Download Chapter 3. The Beginnings of Genomic Biology

Chapter 3. The Beginnings of Genomic Biology –
Molecular Genetics
Contents
3. The beginnings of Genomic Biology – molecular
genetics
3.1. DNA is the Genetic Material
3.2. Watson & Crick – The structure of DNA
3.3. Chromosome structure
3.3.1. Prokaryotic chromosome structure
3.3.2. Eukaryotic chromosome structure
3.3.3. Heterochromatin & Euchromatin
3.4. DNA Replication
3.4.1. DNA replication is semiconservative
3.4.2. DNA polymerases
3.4.3. Initiation of replication
3.4.4. DNA replication is semidiscontinuous
3.4.5. DNA replication in Eukaryotes.
3.4.6. Replicating ends of chromosomes
3.5. Transcription
3.5.1. Cellular RNAs are transcribed from DNA
3.5.2. RNA polymerases catalyze transcription
3.5.3. Transcription in Prokaryotes
3.5.4. Transcription in Prokaryotes - Polycistronic mRNAs are
produced from operons
3.5.5. Beyond Operons – Modification of expression in
Prokaryotes
3.5.6. Transcriptions in Eukaryotes
3.5.7. Processing primary transcripts into mature mRNA
3.6. Translation
3.6.1. The Nature of Proteins
3.6.2. The Genetic Code
3.6.3. tRNA – The decoding molecule
3.6.4. Peptides are synthesized on Ribosomes
3.6.5. Translation initiation, elongation, and termnation
3.6.6. Protein Sorting in Eukaryotes
CONCEPTS OF GENOMIC BIOLOGY
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 CHAPTER 3. THE BEGINNINGS OF GENOMIC
BIOLOGY – MOLECULAR GENETICS (RETURN)
As the development of classical genetics
proceeded from Mendel in 1866 through the
early part of the 20th century the understanding
that Mendel’s factors that produced traits were
carried on chromosomes, and that there were
infinite ways that the genetic information from 2
parents could assort in each generation to
produce the genetic variety demanded by
Darwin’s theories on “origin of species” on which
natural selection acted. This gave rise to the
study of gene behavior of more complex traits
and an understanding of genes in populations.
At the same time a quest for the material
inside a cell, perhaps a subcomponent of a
chromosome, that carried the genetic instructions
to make organisms what they are was ongoing.
3.1. DNA IS THE GENETIC MATERIAL.
(RETURN)
In 1928, a British scientist,
Frederick Griffith, published his
work showing that live, rough,
avirulent bacteria could be
transformed by a “principle”
found in dead, smooth, virulent
bacteria into smooth, virulent
bacteria. This meant that the
bacterial traits of rough versus
smooth and avirulence versus
Frederick Griffith
viru(1879-1941)
virulence were controlled by a substance that could
carry the phenotype from dead to live cells.
Griffith’s observations on Pneu-mococcus were
controversial to say the least, and inspired a spirited
debate and much experimentation directed at proving
whether the “transforming principle” was protein or
nucleic acid, the two main components of
CONCEPTS OF GENOMIC BIOLOGY
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chromosomes identified early in the 20th century, well
before Griffith’s experiments. This debate continued
until Oswald Avery and his colleagues, Colin MacLeod,
and Maclyn McCarty published their work in 1944
unequivocally showing that DNA was, in fact, Griffith’s
transforming principle. This completely revolutionized
genetics and is considered the founding observation of
molecular genetics.
Oswald T. Avery
Colin MacLeod
Maclyn McCarty
In 1953, more evidence supporting DNA being the
genetic material resulted from the work of Alfred
Hershey and Martha Chase on E. coli infected with
bacteriophage T2. In their experiment, T2 proteins
were labeled with the 35S radioisotope, and T2 DNA was
labeled with was labeled with the 32P radioisotope. Then
the labeled viruses were mixed separately with the E.
coli host, and after a short time, phage attachment was
disrupted with a kitchen blender, and the location of
the label determined. The 35S-labeled protein was found
outside the infected cells, while the 32P-labeled DNA
was inside the E. coli, indicating that DNA carried the
information needed for viral infection.
Figure 3.1. An electron micrograph of bacteriophage T2 (left),
and a sketch showing the structures present in the virus
(right). The head consists of a DNA molecule surrounded by
proteins, while the core, sheath, and tail fibers are all made of
protein. Only the DNA molecule enters the cell.
Once it was established that DNA was the genetic
material carrying the instructions for life so to speak,
attention turned to the question of “How could a
molecule carry genetic information?” The key to that
became obvious with a detailed understanding of the
structure of the DNA molecule, which was developed by
two scientists a Cambridge University, James Watson
and Francis Crick.
CONCEPTS OF GENOMIC BIOLOGY
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3.2. WASON & CRICK – THE STRUCTURE OF DNA.
(RETURN)
The basic laboratory observations that lead to the
formulation of a structure for DNA did not involve
biologists. Rather Irwin Chargaff, an analytical, organic
chemist, and physicists, Rosalind Franklin and Maurice
Wilkins made the laboratory observations that led to
the solution of the structure of DNA.
Chargaff determined that there were 4 different
nitrogen bases found in DNA molecules; the purines,
adenine (A) and guanine G), and the pyrimidines,
cytosine (C) and thymine (T), and he purified DNA from
a number of different sources so he could examine the
quantitative relationships of A, T, G, and C. He concluded that in all DNA molecules, the mole-percentage
of A was nearly equal to the mole-percentage of T,
while the mole-percentage of G was nearly equal to the
mole-percentage of C. Alternatively, you could state
this as the mole-percentage of pyri-midine bases
equaled the mole-percentage of purine bases. These
observations became known as Chargaff’s rules.
Rosalind Franklin a young x-ray crystallographer
working in the laboratory of Maurice Wilkins at
Cambridge University used a technique known as x-ray
diffraction to generate images of DNA molecules that
showed that DNA had a helical structure with repeating
structural elements every 0.34 nm and every 3.4 nm
along the axis of the molecule.
Rosalind Franklin
Figure 3.2. X-ray diffraction image
of DNA molecule showing helical
structure with repeat structural
elements.
Maurice Wilkins
CONCEPTS OF GENOMIC BIOLOGY
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These astute observations allowed Watson and Crick
to synthesize together a 3-dimentional structure of a
DNA molecule with all of these essential features. This
structure was published in 1953, and immediately
generated much excitement, culminating in a Nobel
Prize in Physiology and Medicine, in 1962 awarded to
Franklin, Wilkins, Watson, and Crick.
Figure 3.3. Watson & Crick’s DNA structure. Their model consisted of
a double helicical structure with the sugars and phosphates making
the two hlices on the outside of the structure. The sugars were held
together by 3’-5’-phosphodiester bonds. The bases pair on the inside
of the molecule with A always pairing with T, and G always pairing
with C. This pairing leads to Chargaff’s observations about bases in
DNA.
The key elements of this structure are:
• Double helical structure – each helix is made from
the alternating deoxyribose sugar and phosphate
groups derived from deoxynuclotides, which are the
monomeric units that are used to make up
polymeric nucleic acid molecules. Each nucleotide
in each chain consists of a nitrogen base of either
the purine type (adenine or guanine) or the
pyrimidine type (cytosine or thymidine) attached to
the 1’-position of 2’-deoxyribose sugar, and a
phosphate group, esterified by a phospho-ester
bond to the 5’-position of the sugar.
Figure 3.4. Structures of purine and pyrimidine bases in DNA,
and structure of 2’-deoxyribose sugar.
CONCEPTS OF GENOMIC BIOLOGY
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Figure 3.5. The building bocks of nucleic
acids are nucleotides and nucleosides. Any
base together with a deoxyribose sugar
forms a deoxyribonucleoside, while if the
sugar is ribose a ribonucleoside is formed
(not shown). Addition of a phosphate on
the 5’ position of the sugar froms
nucleotides from nucleosides.
• The nucleotides are held together in sequence order
along the length of the polynucleotide chain by 3’-5’phosphodiester bonds, and the strands demonstrate
a polarity as the 5’-OH at one end of a polynucleotide strand is distinct from the 3’-OH at the other
end of the strand. Often, but not always, the 5’strand end will have a phosphate group attached.
• Each of the 2 polynucleotide chains of the double
helix are held together by hydrogen bonds beween
the adenosines in one strand and the thymidines in
the other strand, and between the guanosines in one
strand hydrogen bonded to the cytosines in the other
strand.
Figure 3.6. Base pairing between A and T involves two
hydrogen bonds, and pairing between G and C involves 3
hydrogen bonds. This means that the forces holding strands
together in G=C base pair-rich regions are stronger than in A=T
base pair-rich regions.
Figure 3.7. Strand of DNA
showing the 3’,5’-phosphodiester
bonds
holding
nucleotides together.
CONCEPTS OF GENOMIC BIOLOGY
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In order to get a uniform diameter for the molecule
and have proper alignment of the nucleotide pairs in
the middle of the strands, the strands must be oriented in antiparallel fashion, i.e. with the strand polarity of
each strand of the double helix going in the opposite
direction (one strand is 3’-> 5’ whie the other is 5’ ->
3’).
The truly elegant aspect of this solution to DNA
structure produces a spacing of exactly 0.34 nm
between nucleotide base pairs in the molecule, and
there are 10 base-pairs per complete turn of the helix.
This corresponds precisely with Rosalind Franklin’s x-ray
diffraction measurements of repeating units of 0.34 nm
and 3.4 nm, and with her measurements of 2 nm for the
diameter of the double helix.
It is also noteworthy that Watson and Crick
suggested that the structure they proposed produced a
clear method for the two strands of the DNA molecule
to duplicate and maintain the fidelity of the sequence of
bases along each chain as DNA was synthesized inside a
cell. Thus, providing a mechanism for the fidelity of
information transfer from cell generation to cell
generation.
CONCEPTS OF GENOMIC BIOLOGY
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3.3. CHROMOSOME STRUCTURE.
(RETURN)
The DNA inside a cell seldom exists as a simple,
“naked” DNA molecule. Because DNA molecules are
long linear molecules with an overall negative charge
deriving from the phosphate groups making up the
helices, positively charged ionic species within cells are
attracted to these molecules. These positively charged
molecules can be small ions such as K+ and Mg++, or they
can be larger positively charged proteins, and/or other
larger molecular species. These ionic interactions play
an important role producing the folding and packaging
that is required to keep the large linear molecule
packaged inside the microscopic cell.
In the case of proteins it is clear that the positively
charged proteins can interact both by general ionic
interactions, but they can also ingeract in sequence
specific ways; i.e. specific proteins only bind to specific
sequences of bases in the DNA strand. Thus, the types
of molecular interactions that ionic substances,
particularly proteins, have with DNA molecules play
important roles in determining the expression of
information that is carried in the DNA molecule. It will
be obvious as we proceed through our study of genomic
biology, that such DNA-protein interactions are as
critical to describing “genetic information” as are the
base sequences of the DNA molecules themselves. This
was obvious well before we btained the first genomic
DNA sequences, but has become even more apparent
and significant now that we have the DNA sequences of
many genomes. Thus, genomic biology is not merely
the study of DNA nucleotide sequences, but involves
the study of the structure of the genetic material such
as chromosomes and chromatin.
3.3.1. Prokaryotic chromosome structure (return)
Most Prokaryotes (e.g. bacteria) have a single,
circular chromosome although some have more than
one chromosome, and some have linear chromosomes
rather than circular chromosomes. Certainly, the most
well studied bacteria, e.g. Escherichia coli, has a single
circular chromosome that can exist in either a relaxed or
supercoiled state.
Supercoiling involves breaking one of the 2 circular
helical strands and then rotating the broken ends either
in the direction of the helix (+ supercoil) or in the
opposite direction of the helix (- supercoil). As
supercoiling is added to the DNA molecule it becomes
“tightly” coiled (see Figure 3.8.), and therefore can be
compacted more easily. This permits the packaging of
the large DNA molecule into the relatively small cells in
which it must exist and function.
CONCEPTS OF GENOMIC BIOLOGY
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Figure 3.9. Diagram of DNA organizational structure in
prokaryotes. Supercoiled DNA is looped and attached
to scaffold proteins.
Figure 3.8. An E. coli cell lysed open showing the expanse of its DNA
molecule (left). Note that this entire molecule must be folded and
packaged inside the cell in the picture. On the right are two electron
micrographs showing circular DNA molecules either in a relaxed (top) or
supercoiled (bottom) state.
Additional packaging results from the supercoiled
DNA being carefully looped onto a scaffold of proteins
leading to an organized intracellular structure that can
be easily accessible but also keep DNA from twisting
and being damaged du-ring normal cellular processes.
3.3.2. Eukaryotic chromosome structure
(return)
In general Eukaryotes have much larger genomes
than do Archea and other Prokaryotes. This difference
in relative genome size compared to the complexity of
the organism does not appear to be as true for species
within the Eukaryota. This lack of correlation between
organismal complexity and genome size (called the Cvlaue) is referred to as the C-value paradox (Table 3.1)
The C-value paradox results from great variation in the
nature of DNA in different Eukaryotes.
Some
eukaryotes contain substantial amounts of DNA that
appears to have limited or at have a gene density in
their genomes resembling the Prokaryotes (e.g. the
yeasts and malarial parasite in the table above). The
CONCEPTS OF GENOMIC BIOLOGY
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majority of Eukaryotes fall somewhere in between
these extremes, but are highly variable in their DNA
contents. For now we need to appreciate that this
variation in DNA content and type appears to have a
relationship to chromosome structure. But the nature
of this relationship will be considered further once we
learn more about DNA sequencing and examine fully
sequenced genomes.
Figure 3.10. Electron micrograph showing the nucleosome structure
of Eukaryotic DNA. The DNA molecule is barely visible, but connects
the beads of proteins that the DNA wraps around creating the
appearance of beads on a string.
In eukaryotes, there are multiple levels of
chromosomal organization that we will need to
consider. Observations using powerful electron
CONCEPTS OF GENOMIC BIOLOGY
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microscopes demonstrated that in Eukaryotes, the DNA
molecules in chromosomes are organized like beads on
a string. These structures have subsequently been
named nucleosomes. Investigation of the nature of
nucleosomes has shown that they are made from
several types basic proteins (positively charged) found
in cells called histone proteins.
The basic nucleosome consists of a combination of
histones H2A, H2B, H3, and H4. DNA is subsequently
wrapped around these structures producing the beadlike appearance observed in the electron microscope.
Once the nucleosomes are formed, they can condense
or decondense based on interaction with another
histone, histone H1.
Figure 3.11. Nucleosomes are formed when DNA wraps
around a histone complex. Nucleosomes can exist in either a
more condensed or a decondenses state depending ot the
state of the genetic material in a cell.
During prophase of mitosis or meiosis, the
nucleosome structure of chromatin further condenses
into a so-called solenoid structure, which is approximately 30 nm in diameter. This solenoid from is not
visible in a light microscope but can be viewed in an
electron microscope. This appears to be the form DNA
assumes when chromosomes condense during during
mitosis, but the DNA is not as accessible for use in the
cell as it is during interphase, when the chromatin is
decondensed.
CONCEPTS OF GENOMIC BIOLOGY
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Figure 3.13. Loop-folding of the 30 nm solenoid structure yields a
packaged DNA that is visible in a ligh microscope in each Eukaryotic
chromosome.
structure appears to be required to allow for the
appropriate assembly and assortment of the genetic
material during the cell cycle in mitosis. Without this
structural organization, it
Figure 3.12. Condensation of chromatin leads to
the careful packaging of DNA into so called
solenoid sturctures. These structures ultimately
form chromosomes.
The solenoid structures are subsequently looped and
fastened to chromosome scaffold proteins generating a
structure that is visible in a light microscope that we
know as a chromosome.
While this may seem like an elaborate structure
involving several sets of structural proteins, such a
is likely that cellular DNA would become a hopeless
tangle, and cellular reproduction would be severely
hampered, and would likely require too much time and
effort to ultimately be successful.
3.3.3. Heterochromatin & Euchromatin.
(return)
The cell cycle affects DNA packing into chromatin
with chromatin condensing for mitosis and meiosis and
then decondensing during interphase while being most
dispersed at S-phase. However, cytogeneticists have
observed that there can be two differently staining
forms of chromatin, called Euchromatin and
CONCEPTS OF GENOMIC BIOLOGY
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heterochromatin.
Euchromatin condenses and
decondenses with the cell cycle. Euchromatin accounts
for most of the active genome in dividing cells and bears
most of the protein-coding DNA sequences.
Heterochromatin remains condensed throughout the
cell cycle and is believed to be relatively inactive. There
are two types of heterochromatin based on activity, ie.
constitutive heterochromatin that is tightly condensed
in virtually all cell types and facultative
heterochromatin which varies between cell types
and/or developmental stages.
Other methods of characterizing types of DNA
suggest that there are sequences of DNA that can occur
in may copies in the genome. These types of sequences
can be repeated only once in the genome or they can
occur 10’s of thousans of times or more in genomes.
Sequences can be categorized into:
• Unique-sequence DNA, present in one or a few copies
per genome.
• Moderately repetitive DNA, present in a few to 105
copies per genome
• Highly repetitive DNA, present in about 105–107
copies per genome
Observations about repetitive DNA sequences as
described above have been known for decades, and
initially it was shown that Prokaryotic DNA was mostly
unique-sequence DNA, and Prokaryotes had little or no
repetitive sequences. However, Eukaryotes have a mix
of unique and repetitive sequence types of DNA.
• Unique-sequence DNA includes most of the genes
that encode proteins, and Euchromatin is rich in
unique-sequence DNA.
• Repetitive-sequence DNA includes the moderately
and highly repeated sequences. They may be
dispersed throughout the genome or clustered in
tandem repeats. Heterochromatin is rich in moderate
and highly repetitive DNA.
•
Human DNA contains about 65% unique sequences
while unque sequence DNA makes up a much lower
percentage of the genome of organisms that have
unexpectedly large genomes (C-values) that were
discussed earlier in this section.
CONCEPTS OF GENOMIC BIOLOGY
3.4. DNA REPLICATION.
Page 13
(RETURN)
As Watson and Crick were solving the structure of
DNA, they realized the general mechanism by which the
molecule could be copied and maintain fidelity in
copying the DNA molecule. From that beginning,
interest in understanding the duplication of the DNA
molecules of a cell became a subject of investigation,
and led to a number of Nobel Prize awards. However,
understanding DNA replication was critical to the
development of the technologies needed for molecular
genetics and ultimately genomic biology research.
3.4.1. DNA Replication is semiconservative.
but it is possible to find atoms with 7 protons, and 8
neutrons, having an atomic mass of 15 (written as 15N).
It turns out that if you grew bacterial cells on a nitrogen
source enriched in a 15N enriched nitrogen source, the
DNA molecules purified from such cells have a greater
density (they are heavier). By synchronizing cells and
purifying DNA after each round of DNA replication and
then determining the density of the newly made DNA
molecules using density gradient centrifugation,
Meselson and Stahl were able to show that the first
round of DNA synthesis produced molecules having a
hybrid density between light and heavy DNA. While
after a subsequent round of DNA replication they
produced light and hybrid molecules. Such a pattern of
(return)
Among the earliest experiments concerning the
nature of how DNA replicates were the studies of
Mathew Meselson and Frank Stahl. Meselson, while a
Ph.D. student designed an experiment that utilized so
called “heavy” isotopes nitrogen. Elemental isotopes
consist of atoms having the same number of proton, but
with more than the average number of neutrons. For
example, nitrogen normally has 7 protons, and 7
neutrons, giving it an atomic mass of 14 (written 14N)
Figure 3.14. Diagram showing the predicted outcome
of conservative, semiconservative, and dispersive DNA
replication. Original strands are shown in red while
newly made DNA is shown in blue.
CONCEPTS OF GENOMIC BIOLOGY
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15N
labeling was consistent only
semiconservative replication of DNA.
3.4.2. DNA Polymerases.
with
the
(return)
The enzyme that replicates the DNA double helix is
called DNA polymerase. The enzyme is difficult to work
with because there are but a few copies of it needed
per cell, and then they are required only in S-phase of
the cell cycle. In spite of these limitations, Arthur
Kornberg, won the Nobel Prize in 1959 for the first
purification and characterization of an enzyme that
makes DNA. Kornberg’s enzyme was purified from the
bacterium E. coli, and beside the enzyme 4 additional
components were required to make DNA in a test tube.
These factors included a template DNA (Kornberg used
E. coli DNA), the four deoxy nucleotide triponosphates
(dNTP), i.e. dATP, dGTP, dCTP, and dTTP. Note that
these are the deoxy NTP, and not the ribose containing
NTP’s.
The remaining requirements for DNA
polymerase are magnesium ion (Mg++) and a primer
single strand of DNA. This primer requirement involves
a single strand of DNA that will form a short doublestranded region of DNA. DNA polymerase then adds
nucleotides to the free 3’-end of this primer, but
without the primer DNA polymerase is unable to make a
DNA strand. As the nucleotides are added they are
added from the 5’-end to the growing 3’-end of the
strand according to the sequence of the corresponding
strand being copied. This copied strand is referred to as
the template strand.
All DNA polymerases studied to date make DNA
using the general principles established for Kornberg’s
Figure 3.15. Note that the template strand is read from it’s 3’-end
to its 5’-end while the antiparallel, new DNA strand is made from
the 5’-end to the 3’-end.
CONCEPTS OF GENOMIC BIOLOGY
Page 15
enzyme, but there are significant differences between
them in other respects. For example, in E. coli there are
five different DNA polymerases. Kornberg’s enzyme is
now known as DNA polymerase I, but there are also
DNA polymerases II, III, IV, and V. DNA polymerases II,
IV, and V are not involved in the DNA replication
process, and they have specialized functions in repairing
damaged DNA under specific circumstances. DNA
polymerases I and III are the DNA polymerases involved
in the replication of cellular DNA. Both of these DNA
polymerases contain a 3’ -> 5’ exonuclease activity that
is involved in proof-reading the recently made DNA
strand and removing any mistakes that are made. Only
DNA polymerase I has a 5’ -> 3’ exonuclease activity and
we will visit this function again below when the role of
DNA polymerase I in DNA replication is considered.
3.4.3. Initiation of replication.
• A minimal sequence of about 245 bp required for
initiation.
• Three copies of a 13-bp AT-rich sequence.
• Four copies of a 9-bp sequence.
(return)
Replication initiates at a specific sequence in the
genome that is often called an origin of replication. E.
coli has one origin, called oriC, where replication starts
when the strands of the helix are forced apart to expose
the bases, creating a replication bubble with two
replication forks. Replication is usually bidirectional
from the origin using the two forks to enlarge the
bubble in both directions. E. coli has one origin, oriC,
with the following properties:
Figure 3.16. Initiation of DNA replication in E.
coli. at oriC. Noote the 9 and 13 bp repeats
where DNA helicase binds and activates
replicatlion throught the action of DNA primase.
CONCEPTS OF GENOMIC BIOLOGY
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From a series of in vitro studies it has been shown in E.
coli that the following steps are involved in initiating
replication:
1) Initiator proteins attach to oriC (E. coli’s initiator
protein is the DnaA protein derived from the dnaA
gene.
2) DNA helicase (from dnaB gene) binds initiator
proteins on the DNA and denatures the AT-rich 13bp region using ATP as an energy source.
3) DNA primase (from the dnaG gene) binds helicase
to form a primosome, which synthesizes a short
(5–10 nt) RNA primer.
3.4.4. DNA Replication is Semidiscontinuous
(return)
When DNA denatures (strands separate) at the ori,
replication forks are formed. DNA replication is usually
bidirectional, but we will consider events at just one
replication fork, but don’t forget that a similar set of
events are occurring at the other replication fork in the
bubble. The events occurring at each fork are:
1) Single-strand DNA-binding proteins (SSBs) bind the
ssDNA
formed
by
helicase,
preventing
reannealing.
2) Primase synthesizes a primer on each template
strand.
3) DNA polymerase III adds nucleotides to the 3’-end
of the primer, synthesizing a new strand
complementary to the template and displacing the
SSBs. DNA is made in opposite directions (at each
fork) on the two template strands since DNA
polymerase only adds nuclotides to the free 3’end.
4) The new strand made 5’-to-3’ in the same
direction as movement of the replication fork, i.e.
DNA polymerase III is continuously moving toward
the fork on one strand of the bubble at each fork.
This defines the “leading strand”. On the other
strand the new strand must be made in the
opposite direction as it must be made 5’ -> 3’.
5) This means that on this “lagging strand” primase
must add the RNA primer very close to the
replication fork, and the DNA polymerase III
moves away from the fork rather than toward the
fork like it was on the leading strand.
6) The Leading strand needs only one primer and
continuously makes the new DNA strand, while on
the lagging strand a series of RNA primers are
required and only a limited number of DNA
nucleotides are added by DNA polymerase III
before the previously made fragment is
encountered.
CONCEPTS OF GENOMIC BIOLOGY
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ments. DNA replication is therefore semidiscontinuous.
8) As the bubble enlarges and DNA helicase denatures
(untwists) the strands, this causes tighter winding
in other parts of the circular chromosome. A
protein called DNA Gyrase relieves the tension
created in the molecule.
9) As Okazaki fragments accumulate on the lagging
strand, DNA polymerase I binds and the 5’ -> 3’
exonuclease activity removes the RNA primers, and
replaces them with DNA nucleotides.
Figure 3.17. DNA replication at a replication fork showing
continuous DNA synthesis on the lower strand and discontinuous
DNA synthesis on the upper strand where Okazaki fragments are
d d
7) Thus, the leading strand is synthesized continuously, while the lagging strand is synthesized discontinuously in the form of shorter pieces of DNA with
interspersed RNA primers called Okazaki frag-
Figure 3.18. Removal of the RNA primers by the 5’-> 3’
exonuclease of DNA polymerase I, and replacement with DNA
nucleotides on the lagging strand.
CONCEPTS OF GENOMIC BIOLOGY
Page 18
10) The DNA fragments lacking RNA primers are now
fastened together using an enzyme called DNA
ligase that closes the remaining gaps on the lagging
strand.
Primer removal differs from that in prokaryotes. Pol
continues extension of the newer Okazaki fragment,
displacing the RNA and producing a flap that is removed
by nucleases, thus allowing the Okazaki fragments to be
joined by DNA ligase.
Other DNA polymerases replicate mitochondrial or
chloroplast DNA, or they are used in DNA repair. These
are all similar to the prokaryotic system described in
detail above.
3.4.6. Replicating ends of chromosomes.
Figure 3.19. DNA ligase joins an opening in a DNA strand remaking
acomplete phosphodiester-linked polynucleotide chain.
3.4.5. DNA replication in Eukaryotes.
(return)
Enzymes of eukaryotic DNA replication are not as
well characterized as their prokaryotic counterparts.
Fifteen DNA polymerases are known in mammalian
cells, for example. Three DNA polymerases are used to
replicate nuclear DNA. Pol extends the 10-nt RNA
primer by about 30 nt. Pol and Pol extend the
RNA/DNA primers, one the leading strand and the other
on the lagging stand, but it is not clear which
synthesizes which.
(return)
Replicating the ends of chromosomes in organisms
without circular chromosomes presents unique
problems. Removal of primers at the 5’-end of the
newly made strand will produce shorter strands that
cannot be extended with existing DNA polymerases, and
if the gap is not addressed chromosomes would become
shorter each time DNA replicates.
Thus a new
mechanism for the completion of the ends of the
chromosome is required. This is accomplished using the
telomerase system.
Most eukaryotic chromosomes have short, speciesspecific sequences tandemly repeated at their
telomeres. It has been shown that chromosome lengths
are maintained by telomerase, which adds telomere
repeats without using the cell’s regular replication
CONCEPTS OF GENOMIC BIOLOGY
Page 19
machinery. In humans, the telomere repeat sequence is
5’-TTAGGG-3’.
Figure 3.20. The dilemma of how the 3’ overhangs are
replicated at each end of the chromosome to duplicate
a chromosome and make sister chromatids.
Telomerase, an enzyme containing both protein and
RNA, includes an 11-bp RNA sequence used to
synthesize the new telomere repeat DNA. Using an RNA
template to make DNA, telomerase functions as a
reverse transcriptase called TERT (telomerase reverse
transcriptase). The 3’-end of the telomerase RNA
contains the sequence 3’-CAUC, which binds the 5-GTTAG-3’ overhang on the chromosome, positioning
telomerase to complete its synthesis of the GGGTTAG
telomere repeat. Additional rounds of telomerase
activity lengthen the chromosome by adding telomere
repeats. Ends of telomere DNA usually loop back to
form a D-loop. After telomerase adds telomere
sequences, chromosomal replication proceeds in the
usual way. Any shortening of the chromosome ends is
compensated for by the addition of the telomere
repeats.
Telomere length may vary, but organisms and cell
types have characteristic telomere lengths, resulting
from many levels of regulation of telomerase. Mutants
affecting telomere length have been identified, and
data shortening of telomeres eventually leads to cell
death. Loss of telomerase activity results in limited
rounds of cell division before the cell death.
CONCEPTS OF GENOMIC BIOLOGY
Page 20
Figure 3.21. Replication of chromosome ends using telomerase.
CONCEPTS OF GENOMIC BIOLOGY
3.5. TRANSCRIPTION.
Page 21
(RETURN)
In cells the genetic information carried in the DNA
nucleotide sequence becomes functional information
that gives characteristics to cells ultimately specifying
traits. This conversion of DNA sequence information
into functional information begins with the creation of
cellular RNAs from one of the two strands of DNA
sequence. This process is called transcription. The
mechanism by which these cellular RNAs are
transcribed from DNAs will be presented in this section
while the regulation of these processes will be covered
later.
3.5.1. Cellular RNAs are transcribed from DNA (return)
Ribosomal RNAs
(return 3.6.4.)
The most abundant type of RNA in most cells is a
structural component of the cellular particle that is
involved in the synthesis of proteins called a ribosome.
Since ribosomes have 2 subunits, a large subunit and a
small subunit, they also have two major types of
ribosomal RNA. These are described in detail in Table
3.2. In addition to the largest ribosomal RNAs there are
additional smaller ribosomal RNAs as well. Note that
the size and nature of all of these ribosomal RNAs is
different in Prokaryotes and Eukaryotes.
CONCEPTS OF GENOMIC BIOLOGY
Page 22
In prokaryotes a small 30S ribosomal subunit
contains the 16S ribosomal RNA. The large 50S
ribosomal subunit contains two rRNA species (the 5S
and 23S ribosomal RNAs). Bacterial 16S ribosomal RNA,
23S ribosomal RNA, and 5S rRNA genes are typically
organized as a co-transcribed unit (operon). There may
be one or more copies of the operon dispersed in the
genome (for example, Escherichia coli has seven).
Archaea contains either a single rDNA operon or
multiple copies of the operon.
Mammalian
mitochondria
have
only
two
mitochondrial rRNA molecules (12S and 16S) but do not
contain 5S rRNA. The ribosomal RNAs are transcribed
from the mitochondrial genome. This is also the case for
plant mitochondrial rRNAs although plants contain a
more prokaryotic like ribosomal RNAs, i.e. a 16S, a 26S,
and a 5S rRNA. Plants also contain chloroplast
ribosomal RNAs (16S, 23S, and 5S) produced by
transcription from the chloroplast genome.
In Eukaryotes, the cytoplasmic small ribosomal
subunit (40S) contains an 18S rRNA while the large
ribosomal subunit (60S contains a 28S, 5S, and 5.8S
rRNA. As in Prokaryotes these rRNAs are structural
components of ribosomes where they perform essential
function. In mammals, the 28S, 5.8S, and 18S rRNAs are
encoded by a single nuclear transcription unit (45S).
Two internally transcribed spacers separate the 3 rRNA
species in the 45S transcript. Generally, there are many
copies of the 45S rDNAs organized clusters throughout
the nuclear genome. In humans, for example, each
cluster has 300-400 repeats. 5S rDNA is not made as
part of the 45S transcript, but occurs in tandem arrays
(~200-300 5S genes) interspersed in the mammalian
genome independently of the 45S rDNA genes.
All organisms (and mitochondria and chloroplasts)
produce a type of RNA that codes for the amino acid
sequence of proteins. This RNA is a copy of the DNA
sequence of the gene and is transcribed from one of the
two DNA strands of each gene. By reproducing the DNA
sequence as an mRNA copy the sequence information
for the gene is faithfully maintained allowing the
generation of many gene “copies” that can be used to
produce even more protein copies from each gene.
Messenger RNAs – mRNAs
Transfer RNAs - tRNA
(return 3.6.3.)
Transfer RNAs (tRNAs) are smaller (~90 nt) RNA
molecules that are transcribed from genes scattered
throughout both Prokaryotic and Eukaryotic genomes,
including mitochondrial and chloroplast genomes.
These molecules are the “decoding” molecules that
determine which amino acids are put in proteins in the
CONCEPTS OF GENOMIC BIOLOGY
Page 23
order specified by the nucleotide sequence in the
mRNA. They are highly structured RNA molecules, and
there is at least one, often several, tRNA for each of the
twenty protein-contained amino acids. Each tRNA is
processed from a transcribed precursor-tRNA molecule
coded for by specific tRNA genes, and typically there is
but one tRNA produced per tRNA gene.
In Eukaryotes tRNA are scattered across all
chromosomes, and there are separate sets of tRNA
genes in each of the organelle genomes present in
eukaryotes.
Other Non-protein-coding Transcribed RNAs
More recently additional types of RNAs that perform
vital functions in cells have been described. Most of
these have been described in Eukaryotes once we
described and characterized genomes of Eukaryotes.
Small nuclear RNAs (snRNA) are smaller RNAs
(typically ~ 150 nt) transcribed from nuclear DNA in
eukaryotic cells. snRNAs are structurally part of small
nuclear ribonucleoprotein particles (snRNPs) that are
involved in processing mRNAs in the nucleus of cells.
Typically there are but a handful of different snRNAs
made in each species and these are highly conserved
among eukaryotes.
Small nucleolar RNAs (snoRNAs) are a class of small
RNA molecules that function to guide modification of
other types of RNA, mostly rRNA, tRNA, and snRNA.
One of the main functions of snoRNAs involves
modification of the 45S ribosomal precursor so that it
can be futher processes to generate the 18S, 5.8S, and
28S rRNAs.
Small regulatory RNAs are found in prokaryotes
where they are involved in the regulation of gene
expression, but mostly they are known for the role they
play in transcriptional, posttranscriptional and
translational control of gene expression in Eukaryotes.
These molecules are an array of 20-30 nt RNAs
transcribed in various ways from genes in the genomes
of organisms. Note that although there are primarily 2
types of srRNAs, microRNAs (miRNA) and short
interfering RNA (siRNA) these types are specific to
certain organisms and there are likely thousands of
genes transcribed for such srRNAs.
3.5.2. RNA polymerases catalyze transcription (return)
RNA polymerase is the enzyme responsible for
copying a DNA sequence into an RNA sequence, during
the process of transcription. As complex molecule
composed of protein subunits, RNA polymerase controls
the process of transcription, during which the
information stored in a molecule of DNA is copied into a
molecule of cellular RNA.
CONCEPTS OF GENOMIC BIOLOGY
Page 24
The detailed mechanism of how RNA polymerase
works is shown in Figure 3.22.
Figure 3.22. The chemical reaction catalyzed by RNA polymerases
showing both the reactants and products and the specificity of base
pair addition. Note the antiparallel nature of the RNA strand to the
DNA strand being transcribed.
RNA polymerase makes a
phosphodiester bond between the 5’-phosphate group closest to the
ribose sugar and the 3’-OH on the 3’-end of the growing strand of
RNA.
Multisubunit RNA polymerases exist in all species,
but the number and composition of these proteins vary
across taxa. For instance, bacteria contain a single type
of RNA polymerase that transcribes mRNA, tRNA, and
all rRNAs. Eukaryotes contain three (animals and fungi)
to five (plants) distinct types of RNA polymerases. Each
of these RNA polymerases transcribes different species
of RNA as shown in Table 3.3.
CONCEPTS OF GENOMIC BIOLOGY
Page 25
In spite of these differences, there are striking
similarities among transcriptional mechanisms for all
RNA polymerases. For example, transcription is divided
into three steps for both bacteria and eukaryotes. They
are initiation, elongation, and termination. The process
of elongation is highly conserved between bacteria and
eukaryotes, but initiation and termination are
somewhat different.
All species require a mechanism by which
transcription can be regulated in order to achieve
spatial and temporal changes in gene expression.
Proteins that interact with the core RNA polymerase,
and that recognize specific sequences in the DNA
mediate these initial regulatory steps during
transcription initiation. However the types and nature
of these interacting proteins are quite distinct in
Prokaryotes compared to Eukaryotes. This leads to a
discussion of how transcription initiation at each gene
locus takes place in both Prokaryotes and Eukaryotes.
3.5.3. Transcription in Prokaryotes (return)
For a model of Prokaryotic gene regulation, the
bacterium, Escherichia coli, will be used as a model.
This model is similar to nearly all Prokaryotes.
A prokaryotic gene is a DNA sequence in the
chromosome. The gene has three regions, each with a
function in transcription (see Figure 3.23.). These are:
Figure 3.23. Prokaryotic genes all have promoter regions upstream
(toward the 5’-end of the mRNA) of the protein coding gene and
terminator regions downstream (toward the 3’-end of the mRNA).
These regions are located at the 3’-end (promoter) and the 5’-end
(terminator) of the template strand of DNA. Typically the nucleotide
where RNA polymerase begins transcribing is designaed the +1
nucleotide position, and sequences in the promoter are designated as
(-) nt positions.
1) A promoter sequence that attracts RNA
polymerase to begin transcription at a site
specified by the promoter. Some genes use one
strand of DNA as the template; other genes use
the other strand.
2) The transcribed sequence, called the RNA-coding
sequence. The sequence of this DNA corresponds
with the RNA sequence of the transcript.
3) A terminator region that specifies where transcription will stop.
CONCEPTS OF GENOMIC BIOLOGY
Page 26
The process of transcription initiation in E. coli is
shown in Figure 3.24. The process involves two DNA
sequences centered at -35 bp and -10 bp upstream from
the +1 start site of transcription in the promoter region
of the gene. These two consensus sequences (in E. coli)
are 5’-TTGACA-3’ at the -35 nt region and 5’-TATAAT-3’
at the -10 region (previously known as a Pribnow box,
but they can vary according to the organism and gene
within the organism.
Transcription initiation requires the RNA polymerase
holoenzyme (only one type is found in bacteria) to bind
to the promoter DNA sequence. Holoenzyme consists
of:
a
b
c
1) Core enzyme of RNA polymerase, containing five
polypeptides (two alpha, one beta, one beta’ and
an omega; written as α2ββ’ω).
2) One of several sigma factors (σ-factor) that binds
the core enzyme and confers ability to recognize
specific gene promoters.
RNA polymerase holoenzyme binds promoter in two
steps (Figure 3.24) that involve the sigma factor. First, it
loosely binds to the -35 sequence of dsDNA closed
promoter complex (Figure 3.24a). Second, it binds
tightly to the -10 sequence (Figure 3.24b), untwisting
about 17 bp of DNA at the site. At this point RNA
d
Figure 3.24. Prokaryotic (E. coli) transcription initiation. a) RNA
Polymerase holoenzyme is “recruited to the promoter by a specific
σ-factor (sigma factor); b) strands of the DNA are separated
exposing the sense strand for copying; d) nucleotides are
polymerized as RNA polymerase moves down the strand, and σfactor leaves the complex as; d) elongation continues, the newly
made mRNA exits the enzyme, and the transcription “bubble” moves
CONCEPTS OF GENOMIC BIOLOGY
Page 27
polymerase is in position to begin transcription (open
promoter complex).
Promoters often deviate from consensus the
consensus sequences at -35 and -10, and the associated
genes will show different levels of transcription,
corresponding with σ-factor’s ability to recognize their
sequences. E. coli has several sigma factors with
important roles in gene regulation. Each sigma can bind
a molecule of core RNA polymerase and guide its choice
of genes to transcribe, but has different affinity for
specific promoters.
Most E. coli genes have a σ70 promoter, and σ70 is
usually the most abundant σ-factor in the cell. σ70
recognizes the sequence TTGACA at -35, and TATAAT at
-10. Other sigma factors may be produced in response
to changing conditions, and each can bind the core RNA
polymerase, enabling holoenzyme to recognize different
promoters. An example is σ32, which arises in response
to heat shock and other forms of stress and recognizes a
sequence at -39 bp and -15 bp. E. coli has additional
sigma factors with various roles (Table 3.4), and other
bacterial species also have multiple similar and
additional sigma factors.
Many bacterial genes are controlled by regulatory
proteins that interact with regulatory sequences near
the promoter. There are two classes of regulatory
TABLE 3.4.
E. coli σ-factors and their function
s-factors
Function
σ70 (rpoD) = σA
the "housekeeping" sigma factor or also
called as primary sigma factor, transcribes
most genes in growing cells. Every cell has
a “housekeeping” sigma
σ19 (fecI)
the ferric citrate sigma factor, regulates
the fec gene for iron transport
σ24 (rpoE)
the extracytoplasmic/extreme heat stress
sigma factor
σ28 (rpoF)
σ32 (rpoH)
the flagellar sigma factor
the heat shock sigma factor; it is turned
on when the bacteria are exposed to heat.
Due to the higher expression, the factor
will bind with a high probability to the
polymerase-core-enzyme. Doing so, other
heatshock proteins are expressed, which
enable the cell to survive higher
temperatures. Some of the enzymes that
are expressed upon activation of σ32 are
chaperones, proteases and DNA-repair
enzymes.
the starvation/stationary phase sigma
factor
the nitrogen-limitation sigma factor
σ38 (rpoS)
σ54 (rpoN)
proteins, i.e. activators that stimulate transcription by
facilitating RNA polymerase activity, and repressors that
CONCEPTS OF GENOMIC BIOLOGY
Page 28
inhibit transcription by decreasing RNA polymerase
binding or elongation of RNA.
Once initiation is completed, RNA synthesis begins,
and the sigma factor is released and reused for other
initiations (Figure 3.24c). Core enzyme completes the
transcript. Core enzyme untwists DNA helix locally,
allowing a small region to denature. Newly synthesized
RNA forms an RNA–DNA hybrid, but most of the
transcript is displaced as the DNA helix reforms (Figure
3.24d).
Terminator sequences are used to end transcription.
In E. coli there are two types of transcript termination:
1) Rho-independent (ρ-independent) or type I
terminators (Figure 3.25, upper) have twofold
symmetry that would allow a hairpin loop to form
(Figure 3.25). The palindrome is followed by 4–8 U
residues in the transcript, and when these
sequences are transcribed, they form a stem-loop
structure and cause chain termination.
2) Rho-dependent
(ρ-dependent) or type II
terminators (Figure 3.25, lower) require the
protein ρ for termination. Rho binds to the C-rich
sequence in the RNA upsteam of the termination
site and moves with the transcript until
encountering a stalled polymerase. It then acts as
a helicase, using ATP hydrolysis for energy to
move along the transcript and destabilize the
RNA–DNA hybrid at the termination region,
terminating transcription.
Figure 3.25. Simplified
schematics of the mechanisms
of prokaryotic transcriptional
termination. In Rhoindependent termination, a
terminating hairpin forms on the
nascent mRNA interacting with
the NusA protein to stimulate
release of the transcript from
the RNA polymerase complex
(top). In Rho-dependent
termination, the Rho protein
binds at the upstream rut site,
translocates down the mRNA,
and interacts with the RNA
polymerase complex to
stimulate release of the
transcript.
3.5.4. Transcription in Prokaryotes – polycistronic
mRNAs from operons (return)
While we have considered the structure of a
prokaryotic gene as having a promoter, a coding region,
and a termination region (see Figure 3.23), in most
cases multiple protein-coding regions are under the
control of a single promoter. This genetic structure is
CONCEPTS OF GENOMIC BIOLOGY
Page 29
referred to as an operon, and the mRNA transcribed
from each operon is in fact an RNA capable of producing
multiple peptides. This type of mRNA, typical of
prokaryotes, and Eukaryotic mitochondria and
chloroplasts, is referred to as a polycistronic mRNA.
Thus, the proteins binding to promoter and regulatory
regions of genomes that regulate gene expression in
prokaryotes regulate the production of multiple
peptides simultaneously. Typically, these peptides are
functionally related, e.g. the proteins required to
catabolize lactose as a carbon source [lac operon] (see
Figure 3.26.), or the proteins required to make the
amino acid tryptophan [trp operon] (see Figure 3.27.).
The lac operon is an example of an inducible
(positively regulated) operon. The repressor protein
does not bind to the operator and stop transcription in
the presence of the effector (lactose), while the
tryptophan operon is an example of a repressible
(netatively regulated) operon. The repressor protein
only binds to the operator in the presence of the
effector molecule (tryptophan). Thus, using the similar
types of regulatory proteins and genes, and similar
operon structure almost any type of gene regulation can
be obtained.
Additionally, it should be noted that the proteins for
related critical cellular functions can be coordinately
Figure 3.26. The lac operon in E. coli. Three lactose metabolism genes
(lacZ, lacY, and lacA) are organized together in a cluster called
the lac operon. The coordinated transcription and translation of
the lac operon structural genes is controlled by a shared promoter,
operator, and terminator. A lac regulator gene (lacI) with its separate
promoter is found just outside the lac operon. The lacI gene produces
a regulatory protein, the lac repressor protein that binds to the
“inducer”, which is lactose (or a derivative, allolactose) when it is
present in a cell. The lacI protein also can bind to a region of the
operon between the lac promoter and the structural genes referred to
as the lac operator (lacO). In the absence of lactose (allolactose) the
lacI protein tightly binds to the operator and prevents RNA polymerase
from transcribing the polycistronic mRNA. When lactose binds to the
lacI protein, the lacI protein cannot bind to the lacO gene, and RNA
polymerase proceeds to produce the polycistronic mRNA
corresponding to the lacZ, lacY, and lacA genes.
© 2013 Nature Education Adapted from Pierce, Benjamin. Genetics: A Conceptual Approach,
2nd ed. All rights reserved.
regulated as a consequence of the production of
polycistronic mRNAs.
CONCEPTS OF GENOMIC BIOLOGY
Page 30
A
B
Figure 3.27. The tryptophan operon of E. Coli consists of five
structural genes (trpE, trpD, trpC, trpB, and trpA) with a common
promoter, operator, and terminator. A separate promoter regulates
the trpR regulatory protein (trp repressor). Transcription of the trp
operon produces a polycistronic mRNA that contains a leader peptide
and coding sequences for the 5 structural genes that produce the 5
enzymes required to make tryptophan. Since tryptophan is an amino
acid required for cell growth, the trp operon is “repressed” when
cells have access to an abundant supply of tryptophan (panel A), and
becomes “derepressed” when cells are starving for tryptophan (panel
B). A) Tryptophan present, repressor bound to operator, operon
repressed. When complexed with tryptophan, the repressor protein
binds tightly to the trp operator, thereby preventing RNA polymerase
from transcribing the operon structural genes. B) Tryptophan absent,
repressor not bond to operator, operon derepressed. In the absence
of tryptophan, the free trp repressor cannot bind to the operator
site. RNA polymerase can therefore move past the operator and
transcribe the trp operon structural genes, giving the cell the
capability to synthesize tryptophan.
3.5.5. Beyond Operons - Modification of expression
of prokaryotic genes (return)
Additional regulation of operons is often used to
produce further fine-tuning of transcription. This can
vary with each operon in Prokaryotic genomes. A
common type of additional regulation has been shown
for the lac operon and many other catabolic operons.
Glucose is the preferred carbon source in E. coli. In the
presence of glucose, lactose will not be utilized. This
means that if an abundant supply of glucose and lactose
are both available, the lac operon will not be induced
until the glucose is used up. This phenomenon is often
referred to as catabolite repression, and the critical
components of catabolite repression in the lac operon
are shown in Figure 3.28.
When the concentration of intracellular glucose is
low (Figure 3.28, upper panel) the levels of the signal
molecule cAMP are high, and cAMP binds to CAP
protein. The association between RNA polymerase and
promoter DNA is enhanced when the CAP-cAMP
complex is present. Enhanced RNA polymerase binding
leads to a high rate of transcription (provided that the
operator is free) and translation of the lac operon
polycistronic mRNA. The resulting mRNA transcripts are
translated into the enzymes beta-galactosidase,
permease, and transacetylase, and these enzymes are
CONCEPTS OF GENOMIC BIOLOGY
Page 31
used to break down lactose into glucose and Galactose.
The latter can subsequently be converted into glucose.
When the glucose concentration in the cell is high
(Figure 3.28, lower panel), low concentrations of cAMP
result in decreased binding of cAMP to CAP. Therefore,
the cAMP-CAP complex is not bound to the bacterial
DNA, and as a result, neither is RNA polymerase. This
lowers the rate of transcription and polycistronic mRNA
production is decreased for the lacZ, lacY, and lacA
genes. The absence of these proteins reduces glucose
production from lactose, leading to the use of the
available glucose prior to the use of any lactose.
The interaction of CAP with DNA and with cAMP
directly regulates the production of mRNA. Some type
of interaction of proteins with regulatory regions in the
DNA mediates the phenomenon of catabolite
repression in operons associated with carbon source
utilization in prokaryotes.
In anabolic operons (typical of amino acid synthesis),
a phenomenon of additional regultation referred to as
attenuation has been documented. The example most
commonly considered involves the trp operon discussed
above.
Figure 3.28. Diagram showing the major effects of low glucose
(upper panel) and high glucose (lower panel) on the expression
of lac operon genes.
© 2013 Nature Education Adapted from Pierce, Benjamin. Genetics: A Conceptual Approach,
2nd ed. (New York: W. H. Freeman and Company), 446. All rights reserved.
The leader sequence in the polycistronic mRNA of the
trp operon contains several trp codons, and can form 3
different stem-loop structures. Depending on the
CONCEPTS OF GENOMIC BIOLOGY
Page 32
when trp is abundant. While the other structure does
not terminate transcription, and the polycistronic mRNA
is produced.
Several amino acid synthetic operons (e.g. phenylalanine, histidine, leucine, threonine, and isoleucinevaline) demonstrate this same type of attenuation.
Consequently, this mechanism is relatively widespread
as a means of modulating and fine-tuning pathways for
amino acid biosynthesis.
3.5.6. Transcription in Eukaryotes
Figure 3.29. Attenuation of the trp operon. The diagram at the center
shows the general folding of the leader sequence of the trp
polycistronic mRNA and labeling of strands. The mRNA is folded in four
parallel strands connected at the bottom by two small hairpin loops
between strands 1 and 2 and strands 3 and 4 and by one large hairpin
loop at the top between strands 2 and 3. In the structure on the left,
strands 1 and 2 and strands 3 and 4 are stabilized by base pairing. This
structure terminates transcription of the trp operon in the presence of
high tryptophan. In contrast, strands 2 and 3 are stabilized by base
pairing in the structure on the right, which allows transcription of the
trp operon to continue in the presence of low tryptophan.
© 1981 Nature Publishing Group Yanofsky, C. Attenuation in the control of expression of bacterial
operons. Nature 289, 753 (1981). All rights reserved.
amount of available tryptophan, one of two structures
can be produced (Figure 3.29) One structure leads to
termination of transcription in the leader sequence
(return)
Although transcription in Eukaryotes follows the
general principles outlined above for Prokaryotes, there
are many specific details that are different. Recall that
there are as many as five Eukaryotic RNA polymerases.
While each of these transcribes different types of RNA,
they are all Multisubunit RNA polymerases that function
in related ways. The mechanism of the important RNA
polymerase II that produces mRNAs will be described
here, but each of the 5 has similar mechanisms for
initiation, elongation, and termination of transcription.
Eukaryotic mRNAs are nearly always monocistronic
mRNAs with a general structure as shown in Figure
3.30. The key transcribed features are a 5’-UTR
(untranslated region), a coding region, and 3’-UTR.
CONCEPTS OF GENOMIC BIOLOGY
Page 33
Other nontranscribed features that are typical of
mRNAs in
upstream
Enhancers
5’ UTR
TATA box
Promoter
Exon 1
5’
Coding Region
3’ UTR
Exon 2
Intron 1
Exon 3
DNA
3’
Intron 2
Gene Transcription
by RNA Polymerase II
Nuclear Processing – 5’ Capping &
poly-A tail addition
3’ Poly-A tail
G
5’ Cap
7-Me
AAAAAA
Nuclear Processing – Intron removal
& transport to the cytoplasm
G
7-Me
Final mRNA
5’ UTR
5’ Cap
Protein Coding Region 3’ UTR
Core promoter elements are located near the
transcription start site and specify where transcription
begins. Examples include:
1)
2)
Primary Transcript
Pre-mRNA
Eukaryotic promoters, core promoter elements and
promoter proximal elements.
3’ Poly-A tail
AAAAAA
Figure 3.30. Diagram showing the elements and structure of a typical
eukaryotic mRNA-producing gene. Note that a primary transcript is
produced which is subsequently modified by the addition of a 7-methyl
guanosine (Cap), and the poly-A tail. Subsequently, introns are spiced
from the transcript to make a finished mRNA ready to exit the nucleus.
Eukaryotic cells include a 5’-Cap structure and a poly-A
tail that will be described in more detail below.
Promoters in many Eukaryotes have been analyzed
either by the use of directed mutations within promoter
sequences or by comparative analysis of multiple genes
from different organisms. These studies have revealed
that there are two types of elements found in
The initiator element (Inr), a pyrimidine-rich A
that spans the transcription start site;
The TATA box (also known as a TATA element
or Goldberg–Hogness box) at -30 nt (full
sequence is TATAAAA). This element aids in
local DNA denaturation and sets the start point
for transcription.
Promoter-proximal elements are required for high
levels of transcription. They are further upstream from
the start site, at positions between -50 and -200. These
elements generally function in either orientation.
Examples include:
1) The CAAT box, located at about -75.
2) The GC box, consensus sequence GGGCGG,
located at about -90.
Various combinations of core and proximal elements
are found near different genes. Promoter-proximal
elements are key to understanding the rate at which
transcription initiation occurs and thus the level of gene
expression.
CONCEPTS OF GENOMIC BIOLOGY
Page 34
Eukaryotic Transcription initiation requires assembly
of RNA polymerase II and binding of general
transcription factors (GTFs) on the core promoter at the
TATA box (see Figure 3.31) forming a preinitiation
Figure 3.31. Eukaryotic transcription begins with the formation of a
transcription preinitiation complex (PIC) on the TATA box in the
promoter of the gene. The PIC is a large complex of proteins that is
necessary for the transcription of protein-coding genes in eukaryotes.
The preinitiation complex helps position RNA polymerase II over gene
transcription start sites, denatures the DNA, and positions the DNA in
the RNA polymerase II active site for transcription. The minimal PIC
includes RNA polymerase II and six general transcription factors: TFIIA,
TFIIB, TFIID, TFIIE, TFIIF, and TFIIH. Additional regulatory complexes (coactivators and chromatin-remodeling complexes) could also be
components of the PIC.
complex (PIC). Note that the PIC is sometimes referred
to simply as the transcription initiation complex. GTFs
are needed for initiation by all RNA polymerases and are
numbered to match their corresponding RNA
polymerase and lettered in the order of discovery (e.g.,
TFIID was the fourth GTF discovered that works with
RNA polymerase II). The general transcription factors
along with other proteins forming specific PICs at a
particular promoter poise RNA polymerase to begin
transcription of the gene behind the promoter.
Once the PIC forms, RNA polymerase will initiate
transcription. However, the rate at which transcription
initiation occurs at a particular gene depends on 2
factors. The first factor is the number and types of
enhancer/silencer sequence elements found in the
promoter. These sequence elements can be from 50 nt
to over 1,000 nt in length. Enhancer/silencer elements
must be located in cis (meaning close to) to
promoter/coding sequence in order to effect the
expression of a gene.
Some enhancer/silencer
sequences have been found that are as much as 1
megabase (1,000,000 nt) away from the transcription
start site (TATA box), but most are within a few
thousand bases or less of the TATA box.
The second factor regulating the rate of transcript
initiation is proteins that can bind to specific enhancer
or silencer sequence elements. Activators are proteins
CONCEPTS OF GENOMIC BIOLOGY
Page 35
that bind to enhancer sequences. Activator proteins
also contain protein-protein interaction domains that
allow them to bind to and affect the behavior of other
proteins. These other proteins could be RNA polymerase itself; other general transcription factors in the PIC;
or other adapter proteins that interact with the PIC (see
Figure 3.32).
Figure 3.32. An activator protein binding to a promoter-proximal
enhancer seuqence, interacting with an adapter protein, and the
PIC to enhance transcription initiation.
Repressor proteins can either bind to silencer or
enhancer sequence elements in the promoter. In so
doing they reverse the effect of activator proteins by
either interfering with the critical protein-protein
interactions of activators or by binding tightly to
enhancer sequences keeping activators from binding.
Thus, activator and repressor proteins are important
in transcription regulation. They are recognized by
promoter-proximal elements and other enhancer/
silencer sequence elements found upstream of the
promoter, and they are specific for groups of similarly
regulated genes. These proteins mediate the rate of
transcription initiation for genes that contain recognized
sequence elements. The presence or absence of specific
activator and repressor sequences in a specific cell
either because of cell type or because of environmental
factors can mediate the initiation of transcription. For
example, housekeeping genes (used in all cell types for
basic cellular functions) have common promoterproximal elements and are recognized by activator
proteins found in all cells. Examples of genes with
housekeeping functions include: actin, hexokinase, and
Glucose-6-phosphate dehydrogenase.
Genes expressed only in some cell types or at
particular times have promoter-proximal elements
recognized by activator proteins found only in specific
cell types or times. Enhancers are another cis-acting
element. They are required for maximal transcription of
a gene.
CONCEPTS OF GENOMIC BIOLOGY
Page 36
Enhancers/silencers are usually upstream of the
transcription initiation site but may also be
downstream. They may modulate from a distance of
thousands of base pairs away from the initiation site.
Because there are similar enhancer and silencer
sequences in front of several genes that are
coordinately regulated, and each gene promoter has its
own unique spectrum of such sequences, Eukaryotic
cells can avoid the necessity of contiguous organization
of genes into operons as is common in prokaryotes.
Additionally, each tissue produces a set of tissuespecific and general activator and repressor proteins,
and the spectrum of these proteins can be influenced by
environmental factors such as cellular surroundings,
temperature, chemical environment, etc. This affords
the ability of each cell to “customize” the expression of
genes depending on the protein functions that are
required in each cell based on cell type and cellular
environment. This phenomenon is referred to as
combinatorial gene regulation and is illustrated in
Figure 3.33.
Once transcription initiation has occurred, the RNA
polymerase moves away from the TATA box as the
transcript is elongated. This is fundamentally the work
of RNA polymerase, and the other proteins of the PIC
now leave the complex to be recycled to form new PICs
while RNA polymerase elongates the primary transcript
nucleotide chain to complete the formation of the
primary transcript.
3.5.7. Processing the primary transcript into a
mature mRNA (return)
Figure 3.33. Combinatorial gene regulation leads to the coordinate
regulation of batteries of genes in Eukaryotes. The types of enhancer
and silencer sequences in front of each gene determine the level of
transcription of each gene based on the activator and repressor
proteins present in each cell/tissue type and the environment
surrounding each cell.
As shown in Figure 3.30, the primary transcript must
be processed in 3 significant ways to become a mature
mRNA. This processing all takes place in the nucleus of
the cell and prepares the mRNA for transport to the
CONCEPTS OF GENOMIC BIOLOGY
Page 37
cytosol of the cell where it will subsequently be
translated to produce a protein.
First, the primary transcript must acquire a cap at its
5’- end. The cap prepares the transcript for transport
from the nucleus, provides stability to attack by
exonucleases in the cytoplasm, and aids in the initiation
of the translation process. Structurally, a cap consists of
a 7-methyl guanosine attached by 3 phosphate groups
to the 5’-end of the transcript. Note that the cap is
reversed compared to the RNA strand, i.e. it is attached
5’ to 5’ not 5’ to 3’ as are the other nucleotides in the
transcript. The cap can be attached to the transcript
during transcription before completion of the primary
transcript, but it is critical to efficient transport of the
mRNA from the nucleus so it must be attached in the
nucleus.
The second processing step occurs at the 3’-end of
the transcript (Figure 3.35), and is involved in transcript
termination of elongation by RNA polymerase II. Note
that other eukaryotic RNA polymerases may have other
mechanisms of transcript termination since they do not
produce poly adenylated transcripts.
The process for addition of the poly-A tail involves a
complex of proteins that assembles at a poly-A addition
consensus sequence (AAUAAA). The proteins involved
in the cleavage step of the termination process include:
1) CPSF (cleavage and polyadenylation specificity
factor).
2) CstF (cleavage stimulation factor).
3) Two cleavage factor proteins (CFI and CFII).
Figure 3.34. Structure of the 5’-Cap added to Eukaryotic primary
RNA transcripts. The cap consists of a 7-methyl guanosine
residueattached 5’ to 5’ at the 5’ end of the transcript by 3
phosphate groups (a phosphotetraester).
Following cleavage, the enzyme poly(A) polymerase
(PAP) adds A nucleotides to the 3’ end of the cleaved
transcript RNA, using ATP as a substrate. PAP is bound
to CPSF during this process. Typically, about 200-250 A’s
CONCEPTS OF GENOMIC BIOLOGY
Page 38
A. Cleavage
B. Poly-A tail addition
are added. PABII (poly-A binding protein II) binds the
poly-A tail as it is produced. Upon completion of the
poly-A tail, further transcription is terminated with the
release of the pre-mRNA transcript from the protein
complex.
The third step in the process of producing a mature
mRNA from a pre-mRNA involves removal of sequences
that are found in the DNA coding sequence and premRNA that are absent from the mature mRNA that is
found in the cytoplasm of the cell. These removed
sequences are called introns. The parts of the premRNA that remain in the mature mRNA are called exons
(see Figure 3.30).
The removal of introns from the primary transcript to
is a process referred to as splicing, and it typically
involves a protein RNP particle referred to as a
spliceosome.
Figure 3.35. The addition of a poly-A tail to the
transcript terminates transcription of the pre-mRNA.
The process involves 2 steps: A) cleavage of the
growing primary transcript by a complex of proteins
that recognize a poly-A addition signal in the
transcript; B) Addition of 200-300 A’s to the 3’ end of
the transcript by PolyA polymerase (PAP).
Spliceosomes are small nuclear ribonucleoprotein
particles (snRNPs) associated with pre-mRNAs. snRNAs
that were previously discussed are structural parts of
spliceosome RNPs. The principal snRNAs involved are
U1, U2, U4, U5, and U6. Each of these snRNAs is
associated with several proteins; e.g. U4 and U6 are
part of the same snRNP. Others are in their own
snRNPs. Each snRNP type is abundant (~105 copies per
CONCEPTS OF GENOMIC BIOLOGY
Page 39
nucleus) consistent with the critical role that these
snRNPs play in nuclear processes.
Figure 3.36. The process of intron spicing
conducted by U2-dependent spiceosomes. Note
that there are other types of spiceosomes, and
that there are a few introns that are spliced
independent of spliceosomes. The binding of at
least 5 RNP complexes containing snRNAs and
proteins ultimately produce a structure that holds
the transcript cleaved ends together while the
intron is spliced out producing a “lariat” structure.
The exon ends of the transcript are then ligated
together producing a mature mRNA with the
intron removed from the sequence.
The steps of RNA splicing are outlined in Figure 3.36:
1) U1 snRNP binds the 5’ splice junction of the intron,
as a result of base-pairing of the U1 snRNA to the
intron RNA.
2) U2 snRNP binds by base pairing to the branchpoint sequence upstream of the 3’ splice junction.
3) U4/U6 and U5 snRNPs interact and then bind the
U1 and U2 snRNPs, creating a loop in the intron.
4) U4 snRNP dissociates from the complex, forming
the active spliceosome.
5) The spliceosome cleaves the intron at the 5’ splice
junction, freeing it from exon 1. The free 5’ end of
the intron bonds to a specific nucleotide (usually
A) in the branch-point sequence to form an RNA
lariat.
6) The spliceosome cleaves the intron at the 3’
junction, liberating the intron lariat. Exons 1 and 2
are ligated, and the snRNPs are released.
One of the most interesting aspects of intron splicing
is that there can be different transcripts created based
on how introns are spliced. This is referred to as
alternative splicing can be used to produce different
polypeptides from the same gene as shown in Figures
3.37 and 3.38.
CONCEPTS OF GENOMIC BIOLOGY
Page 40
Figure 3.38 Alternative splicing of 1 primary transcript to produce 3
different proteins.
Figure 3.37. A schematic representation of alternative splicing. The
figure illustrates different types of alternative splicing: exon inclusion
or skipping, alternative splice-site selection, mutually exclusive exons,
and intron retention. For an individual pre-mRNA, different alternative
exons often show different types of alternative-splicing patterns.
© 2002 Nature Publishing Group Cartegni, L., Chew, S. L., & Krainer, A. R. Listening to
silence and understanding nonsense: exonic mutations that affect splicing. Nature
Reviews Genetics 3, 285–298 (2002). All rights reserved.
From the above discussion it is clear that processing
of a mature mRNA from the primary RNA transcript, and
the transport of the mature mRNA from the nucleus to
the cytoplasm are steps that can influence the amount
of translatable mRNA for a particular protein that exists
in a cell. The details of the steps we have discussed
have emerged from a series of original molecular
genetic studies, and have been greatly embellished
more recently by functional genomic studies that we
will investigate further in subsequent chapters.