ROSTOV STATE MEDICAL UNIVERSITY

DEPARTMENT OF THE GENERAL BIOLOGY AND ANATOMY

РОСТОВСКИЙ ГОСУДАРСТВЕННЫЙ МЕДИЦИНСКИЙ УНИВЕРСИТЕТ

КАФЕДРА ОБЩЕЙ БИОЛОГИИ И АНАТОМИИ

Lecture 1
Molecular bases
of a heredity
Lectures to medical biology and genetics
for foreign students of the
first year of education
 The plan of the lecture:
• 1. Structure of DNA.
• 2. Properties of DNA: replication and reparation.
• 3. The organization of the hereditary information in
prokaryote.
• 4. Structure of RNA. Comparative characteristic of DNA
and RNA.
• 5. A stage of synthesis of protein.
• 6. The transcription in prokaryote and eukaryote.
• 7. Posttranscriptional modification. mRNA processing.
• 8. The genetic code.
• 9. The translation: initiation, elongation, and termination.
• 10. DNA Fractions
• 11. Controlling of transcription
 The biology as a natural science
about the life.
• The biology - is a science, which studies life as a special
form of matter being having its own laws of existence and
development. The subject of biology study is live organisms
and their natural communities.
• Biology is a complex science. It includes more than 50
disciplines. There are following among them:
• • Morphological disciplines (anatomy, histology)
describing organism structure.
• • Physiological disciplines (cell physiology, plant
physiology, animal physiology).
• • General biological disciplines (cytology, genetics,
evolution etc.).
• • Ecological disciplines (biogeography, parasitology).
• • Bordering disciplines (biochemistry, biophysics,
anthropology, molecular biology, space biology etc.).
• Biology is a leading natural science. The high level of
biological research is necessary condition for modern
medicine progress.
 Biology
• The term biology was formed by Lamarck
in 1802 from two Greece words bios-life
logos- science.

• Four unifying principles form the

foundation of modern biology:
• cell theory,
• evolution,
• genetics and
• homeostasis.

Jean-Batiste Pierre Antoine

de Monet, chevalier de
Lamarck; (1744 —1829)
 The biology role in
doctors training
• The biology is theoretic base of medicine, that why it is
important to study biology for future doctor.
• Morphological, biochemical, genetical and
physiological disciplines give a base for pathology.
• Such practical branches of medicine as therapy and
surgery are based on anatomy, physiology and
biochemistry.
• Epidemiology is based on achievements of ecology,
zoology, parasitology, microbiology and virology.
 Properties of Life
•All living organisms share basic characteristics:
•1. Order. All organisms consist of one or more cells with highly
ordered structures.
•2. Irritability. All organisms respond to stimuli. Plants grow toward a
source of light, and your pupils dilate when you walk into a dark room.
•3. Growth, development, and reproduction. All organisms are
capable of growing and reproducing, and they all possess hereditary
molecules that are passed to their offspring, ensuring that the offspring
are of the same species. Although crystals also "grow," their growth
does not involve hereditary molecules.
• 4. Heredity and diversity. The heredity provides material succession
between generations. Traits, which are inherited, provide adaptation to
environment. The storage and transmitting hereditary information is a
function of nucleic acids.
•5. Substance and energy exchange. The main property of life is
metabolic exchange. Each organism can be presented as an open
system supporting constant substance and energy exchange with
environment.
 Properties of Life
•6. Self-Regulation. All organisms have regulatory
mechanisms that coordinate the organism's internal
functions. These functions include supplying cells with
nutrients, transporting substances through the organism,
and many others.
•7. Homeostasis. All organisms maintain relatively
constant internal conditions, different from their
environment, a process called homeostasis.
•8. Adaptation

•All organisms are cells or aggregates of cells.

 Life organization levels
• Modern biology study life processes on different levels.
Molecular-genetic level. Elementary structures of this
level are central regulating systems - codes of
hereditary information, transmitted from generation to
generation. Elementary events are codon reproducing
and protein synthesis on a gene matrix. DNA
reduplication preserves genetic information, placed in
genes, for next generation.
• Cellular level. Elementary structure of this level is a cell.
Elementary event is cell division and cell development.
On this level all organisms look kind of similar. The
genetic information is realized in particular proteins on
this level too. Protists cellular level coincides with
organism level.
 Life organization levels
• Ontogenetic level. Elementary structures are organisms.
Elementary events are ontogenesis, differentiation and still
unknown mechanism that direct all that processes.
• Population level. Elementary structures are populations of
any life species. Elementary event is directed changes in
their genofond.
• Biospheral level. Elementary units on this level are
biogeocenosis. Biogeocenos is open system for energy
flow and substance flow, as well. All biogeocenosis even
different in structure are united to one complex - called
biosphere.
 The cell is the
fundamental unit of life
•Cell theory states that all living things are composed
of one or more cells, or the secreted products of
those cells.
•Eucaryotic cells contain membrane-bounded
organelles that carry out specialized functions.
•Cells arise from other cells through cell division, and
in multicellular organisms, every cell in the organism's
body is produced from a single cell in a fertilized egg.
Furthermore, the cell is considered to be the basic
part of the pathological processes of an organism.
 DNA
• The molecule of DNA enables the living organisms to
reproduce their complex components from one
generation to the next. The structure of DNA provides
mechanisms for own replication, also directs the RNA
synthesis and, through RNA, controls protein synthesis.
 History of
discovering

• A German chemist, Friedrich Miescher, discover DNA in

1869. Miescher extracted a white substance from the
nuclei of human cells and fish sperm. He called this
substance “nuclein”, because it seemed to be
specifically associated with the nucleus. Because
Micscher's nuclein was slightly acidic, it came to be
called nucleic acid.
 • In 1920s, the basic structure of nucleic acids
was determined by biochemist Levene, who
found that DNA contains three main
components,
• phosphate group,
• five-carbon sugars, and
• nitrogen-containing bases called purines
(A,G) and pyrimidines (T, C, U).
• The base is bound to first carbon atom in the
sugar and phosphate group is bound to fifth
carbon atom in the sugar. Third atom of sugar
always has a hydroxyl (-OH) group.

Phoebus Levene
(1869 –1940)
• In 1952 Rosalind Franklin carried out
an X-ray diffraction analysis of DNA.
She yielded information about the
three-dimensional structure of a
molecule.

Rosalind Franklin
(1920 –1958)

• X-ray of the crystalline A- • X-ray of B-form DNA

form of DNA. obtained by Rosalind
Franklin in late 1952
• On the bass on this knowledge's James Watson and
Francis Crick first proposed the double helix as the
three-dimensional structure of DNA in 1953.

James Watson and Francis Crick

Nucleic acid exists in two
forms:
• desoxyribonucleic acid
(DNA) and
• ribonucleic acid (RNA).
• DNA - is the storage of
genetic information. It is in
the nucleus chromosomes,
in the mitochondria, in the
chloroplasts of eukaryotic
cells, in prokaryotic cells, in
many viruses.
• RNA serves for transmitting
and realization of hereditary
information in prokaryotic
and eukaryotic cells. In
many viruses RNA work as a
primary storage of hereditary
information.
• The new nucleotide
can be attached to
the chain only to 3'
hydroxyl group of
the polymer.
• A nucleotide
without phosphate
group has a name
nucleoside.
• Organic bases are
purine - adenine
and guanine or
pyrimidine -
thymine, cytosine
and uracil.
• DNA consists of
2x10-9 and more
nucleotides.
• The bonding results in backbone with a repeating pattern of sugar-
phosphate-sugar-phosphate (phosphate group binds to 3-carbon
of previous nucleotide sugar and to 5- carbon of next nucleotide
sugar).
• The nitrogenous bases are paired in the interior of the helix by
hydrogen bonds, by which the secondary structure of DNA is
formed.
• Only certain bases in the double helix are compatible with each
other,
• A always pairs with T (2 hydrogen bonds), and
• G - with C (3 hydrogen bonds).
• The two strands of the double helix
are complementary, each the
predictable counterpart of the
other. The tertiary structure of DNA
is presented by double helix of
three dimensional molecule.
• Two strands are intertwinerd in a
clockwise direction and run in
opposite directions - they are
antiparallel, i.e. the 5' end of one
strand in iront of the З'-end of the
other.
• The spiral of DNA completes a turn
in 3.4 nm.
• Each nucleotide occupies 0,34 nm.
Thus, there are 10 nucleotides per
turn.
• The width of the DNA - 2 nm.
 Chargaff rules.
• The four nucleotides were not present
in equal proportions in DNA molecules.
A careful study carried out by Erwin
Chargaff showed that the nucleotide
composition of DNA molecules varied
in complex way, depending on the
source of the DNA. This strongly
suggested that DNA was not simple
repeating polymer and might have the
information- encoding properties
genetic material must have.
• Chargaff observed an important
underlying regulation in double-
stranded DNA, called Chargaff rules.
• The amount of adenine present in DNA
Erwin Chargaff
always equals the amount of thymine,
and amount of guanine always equals
the amount of cytosine.
 Conformational Forms of
DNA
• DNA can exist in A, В and Z conformational forms. Sugar
puckering is the most important characteristic for
distinguishing the DNA forms.
• The В form (B-DNA) is the structure proposed by Watson
and Crick, and is the native conformation of DNA in
solution. It consists of a right-handed antiparallel double
helix of sugar-phosphate backbone, with purine-
pyrimidine base pairs roughly perpendicular to the axis
of the helix. The В form is the metabolically stable
configuration.
• One turn of the helix makes 10 base pairs (10-fold helix),
its length is 3.4 nm.
• The A form (A-DNA) has 11 base pairs (11-fold helix).
• The Z form (Z -DNA) is left handed conformations of
DNA, which is 1.8 nm in diameter and the turn
contains 12 base pairs, its length is 0.37 nm. The
phosphates in the backbone are zigzagged, hence is
the name of Z-DNA. The zigzagging is a consequence
of a fact that the repeating unit is a dinucleotide
rather than a mononucleotide.
 DNA REPLICATION
•The DNA model proposed by
Watson and Crick, is in fact, is
pair of templates, each of
which is complementary to the
other.
•When the double helix
replicates, each of the
daughter molecules will have
one old (parent) strand and one
newly made strand.
• The replication of a DNA begins at special site
called origin replication. The bacterial circular DNA
has only single origin, in contrast - the eukaryotic
chromosome has hundreds or thousands of
implication origins.
• The helicase that initiates the replication recognize this
sequence and attaches to DNA. It untwists the double
helix by cutting the hydrogen bonds between
complementary nucleotide. The Y-shaped region where
the new strands of DNA are elongating is called a
replication fork. To maintain the untwisted condition of the
already untwisted single strands of special proteins called
single-strand binding proteins (SSBP), or double helix
destabilizing proteins, line along the separated strands
and prevent then joining.
• Along with separation of the double helix by helicase, supercoil
appear ahead of the replication fork. These knots can hinder
the further advancing of helicase. Therefore a special enzyme
known a topoisomerase cuts (binds reversibly to) the one
strand ahead of helicase. The cut strand rotates round the
second one and thus the tension is released.
• The main enzyme of replication is the DNA-polymerase, which
adds a new nucleotide to 3-OH'-group of the previous nucleotide
forming a phosphodiesther covalent bond. Each added
nucleotide is complementary to the one in the template strand.
Peculiarity of the DNA-polymerase is that it cannot start the
replication (attaching the first nucleotide) from the beginning,
since it needs a free 3-OH'-group already bound to the template.
This is provided by primer which is a RNA-stretch (10 nucleotides).
It is synthesized by primase.
• DNA-polymerase is able to elongate the chain only in one direction - from 5' to
3' end of a new strand. It moves along the template as the replication fork
progresses. The DNA strand made by this mechanism is called the leading
strand.
• To elongate the other new strand of DNA, polymerase must work along the
template away from the replication fork and synthesize short segments of DNA
called Okazaki fragments (figure). Each of them is directed to the 5' to 3’ end of
the second new strand and every time starts from a primer. Then all the primers
are removed, and the oligonucleotide fragments are joined by ligase. This
strand is referred to as a lagging strand. The length of the Okazaki fragment in
prokaryotes is about 1000-2000 nucleotides, and in eukaryotes - about 100- 200.
• The fragment of DNA beginning from the origin of replication up to the end of
replication is known as a replicon - the unit of replication.
 DNA REPAIR
•All biological polymers, including DNA, are subject to damage.
Any damage to DNA might alter its information content and
products disastrous changes in the cell's proteins. Although
thousands of changes occur in a cell’s DNA every day, the cell
accumulates only about two or three stable changes (mutations)
each year. 20 or more kinds of DNA repair enzymes eliminate
the changes. These enzymes, as endonuclease recognize and
exonuclease remove a damaged area of a DNA strand,
replacing it with nucleotides complementary to those on the
opposite, undamaged strand.
• DNA repair depends
on the existence of
two copies of the
genetic information,
one in each strand
of the double helix
(fig). As long as one
strand remains
undamaged, the
repair enzymes can
use it as a template
to replace a
damaged segment
in its partner. The
double helix is,
therefore, vital to
genetic stability.
• In humans, the rare inherited
disease xeroderma
pigmentosum results from
having defective genes for
DNA repair enzymes. As a
result, skin cells cannot repair
DNA damaged by the sun's
ultraviolet rays. People with
this condition often develop
fatal skin cancer.
 The three types of RNA
• The information necessary for the synthesis of protein is
stored in the cells DNA. The cell uses this information by
first making an RNA transcript of it.
• Like DNA, RNA molecules are long polymers made up of
nucleotides. Each nucleotide subunit consists of a sugar
molecule, a nitrogenous base, and a phosphate group.
• RNA differs from DNA in several respects:
1. The sugar in RNA is ribose, whereas that in DNA is
deoxyribose.
2. RNA and DNA contain different sets of nitrogenous bases.
RNA contains uracil (U) instead of thymine (T). Like thymine,
uracil base-pairs with adenine.
3. RNA usually consists of a single strand of nucleotides,
whereas DNA is usually double-stranded, with two
complementary chains of nucleotides wound into a double
helix.
• I The class of RNA found in ribosomes is called ribosomal RNA.
During the polypeptides synthesis, rRNA provides the sit where
polypeptides are assembled.
• II Transfer RNA molecules transport the amino acids to the
ribosomes for use in building of polypeptides. Human cells contain
about 45 different kinds of tRNA. The primary structure of tRNA is a
sequence of about 76 nucleotides. The secondary structure is formed
by hydrogen bonds between complementary nucleotides within this
strand so that it resembles a clover leaf. The tRNA actually twists and
folds into a compact three-dimensional tertiary structure that is L-
shaped. The clover-leaf like molecule of tRNA has also an acceptory
site for certain aminoacid which matches the anticodon:
• III Messenger RNA molecules are long strand of RNA that are
transcribed from DNA.
TRANSCRIPTION
Transcription is the process of transfer of
the information encoded in DNA to
RNA.
There are 3 types of RNA- Polymerases,
which can transcribe the DNA:

RNA-polymerase I - for
synthesis of the rRNA of the big subunit
of ribosomes;
RNA-polymerase II - for mRNA
synthesis;
RNA-polymerase III - for the
rRNA of the small subunit of ribosomes
and tRNA.
The whole process of transcription
consists of 3 phases: initiation,
elongation and termination.
 Initiation
• Transcription is initiated by binding of the RNA-polymerase to the
promoter on the DNA. In addition to determining where transcription
starts, the promoter determines which of the two strands of the DNA
helix is used as a template which is a special sequence to begin the
process.
• In eukaryotes first the transcription factors, which are special proteins,
bind to the promoter and mediate binding of RNA-polymerase to the
promoter site. The association of the promoter, proteins and RNA-
polymerase is called a transcription initiation complex.
• The interaction between RNA-polymerase and transcription factors is
an example of regulation of eukaryotic transcription.
 Elongation
Like the DNA-polymerase adds the nucleotides also in 5 to 3 direction. The
stretch of DNA that is transcribed into mRNA molecule is called a transcription
unit. As RNA-polymerase moves along the DNA, it continues to untwist the
double helix, exposing about 10-20 bases at a time for pairing with RNA
nucleotides. While the transcription progresses, the double helix re-forms again,
and the new RNA molecule peels away from its template.
 Termination
• The transcription proceeds
until shortly after the RNA-
polymerase transcribes a
DNA sequence called a
terminator. This is a special
sequence of nucleotides
AAUAAA. At a point at about
10-35 nucleotides farther
along, the pre-mRNA is cut
free from the enzyme.
• The complex of promoter,
transcription unit and
terminator is known as a
transcripton.
• The primary transcript of
mRNA is called a pre-mRNA
or heterogenic nuclear RNA
(hn-RNA), which is not yet
the mature form to realize
the translation. Therefore it
needs in undergo some
changes alter transcription.
 Posttranscriptional modification.
mRNA processing
In the eukaryotic nucleus pre-mRNA is modified in various ways before it is
dispatched to the cytoplasm.
Each end of a pre-mRNA molecule is modified in aparticular way. The 5’ end, the
end made test during transcription, is immediately capped off by a modified
form of guanine nucleotide (methylated guanosine triphosphate - G-ppp). This
5`cap helps to attach the small subunit of ribosome in cytoplasm.
The 3 end is added by poly(A) tale consisting of 30-200 adenine nucleotides.
Poly(A) tail inhibits the degradation of mRNA and facilitates its export to
cytoplasm.
• The most remarkable stage of
RNA processing in eukaryotic
nucleus is the removal of a
large portion of non-
informative fragments called
initrons, which are initially
synthesized during
transcription and are
noncoding sequences lied
between coding segments
called exons. They are
translated into amino acid
sequences. Alter removal of
introns the exons are joined
together by ligase enzyme
into one continuous coding
sequence of final mRNA
structure. This ”cut-and paste"
job is known as splicing.
 The genetic code
• DNA is a code for the production of protein molecules
and the sequence of bases in the DNA must be a
code for the sequence of amino acids in protein
molecules. This relationship between bases and amino
acids is known as the genetic code.
 Features of the genetic
•
•
The code is triplet. code
Three bases in DNA code one amino acid in a protein. The
complementary triplets in the mRNA are referred to as
codons. Each codon is therefore three bases long and is the
code for one amino acid.
• The code is degenerate.
• Some amino acids are coded by several codons. Analysis of
the code also shows that for many amino acids only the first
two letters appear to be significant.
• The code is punctuated.
• Three of the codons act as Tull stops' in determining the end of
the code message. An example is UAA. They are sometimes
described as 'nonsense codons1 and do not code for amino
acids. They act as 'stop signals' for the termination of
polypeptide chains during translation. Certain codons act as
'start signals' for the initiation of polypeptide chains, such as
AUG (methionine).
 Features of the genetic
• The code is universal code
• The same triplets code for the same amino acids in all
organisms.
• The code is non-overlapping.
• For example, an mRNA sequence beginning
AUGAGCGCA is not read AUG/UGA/GAG ... (an overlap
of two bases) or AUG/GAG/GCG ... (an overlap of one
base). (However, overlapping of certain genes does
occur in a 1 few organisms such as the bacteriophage
0X174. This seems likely to be exceptional and may be
an economy measure when there are very few genes.)
TRANSLATION
• Translation is the construction
of an amino acid sequence
(polypeptide) from mRNA
molecule. It really is a
translation from one code,
nucleotide sequence, to
another code, amino acid
sequence. The ribosome is the
site of this action, just as RNA
polymerase was the site of
mRNA synthesis.
TRANSLATION
Translation also consists of initiation, elongation and termination phases.
Aminoacyl-tRNA complex is formed by an enzyme aminoacyl-tRNA
synthetase, a process which requires energy (ATP).
 Initiation
• The mRNA which has undergone processing, is
transported to cytoplasm and with the help of its
cap the ribosomal small subunit gets attached to
mRNA. The first aminoacid that is transferred by
tRNA is Methionine.
 Elongation
• Then next aminoacid -tRNA complex arrives to the ribosome, and a
peptide bond between carboxyl group (COOH) of the previous
aminoacid and the nitro-group (NH2) of next aminoacid is formed by
releasing a molecule of water (H20), After the discharged tRNA leaves
ribosome, the latter shifts its position on mRNA moving on by one
triplet. The ribosome moves along the mRNA, matching 3 base pairs
all a time and adding the amino acids to the polypeptide chain.
• When the ribosome reaches one of the "stop” codes,
the ribosome releases both the polypeptide and the
mRNA. This polypeptide will twist into its native
conformation and begin to act as a protein in the cell
metabolism.
 Polysome (polyribosome)
• A single ribosome can
make an average-sized
polypeptide in less than
a minute. However, a
single mRNA is used to
make many copies of a
polypeptide
simultaneously, because
several ribosomes work
on translating the
message at the same
time. Once a ribosome
moves past the initiation
codon, a second
ribosome can attach to
the mRNA. Such strings of
ribosomes are called
polysomes.
 Posttranslational
modification
• Additional steps may be required before the protein can begin doing its
particular job in the cell. Certain polypeptides can be chemically modified
by the attachment of sugars, lipids, phosphate groups etc. In some cases,
e.g., a protein insulin is first synthesized as a single polypeptide chain but it
becomes active only after an enzyme excises (removes hydrogen ions from
sulfhydryl SH groups) the central part of it leaving a protein made up of 2
polypeptide chains connected by disulfide bridge -S-S-.
 INFORMATION FLOW. CENTRAL
DOGMA OF BIOLOGY
• Beadle and Tatum
proposed the "one gene
one enzyme" theory.
One gene codes for the
production of one
protein. "One gene one
enzyme" has been
modified to "one gene
one polypeptide" since
many proteins (such as
haemoglobin) are made
of more than one
polypeptide.
haemoglobin
• Crick’s central dogma - Information flow (with the
exception of reverse transcription) is from DNA to
RNA via the process of transcription, and hence to
protein via translation. Transcription is the making
of an RNA molecule on a DNA template.
• Although originally called
dogma, this idea has been
tested repeatedly until it
was found that retroviruses
possess reverse
transcriptase enzyme
(revertase or RNA-
dependent DNA
polymerase), which
enables reverse
transcription- synthesis DNA
on template of mRNA.
Recently it was also found
another direction of
information flow RNA-
synthesis on RNA template
in viruses, plants which is
realized by RNA dependent
RNA polymerase enzyme.
• Final expression of DNA genes is manifested as a
certain trait. And gene mutations cause genetic
diseases, which are known also as molecular
diseases, enzymopathies or genopathies.
 DNA Fractions
• In prokaryotes most of the DNA in a genome codes for a
protein (or tRNA and rRNA), with the small amount ofDNA
consisting mainly of regulatory sequences, such as
promoters. The coding sequence of nucleotides along a
prokaryotic gene proceeds from start to finish without
interruption.
• In eukaryotic genomes, by contrast, most of the DNA -
about 97% in humans - does not encode protein or RNA.
The sequences that encode proteins are known as unique
sequences fraction, which is presented only in a single
copy. Some of the informative sequences encode for
histone proteins, rRNA and tRNA. These sequences repeat
moderately about 100- 1000 times. This prevents
expression of mutation of these essential genes that are
frequently transcribed.
Some of noncoding sequences are known to
be regulatory sequences and introns -
noncoding sequences that interrupt coding
DNA
sequences (exons). Most of noncoding
sequences consist of repetitive DNA,
nucleotide sequences that are present in
Fractions
many copies in a genome, usually not within
genes. These are of two types: tandemly
repetitive, DNA (or satellite DNA, about 10-
15% of mammalian DNA) and interspersed
repetitive DNA.
The tandemly repetitive DNA sequences are
encountered at chromosomes telomeres
and centromeres, suggesting that this DNA
plays a structural role for chromosomes. The
DNA at centromeres is essential for
separation of chromatids in cell division and
it may help organize the chromatin within the
interphase nucleus. Telomeric DNA protects
the chromosome by binding proteins: that
stop the ends from "fraying” and from
sticking to other chromosomes. In addition, it
protects genes from being lost at each;
round of replication (telomerase cuts a
repetitive DNA at telomere). Also these
repeats aid in recognition of homologous
chromosomes in meiosis.
• The repeated units of
interspersed repetitive
DNA (about 25-40% of
mammalian DNA) are not
next to each other; instead
they are scattered about
the genome. These copies
are usually not identical to
each other. Most such
sequences seem to be
transposons - the mobile
genetic elements.
 Controlling Transcription
Initiation
• How do organisms use regulatory DNA sequences
and the proteins that bind them to control when
genes are transcribed? The same basic controls are
used in bacteria and eukaryotes, but eukaryotes
employ several additional elements that reflect their
more elaborate chromosomal structure. We will
begin by discussing the relatively simple controls
found in bacteria.
 Repressors Are OFF
Switches
• A typical bacterium possesses genes encoding several
thousand proteins, but only some are transcribed at any
one time; the others are held in reserve until needed.
When the cell encounters a potential food source, for
example, it begins to manufacture the enzymes
necessary to metabolize that food.
• Perhaps the best-understood example of this type of
transcriptional control is the regulation of tryptophan-
producing genes (trp genes), which was investigated in
the pioneering work of Charles Yanofsky and his students
at Stanford University.
 Operons
• The bacterium Escherichia coli uses proteins encoded by a cluster of
five genes to manufacture the amino acid tryptophan. All five genes
are transcribed together as a unit called an operon, producing a
single, long piece of mRNA. RNA polymerase binds to a promoter
located at the beginning of the first gene, and then proceeds down
the DNA, transcribing the genes one after another. Regulatory proteins
shut off transcription by binding to an operator site immediately in
front of the promoter and often overlapping it.
• When tryptophan is present in the medium
surrounding the bacterium, the cell shuts off
transcription of the trp genes by means of a
tryptophan repressor, a helix turn-helix regulatory
protein that binds to the operate site located within
the trp promoter. Binding of the repressor to the
operator prevents RNA polymerase from binding to
the promoter. The key to the functioning of this
control mechanism is that the tryptophan repressor
cannot bind to DNA unless it has first bound to two
mol ecules of tryptophan. The binding of tryptophan
to the repressor alters the orientation of a pair of
helix-turn helix motifs in the repressor, causing their
recognition helices to fit into adjacent major grooves
of the DNA.
• Thus, the bacterial cell's synthesis of tryptophan de
pends upon the absence of tryptophan in the
environment. When the environment lacks
tryptophan, there is nothing to activate the repressor,
so the repressor cannot prevent RNA polymerase
from binding to the trp promoter. The trp genes are
transcribed, and the cell proceeds to manufacture
tryptophan from other molecules. On the other hand,
when tryptophan is present in the environment, it
binds to the repressor, which is then able to bind to
the trp promoter. This blocks transcription of the trp
genes, and the cell's synthesis of tryptophan halts.
 Activators Are ON
Switches
• Not all regulatory switches shut genes off — some
turn them on. In these instances, bacterial
promoters are deliberately constructed to be poor
binding sites for RNA polymerase, and the genes
these promoters govern are thus rarely
transcribed—unless something happens to improve
the promoter's ability to bind RNA polymerase. This
can happen if a regulatory protein called a
transcriptional activator binds to the DNA nearby.
By contacting the polymerase protein itself, the
activator protein helps hold the polymerase against
the DNA promoter site so that transcription can
begin.
• A well-understood transcriptional activator is the
catabolite activator protein (CAP) of E. coli, which initiates
the transcription of genes that allow E. coli to use other
molecules as food when glucose is not present. Falling
levels of glucose lead to higher intracellular levels of the
signaling molecule, cyclic AMP (cAMP), which binds to
the CAP protein. When cAMP binds to it, the CAP protein
changes shape, enabling its helix-turn-helix motif to bind
to the DNA near any of several promoters. Consequently,
those promoters are activated and their genes can e
transcribed.
 Combinations of
Switches
• By combining ON and OFF switches, bacteria can create
sophisticated transcrip-tional control systems. A
particularly well-studied example is the lac operon of E.
coli . This operon is responsible for producing three
proteins that import the disaccharide lactose into the cell
and break it down into two monosaccha-rides: glucose
and galactose.
 The Activator Switch.
• The lac operon possesses two regulatory sites. One is a
CAP site located adjacent to the lac promoter. It ensures
that the lac genes are not transcribed effectively when
ample amounts of glucose are already present. In the
absence of glucose, a nigh level of cAMP builds up in the
cell. Consequently, cAMP is available to bind to CAP and
allow it to change shape, bind to the DNA, and activate
the lac promoter. In the presence of glucose, cAMP levels
are low, CAP is unable to bind to the DNA, and the lac
promoter is not activated.
 The Repressor Switch.
• Whether the lac genes are actually transcribed in the absence
of glucose is determined by the second regulatory site, the
operator, which is located adjacent to the promoter. A protein
called the lac repressor is capable of binding to the operator,
but only when lactose is absent. Because the operator and the
promoter are close together, the repressor covers part of the
promoter when it binds to the operator, preventing RNA
polymerase from proceeding and so blocking transcription of
the lac genes. These genes are then said to be "repressed". As
a result, the cell does not transcribe genes whose products it
has no use for. However, when lactose is present, a lactose
isomer binds to the repressor, twisting its binding motif away
from the major groove of the DNA. This prevents the repressor
from binding to the operator and so allows RNA polymerase to
bind to the promoter and transcribe the lac genes. Transcription
of the lac operon is said to have been "induced" by lactose.
• This two-switch control mechanism thus causes the cell to
produce lactoseutilizing proteins whenever lactose is present
but glucose is not, enabling it to make a metabolic decision to
produce only what the cell needs, conserving its resources.
• Bacteria regulate gene expression transcriptionally
through the use of repressor and activator
"switches," such as the trp repressor and the CAP
activator. The transcription of some clusters of
genes, such as the lac operon, is regulated by both
repressors and activators.