryote
A
B
C
Gene Expression in Prokaryotes, continued …
A. Transcriptional Control
• can speed up or slow down transcription of
mRNA
B. Post-transcriptional Control
• the cell may transcribe mRNA or break it sown
before translation
• may lengthen or shorten poly-A tail to control
the time the mRNA remains stable + active
C. Post-translation Control
• the cell may modify the polypeptide chemically
or vary the rate at which the polypeptide
becomes a functional protein
• the cell also break down the polypeptide before
it becomes a functional protein
 The cell can conserve more energy + resources if
the control point is earlier in the synthesis pathway.
The Operon Model
Operon:
 A stretch of DNA that contains a set of one or more
genes involved in a particular metabolic pathway,
along with a regulatory sequence called an
operator.
Operator:
 Is a DNA sequence located in the promoter
sequences.
• it functions as a control element, governing
whether or not RNA polymerase can bind to the
promoter sequences
Negative Gene Regulation
 In negative regulation a repressor molecule
binds to the operator of an operon and
terminates transcription.
• the repressor is a protein that when bound to the
operator prevents RNA polymerase from binding to the
promoter
 Example (for E. coli)
• the lack of lactose causes the repressor to bind to the
operator
• however when lactose is present, some of the lactose
is converted to allolactose,
• some of the allolactose is utilized by the cell and some
binds to the repressor, which then releases from the
operator
• thus the allolactose acts as an inducer
– an inducer blocks the action of the repressor
Positive Gene Regulation
 In positive regulation an activator molecule
turns the operon "on".
• the activator is a protein that binds to a
site close to the promoter enabling RNA
polymerase to bind easier to the
promoter
• this allows for an increased rate of
RNA polymerase binding and thus an
increased transcription rate
• thus an increased rate of gene
expression
Positive Gene Regulation, continued …
Example (for E. coli)
 E. coli will metabolize glucose first if it is present, it
will only metabolize lactose if concentrations of
glucose are low.
 When glucose is not available or low a small
molecule called cyclic AMP (cAMP) is present.
• The amount of cAMP present is inversely
proportional to the amount of glucose present.
 As a result, the absence of glucose results in an
increase in cAMP in the cell.
 cAMP binds to a catabolite activator protein (CAP)
which binds to the promoter to stimulate
transcription.
Negative & Positive Regulation
 These two mechanisms work in
combination with each other.
 Negative control = ignition switch
• turns transcription on/off
 Positive control = gas pedal
• regulates the rate of transcription once
the gene is activated
Co-repression
 Repressors are negative control
mechanisms, shutting down operons.
 The presence in the cell of an essential
metabolite turns off its own manufacture
and thus stops unneeded protein synthesis.
• The presence of the metabolite shuts
down the operon.
• It binds to a site on the repressor and
enables the repressor to bind to the
operator.
• When the metabolite is not present,
the repressor leaves its operator, and
transcription begins.
Prokaryotes vs. Eukaryotes
Prokaryotes
 Since they are single-celled organisms with a small
genome and short lifespan, they are more likely to
express all of their genetic material at some point.
• Therefore prokaryotes rely on mechanisms that
can turn off gene expression when not needed.
Eukaryotes
 As much of the cells are specialized, much of the
hereditary material in a cell may not be expressed
in the lifetime of the cell.
• Therefore eukaryotes are more dependant on
mechanisms that turn on genes when needed.
Gene Expression in Eukaryotes
A
B
C
D
E
Gene Control in Eukaryotes
A. Pre-transcription Control
•
the cell controls the extent to which DNA is
exposed to transcription enzymes
• genes found on the more condensed regions
are less transcribed
B. Transcriptional Control
•
the cell controls whether or not exposed DNA is
transcribed into pre-mRNA
• a transcription factor binds to the promoter
to increase the rate of transcription
Gene Control in Eukaryotes, continued …
C. Post-transcriptional Control
• the cell controls the role of processing of the
pre-mRNA into mRNA
• not add 5’ cap or poly A-tail to the pre-mRNA
• if there is no poly-A tail, the mRNA will not be
transported to the cytoplasm & will also be
unstable
D. Translational Control
• mRNA is made, but the cell controls its
transport to the ribosomes in the cytoplasm
• a regulatory protein binds to the 5’ cap
preventing the mRNA from binding to the
ribosome unit
Gene Control in Eukaryotes, continued …
E. Post-translational Control
•
the polypeptide is made but the cell modifies it
chemically or varies the rate at which it
becomes a functional protein
OR
• it may break down the polypeptide before it
becomes a functional protein
• other regulatory proteins can block the
transport of polypeptides or functional
proteins to destinations outside the cell
Gene Control in Eukaryotes, continued …
 As in prokaryotes, the cell can save energy
if the regulatory control is earlier in the
metabolic pathway.
 But eukaryotes may regulate later on along
the metabolic pathway as the mRNA is
more stable (poly-A tail) in eukaryotes than
prokaryotes (as transcription needs to be
turned off).
• Therefore the mRNA can be stored and
translated later.
Mutations
Definition:
 A personal change in the genetic material
of an organism.
 All mutations are heritable.
i.e. can be copied in DNA replication
 Only the one that affect the reproductive
cells will be passed on, called germ cell
mutations.
 Mutations that arise in an organism’s
lifespan are called somatic cell mutations.
Types of Mutations
Point Mutations:
 just one or a few nucleotides are affected
1. Nucleotide Substitution
• One base is substituted for another in
the sequence of DNA nucleotides.
a. silent mutation
–
if there is no effect on cell metabolism
–
–
can result in altered protein
may be harmful (e.g., sickle cell anemia)
–
unable to code for functional polypeptide
product
b. mis-sense mutation
c. nonsense mutation
Types of Mutations, continued …
2. Nucleotide insertions or deletions
• In these types of mutations one base
pair is removed or added to the DNA
sequence.
• This type of point mutation causes a
shift in the reading frame of the codons,
called frameshift mutation.
• possible for two frameshift mutations
to cancel each other out
– it may result in a mis-sense or nonsense
mutation
Types of Mutations, continued …
3. Chromosomal mutations
• Occurs when an exchange in portions of
chromosomes between sister
chromatids during meiosis.
• The portions can become lost or
duplicated during DNA replication.
• “Jumping genes” or transposons.
• this may explain the rapid change
that leads to the development of new
species of organisms
Causes of Mutations
1. Naturally (Spontaneous Mutations)
 due to molecular interactions that take
place normally
2. External Agents
•
•
said to be induced mutations
substances that increase the rate of mutations
are called mutagens
a. Physical Mutagens
–
–
literally tears DNA strand
e.g., X-rays & gamma rays
b. Chemical Mutagens
–
–
a molecule enters the cell to induce mutation
most are said to be carcinogenic
Causes of Mutations, continued …
3. Cumulative Mutations
• mutations can add up to result in
damage or have no significant effect
Mutation Repair Mechanisms
1. Direct Repair
 cells reverse damage
e.g., proofreading of DNA polymerase
2. Excision Repair
• error recognized, removed & replaced
with newly synthesized correct copy
• the new section is synthesized by
DNA polymerase using the correct
DNA strand as a template then
sealed into place by DNA ligase
Mutation Repair Mechanism, continued …
3. Recombination Repair
• damage to both strands
• may use homologous (similar) portion of
sister chromatics as template to
construct new DNA section
• this is likely to contain errors, but it
is better than no repair at all
 If mutations are very severe, it may trigger
suicide genes that cause the cell to die.
Restriction Endonucleases
 Family of enzymes, in most prokaryotic
organisms, that can recognize a specific
short sequence of nucleotides on a strand
of DNA & cut the strand at a particular
point within the sequence.
• this point is known as a restriction site
• used by researchers to cleave DNA
molecules
DNA Amplification
 The process of generating a large sample
of a target DNA sequence.
 2 ways
1. cloning using a bacterial vector
• splice the target fragment into
bacterial plasmid
2. Polymerase Chain Reaction (PCR)
• utilizes heat resistant DNA
polymerase to replicate the target
sequence
Gel Electrophoresis
 Used to separate molecules according to
their mass & electrical charge.
 This process enables fragments of DNA to
be separated so they can be analyzed.
• this pattern is called a DNA fingerprint
Sequencing DNA
 The process used is chain termination
sequencing.
• used to determine sequence of
nucleotides of an organism’s DNA or
“map” the sequence
• used to map the genome of an organism
e.g., the Human Genome Project (HGP)
The Chimera: From Legend to Lab
 In Greek mythology, the chimera is a firebreathing monster with a lion's head and a
goat's body and a serpent's tail.
 In genetics, the term “chimera” is used to
describe genetically engineered organisms
that contain genes from unrelated
organisms.
Recombinant DNA Technology
 A body of techniques for cutting apart and
splicing together different pieces of DNA.
 The genetic material from the one
organism is then inserted into a foreign cell
in order to mass produce the protein
encoded by the inserted gene(s).
 Thus, these cells become "factories" for
the production of the protein coded for by
the inserted DNA.
 Also called ‘genetic engineering’, ‘gene
splicing’ or ‘genetic modification’.
Applications of Recombinant DNA Technology
1. herbicide-resistant corn
2. human insulin
3. bioremediation: PCB-eating bacteria
4. improved nutrition
Weighing the Risk of DNA Technology
1. environmental threats
2. health effects
3. social & economic issues
Transforming Animal DNA
 In animals, the problem is cell
differentiation.
 In plants, differentiation is not permanent
in most cells.
 In animals, once the cells have
differentiated into specialized cells, it is
usually unable to give rise to other cells.
• as portions of its DNA become
permanently activated or deactivated
Cloning
 Organisms that are genetically identical
are said to be clones.
Examples:
• asexual reproduction of plants
• identical twins
• Dolly the sheep
1. Therapeutic Cloning
•
culturing of human cells for use in treating
medical disorders
2. Reproductive Cloning
•
development of a cloned human embryo for
the purpose of creating a cloned human
being
Gene Therapy
Definitions:
1. The process of changing the function of
the genes in order to treat or prevent
genetic disorders.
2. An approach to preventing and/or treating
disease by replacing, removing or
introducing genes or otherwise
manipulating genetic material.
3. Inserting the normal gene into a person, to
replace a non-working or missing gene.
Gregor Mendel
Source: http://www.jic.bbsrc.ac.uk/germplas/pisum/zgs4f1.gif
Friedrich Miescher
Source: http://www.cityinfonetz.de/das.magazin/2001/29/artikel5_bild1_kl.jpg
Phoebus Levene
Courtesy of the Rockefeller Archive Centre
DNA & RNA Diagram
The structure of the
ribose (found in RNA)
differs at the 2’
carbon, where it is
bonded to a hydroxyl
group, whereas in
deoxyribose (found in
DNA) the 2’ carbon is
bonded to a single
hydrogen molecule.
Source: http://library.thinkquest.org/13373/work/dna-rna.gif
DNA Components
O
O
H
CH3
H
N
O
CH3
N
N
O
O
Bases
N
Sugar (2' deoxy-ribose)
O
O
O
O
O
P
O
O
-
P
Phosphate
deoxyribonucleic acid
O
Source: http://www.owlnet.rice.edu/~chem547/lectures/lecture13.ppt
O
RNA Components
O
O
H
H
N
O
N
N
O
Bases
N
OH
OH
O
O
O
O
Sugar (ribose)
O
O
P
O
O
-
P
ribonucleic acid
O
Source: http://www.owlnet.rice.edu/~chem547/lectures/lecture13.ppt
O
Phosphate
Griffith’s Experiment
Key Experiment
In the 1920's, Griffith's experiments
showed that a harmless strain of
bacteria becomes infectious when
mixed with a virulent (harmful) strain
of bacteria that has been killed. The
dead bacteria apparently provides
some chemical that can transform a
harmless bacteria (rough) into a
harmful bacteria (smooth). This
"transforming principle" appeared to
be a gene.
Griffith's Conclusion:
Something from the harmful heatkilled (smooth) cells was transferred
to the harmless (rough) cells to make
it look and act like the harmful
(virulent) strain.
Source:
http://www.csd99.k12.il.us/slargen/Biology/B300%20Handouts/B%20Unit%20Handouts/B%20Sem%2
0II%20Handouts/DNA/SL%20DNA%20-%20How%20Scientists%20Figured%20it%20Out%2003.htm
Erwin Chargaff
Source: http://post.queensu.ca/~forsdyke/images/chargaf2.jpg
Alfred Hershey & Martha Chase
Courtesy of the Cold Springs Harbor Laboratory Archives.
Hershey-Chase Experiment
Source: http://fig.cox.miami.edu/~cmallery/150/gene/c16x2hershey-chase.jpg
Rosalind Franklin
Source: http://www.physics.ucla.edu/~cwp/images/franklin/franklin2.2.jpg
Maurice Wilkins
Source: http://www.kcl.ac.uk/depsta/iss/archives/dna/image/wilkins.jpg
X-ray Crystallography
Source: http://www.genoscope.cns.fr/externe/HistoireBM/diffr_ADN_B.jpg
James Watson & Francis Crick
Source: http://studentweb.tulane.edu/~rmatz/IMG_0595.jpg
DNA – A Twisted Ladder
Copyright © 2004 Pearson Education, Inc.
DNA – Molecular Representation
hydrogen bond
phosphate bridge
Copyright © 2004 Pearson Education, Inc.
Nucleotides Diagrams
Source: http://www.alumni.ca/~leema3m/
Directionality Diagram
Source: http://www.alumni.ca/~leema3m/
Base Pairs Diagram
Pairing is caused by
hydrogen bonds, weak
links between oxygen
and nitrogen atoms
where one of them has a
hydrogen attached.
A-T pairs have 2 hydrogen
bonds, while G-C pairs
have 3 hydrogen bonds.
G-C pairs are stronger, and
they are more frequent in
high temperature
organisms.
Source: http://www.alumni.ca/~leema3m/
Purines & Pyrimidines Diagrams
single-ringed
double-ringed
Source: http://www.mun.ca/biology/scarr/2250_DNA_biochemistry.htm
Nucleoid
Sources: http://bricker.tcnj.edu/micro/le3/3_1.gif
http://www.cat.cc.md.us/courses/bio141/lecguide/unit1/control/nucleoid/images/u1fig12.gif
Plasmid Diagram
Source: http://www.mmb.usyd.edu.au/MBLG2001/MBLGlectures/MicroImages/PlasmidinCell.jpg
DNA Packing
Sources: http://www.accessexcellence.com/AB/GG/chroma_packg.html
http://fig.cox.miami.edu/~cmallery/150/proceuc/chromosome.jpg
DNA Replication Hypotheses
Source: http://fig.cox.miami.edu/~cmallery/150/gene/sf12x1.jpg
Template Replication Model
Copyright © 2004 Pearson Education, Inc.
Replication Bubble & Forks
Source: http://www.anselm.edu/homepage/jpitocch/genbio/dnareplic.JPG
Helicase
Source: http://www.yangene.com/images/DNA2.jpg
Primer & Primase
Source: http://fig.cox.miami.edu/~cmallery/150/gene/16x14.jpg
Okazaki Fragments
Source: http://fig.cox.miami.edu/~cmallery/150/gene/sf12x7b.jpg
DNA Ligase
Source: http://www.blc.arizona.edu/marty/411/Modules/Weaver/Chap20/Fig.2027ad.jpg
Telomere & Telomerase
Source: http://departments.oxy.edu/biology/Franck/Bio130S_2002/Images/Ch16/fig16_19b.JPG
Telomerase Animation
Source: http://www.exn.ca/news/images/1998/01/14/19980114-enzyme.gif
Replication Machine
Source: http://departments.oxy.edu/biology/Franck/Bio130S_2002/Images/Ch16/fig16_16.JPG
Gene Diagram
Copyright © 1999 Access Excellence @ the National Health Museum
Exons & Introns Diagram
Copyright © 1999 Access Excellence @ the National Health Museum
Introns Diagram
Source: http://www.umbc.edu/bioclass/biol100/powerpoints/lecture06/img015.jpg
Central Dogma Theory
Source: http://allserv.rug.ac.be/~avierstr/principles/cendog.gif
Genetic Code Table
Source: http://www.nyu.edu/classes/ytchang/book/n002/code.gif
Transcription – Promoter Sequence
Source: http://www.sp.uconn.edu/~bi102vc/images/promoter.GIF
mRNA Elongation Diagram
Source: http://io.uwinnipeg.ca/~simmons/cm1503/Image278.gif
mRNA Processing Diagram
Source: http://www.virtuallaboratory.net/Biofundamentals/lectureNotes/AllGraphics/transcriptProcessing-1.jpg
mRNA Splicing Diagram
Source: http://www.accessexcellence.org/AB/GG/rna_synth.gif
tRNA Molecule
Source: http://w3.dwm.ks.edu.tw/bio/activelearner/12/images/ch12c4.gif
aa-tRNA Synthetase
Source: http://fig.cox.miami.edu/~cmallery/150/gene/17x13.jpg
Ribosome Diagram
Source: http://fig.cox.miami.edu/~cmallery/150/cells/ribosome.jpg
Translation – Initiation Figure
Source: http://io.uwinnipeg.ca/~simmons/cm1503/Image282.gif
Translation – Elongation Figure
Source: http://io.uwinnipeg.ca/~simmons/cm1503/Image283.gif
Translation – Termination Figure
Source: http://io.uwinnipeg.ca/~simmons/cm1503/Image285.gif
Polyribosome Diagrams
Source: http://io.uwinnipeg.ca/~simmons/cm1503/Image286.gif
Operon Figure
Source: http://www.blc.arizona.edu/Marty/181/181Lectures/Figures/POHS_Figures/Chap13/Fig13_22.JPG
Repressor Active
Source: http://www.blc.arizona.edu/Marty/181/181Lectures/Figures/POHS_Figures/Chap13/Fig13_23a.JPG
Inducer
Source: http://www.blc.arizona.edu/Marty/181/181Lectures/Figures/POHS_Figures/Chap13/Fig13_23b.JPG
Repressor & Inducer
Source: http://www.msu.edu/course/lbs/145/smith/s02/graphics/campbell_18.20.gif
cAMP-CAP for lac Operon
Source: http://www.msu.edu/course/lbs/145/smith/s02/graphics/campbell_18.21.gif
tryp Absent
Source: http://www.blc.arizona.edu/Marty/181/181Lectures/Figures/POHS_Figures/Chap13/Fig13_24a.JPG
tryp Present
Source: http://www.blc.arizona.edu/Marty/181/181Lectures/Figures/POHS_Figures/Chap13/Fig13_24b.JPG
tryp Absent & Present
Source: http://fajerpc.magnet.fsu.edu/Education/2010/Lectures/28_Gene_Control_files/image016.jpg
Nucleotide Substitution Figure
Source: http://io.uwinnipeg.ca/~simmons/cm1503/mutations.htm
Nucleotide Insertion or Deletion Figure
Source: http://io.uwinnipeg.ca/~simmons/cm1503/mutations.htm
Excision Repair Figure
Source: http://io.uwinnipeg.ca/~simmons/cm1503/mutations.htm
Sources:
http://www.accessexcellence.org/
http://www.bios.niu.edu/johns/genetics/dna.ppt
http://www.dnaftb.org/dnaftb/
http://www.emc.maricopa.edu/faculty/farabee/BIOBK/Bi
oBookDNAMOLGEN.html
http://www.monmouthchurch.org/bio111/ch10.ppt
http://www.wisc.edu/molpharm/Courses/pharm620/Lect
ure_2Central_Dogma.web.ppt