DNA REPLICATION

DNA TRANSCRIPTION
 DNA Replication

 DNA replication is a fundamental process occurring in all living

organisms during the normal cell division cycles. Following DNA
replication, two identical DNA molecules have been produced
from a single double-stranded DNA molecule.
 This process is "semiconservative" in that each strand of the
original double-stranded DNA molecule serves as template for the
reproduction of the complementary strand. The newly
synthesized daughter strands remain associated with their
respective parental template strands.
 A. Initiation of DNA replication

 In a cell, DNA replication begins at specific

locations in the genome, called "origins“
 Origins contain DNA sequences recognized by
replication initiator proteins.
 These initiator proteins recruit other Enzymes
to separate the two strands and initiate
replication:
 Enzymes of Replication
 DNA Helicases - These proteins bind to the
double stranded DNA and stimulate the
separation of the two strands. DNA Helicases
break the hydrogen bonds between
complimentary base pairs using ATP energy.
 This process results in a severe torsional stress
into the DNA ahead. This torsional stress is
relieved by DNA topoisomerases that solve
these physical problems in the coiling of DNA.
 DNA-Topoisomerases (DNA Gyrases) –
Topoisomerases relieve torsional stresses in
duplexes of DNA by introducing either double-
(topoisomerases II) or single-stranded
(topoisomerases I) breaks into the backbone of
the DNA.
 These enzymes catalyze also the formation of
negative supercoils. The nicks are then resealed
by the topoisomerases.
 DNA single-stranded binding proteins (SSB-
proteins) - These proteins bind to the DNA and
stabilize the single-stranded structure that is
generated by the action of the helicases. SSB-
proteins also prevent strands from
recombining.
 Replication fork.
 So, the DNA molecule is unwound and prepared for
synthesis by the action of DNA- helicases, DNA
topoisomerases and the single-stranded DNA binding
proteins.  
 The site of the unwound template strands is termed the
replication fork.
  
 To begin synthesis of a new strand, a short
fragment of RNA, called a primer, must be
created by the primase, that is able to begin the
polymerization. The primase utilizes the DNA
strands as templates and synthesizes a short
stretch of RNA generating a primer
 DNA polymerases are a family of enzymes that
carry out all forms of DNA replication. But,
DNA polymerase can only extend an existing
DNA strand paired with a template strand; it
cannot begin the synthesis of a new strand.
 B. Elongation
 Once a primer pairs with DNA to be replicated,
DNA polymerase begins to synthesize a new
strand of DNA by extending the 3'OH end of
the existing ribonucleotide chain, adding new
deoxynucleotides by the creation of
phosphodiester bonds.
 When a nucleotide is added to a growing DNA
strand, two of the phosphates are removed and
the energy produced creates a phosphodiester
bond
Elongation
 DNA polymerases
 In bacteria have been identified as DNA
polymerase (pol) I, II, and III. This enzymes link
together deoxynucleotide triphosphate monomers.
 DNA polymerases are generally extremely
accurate, making less than one error for every 107
nucleotides added.
 These enzymes also check for errors, and make
corrections.
 There have been 5 distinct eukaryotic DNA
polymerases identified, α, β, γ, δ and ε.
 DNA Polymerases.
DNA Polymerase I.Three activities are
associated with DNA polymerase I:
 5' to 3' elongation (polymerase activity)
 3' to 5' exonuclease (proof-reading activity)
 5' to 3' exonuclease (repair activity)
 DNA Polymerase III (Pol III). Two activities
are associated with DNA polymerase III:
 5' to 3' elongation (polymerase activity)
 3' to 5' exonuclease (proof-reading activity)
  
 When the enzyme topoisomerase unwinds DNA, two
single stranded regions of DNA (the "replication fork")
are formed by the enzyme helicase. The replication fork
moves in one direction, and two antiparallel strands
result, but DNA elongation only goes in the 5' to 3'
direction.
 In order for DNA synthesis one strand is copied
continuously and one strand is copied
discontinuously.
 Replication fork
 The strand of DNA synthesized continuously is termed the
leading strand and the discontinuous strand is termed the
lagging strand.
 The leading strand is defined as the new DNA strand at the
replication fork that is synthesized in the 5'→3' direction. On the
leading strand DNA polymerase III is able to synthesize DNA
using the free 3' OH group donated by a single RNA primer.
 The lagging strand is the DNA strand running in the 3'
to 5' direction. Because DNA polymerase III cannot
synthesize in the 3'→5' direction, the lagging strand is
synthesized in short segments known as Okazaki
fragments.
 The RNA fragments are then removed by exonuclease
action of DNA polymerase I and new
deoxyribonucleotides are added also by DNA
polymerase I to fill the gaps where the RNA was
present.
 DNA polymerase does not have the ability to form the
final bond. This is done by the enzyme DNA ligase.
C. Termination of replication

  Because eukaryotes have linear chromosomes, DNA

replication often fails to synthesize to the very end of
the chromosomes (telomeres), resulting in telomere
shortening. This is a normal process in somatic cells —
cells are only able to divide a certain number of times
before the DNA loss prevents further division
 Within the germ cell line, which passes DNA to the
next generation, the enzyme telomerase extends the
repetitive sequences of the telomere region to prevent
degradation.
 Telomerase can become mistakenly active in somatic
cells, sometimes leading to cancer formation.
Repair of DNA Damage and Replication Errors

 Several kinds of chemical change may cause

damage to DNA:
 • Spontaneous hydrolysis of a nucleoside
removes the heterocyclic base component.
• Spontaneous hydrolysis of cytosine changes
it to a uracil.
• Various toxic metabolites may oxidize or
methylate heterocyclic base components.
• Ultraviolet light may dimerize adjacent
cytosine or thymine bases.
 Repair of DNA Damage and Replication
Errors
 All these transformations disrupt base pairing
at the site of the change, and this produces a
structural deformation in the double helix..
Inspection-repair enzymes detect such
deformations, and use the undamaged
nucleotide at that site as a template for
replacing the damaged unit. These repairs
reduce errors in DNA structure from about one
in ten million to one per trillion.
The Central Dogma of Molecular Biology
 The Central Dogma of molecular biology is a simple linear
progression of information from DNA to RNA to Protein, is
summarized in the following illustration.
 The replication process on the left consists of passing information
from a parent DNA molecule to daughter molecules.
 The middle transcription process copies this information to a
mRNA molecule.
 Finally, this information is used by the chemical machinery of the
ribosome to make polypeptides – translation.
 Reverse transcriptase
 As more has been learned about these relationships,
the central dogma has been refined. It is now known
that an RNA-dependent DNA polymerase enzyme,
known as a reverse transcriptase, is able to transcribe a
single-stranded RNA sequence into double-stranded
DNA. Such enzymes are found in all cells and are an
essential component of retroviruses (e.g. HIV), which
require RNA replication of their genomes.
 RNA Synthesis - Transcription

 The process of synthesizing RNA from the genetic information encoded

by DNA is called transcription.
 The enzymes involved in transcription are called RNA polymerases.
Prokaryotes have one type; eukaryotes have three types of nuclear RNA
polymerases.
 The prokaryotic RNA polymerase consists of a core enzyme and an
auxiliary protein factor called sigma. (s factor). The core consists of four
subunits, two are identical, a, the other two similar, b and b'. The b'
subunit binds the DNA while the b subunit binds the nucleotides that are
to be joined together to form the RNA molecule.
 Sigma factors function in identifying specific DNA sequences known as
promoters. Promoters are sites that tell the RNA polymerase where to
begin transcription.
 Eukaryotes have four different RNA polymerases (RNA pol). Three are
required for transcription of nuclear genes and the fourth for
transcription of mitochondrial genes.
 RNA polymerase I transcribes ribosomal RNA (28S rRNA, 18S rRNA,
5,8S rRNA)
 Eukaryotes RNA Polymerases
 Eucariotes have four different RNA polymerases (RNA
pol). Three are required for transcription of nuclear genes
and the fourth for transcription of mitochondrial genes.
 RNA polymerase I transcribes ribosomal RNA (28S rRNA,
18S rRNA, 5,8S rRNA)
 RNA polymerase II transcribes mRNA
 RNA polymerase III transcribes tRNA and several small
RNA's (5S rRNA).
 The three polymerases consist of ten or more subunits. All
have two large subunits with homology to the b and b'
subunits of the prokaryotic RNA polymerase.
  
 There are three phases of transcription:
initiation, elongation and termination
 A. Initiation
 The initiation of transcription is directed by DNA sequences
called promoters which tell the RNA polymerase where to
begin transcription.
 The subunits that enable RNA polymerases to recognize
and bind promoters are called initiation factors. The
initiating nucleotide can be either a purine or pyrimidine.
 There are numerous eukaryotic promoters with multiple
promoter sequence elements.
 The initiation of transcription in eukaryotes is complicated
and involves numerous factors (proteins) that must interact
with the DNA and with one another to initiate transcription.
 Initiation of Transcription
 Promoters
 Only one strand of the DNA that encodes a
promoter, a regulatory sequence, or a gene needs
to be written.
 The first base on the DNA where transcription
actually starts is labeled +1.
 Sequences that precede, are upstream of the first
base of the transcript, are labeled with negative
numbers. Sequences that follow the first base of
the transcript, are downstream, are labeled with
positive numbers.
 Initiation of Transcription
 RNA pol II promoters are quite diverse. This
enables the cell to choose and regulate the
expression of the 50 to 100 thousand different
genes encoded by its DNA.
 There are some sequence elements that are
conserved and found in most RNA pol II
promoters. There are three "boxes": TATA
usually found 25 to 35 base pairs upstream, the
CAAT box and the GC box both located from
40 to 200 base pairs upstream.
 Initiation of Transcription
 These three elements provide a basal level of transcription
and are found in most "housekeeping" genes.
 Other sequence elements, which are continually being
discovered, serve as regulatory elements. Elements that
enable a cell to specifically turn other non-housekeeping
genes on or off in response to environmental signals such
as hormones
 There is a third type of sequence element that can be
located either upstream or downstream relative to the
initiation site which is called an enhancer or silencer.
Enhancers or silencers affect the rate and frequency of
initiation of transcription
 B. Elongation of Transcription

 RNA polymerase links ribonucleotides together in a 5' to 3'

direction. The polymerase induces the 3' hydroxyl group of the
nucleotide at the 3' end of the growing RNA chain which attacks
the phosphorous of the incoming ribonucleotide. A diphosphate
is released and the 5' carbon of the incoming nucleotide is linked
through a phosphodiester bond to the 3' carbon of the preceding
nucleotide.
 B. Elongation of Transcription

 One of the DNA strands in the double helix holds the genetic
information used for protein synthesis. This is called the sense
strand, or information strand. The complementary strand that
binds to the sense strand is called the anti-sense strand, and it
serves as a template for generating a mRNA molecule that
delivers a copy of the sense strand information to a ribosom
 C. Termination of Transcription

 Prokaryotes use two means for terminating

transcription, factor-independent and factor-dependent.
Certain DNA sequences function as signals that tell the
RNA polymerase to terminate transcription.
 The DNA of a terminator sequence encoded an inverted
repeat and an adjacent stretch of uracils. Factor-
dependent termination involves a terminator sequence
as well as a factor or protein called rho.
 The mechanisms by which eukaryotes terminate
transcription are poorly understood. The excess RNA is
then cleaved from the transcript when the RNA is
processed into its mature
 RNA Processing

 Processing helps stabilize and protect the RNA

so it can function in the cytosol and also
functions in regulating the expression of
certain genes.
 Mature mRNA is formed by extensively
modifying the primary transcript also called
heterogeneous nuclear RNA (hnRNA). The
hnRNA must undergo three major
modifications before maturing into mRNA:
capping, polyadenylation and splicing.
 RNA Processing
 Capping: all mRNA's are capped at their 5'
ends with 7-methylguanylate. Guanylyl
transferase catalyzes the linking of 7'-
methylguanylate to the mRNA through a 5' to
5' triphosphate bridge. The capping protects
RNA from exonuclease activity.
 Polyadenylation: is the addition of a chain of
adenylate residues, known as a poly A tail to
the 3' terminus of mRNA. The poly A tail slows
the exonucleolytic degradation of mRNA.
 Splicing: is the removal of noncoding sequences, derived from the DNA
template, from the hnRNA to form a functional mRNA. The noncoding
sequences are called introns while the coding sequences are known as exons.
 All introns have the sequence GU at their 5' ends and AG at their 3' ends. The
guanyl residue at the 5' end of the intron is linked by a 2' to 5' phosphodiester
linkage to an adenylate residue within the intron. The result is a lariat (loop)
structure and the release of the 3' end of the first exon. The 3' end of the intron
is spliced by an enzyme known as a spliceosome, which releases the loop and
frees the 5' end of the second exon. The exons are then joined together.
 The rRNA of both prokaryotes and eukaryotes are synthesized as large
precursors. The precursor rRNA's are processed into their mature form by
nucleases and methylases.
 The tRNA's of both prokaryotes and eukaryotes are also transcribed as
precursors which are cleaved and extensively modified.
  
  
 Translation

 Translation is the synthesis of a protein under the direction of mRNA.

 During this process the nucleotide sequence of an mRNA (messenger
RNA) is translated into the amino acid sequence of a protein.
 The nucleotide sequence of the mRNA is composed of four different
nucleotides whereas a protein is built up from 20 amino acids. To allow
the four nucleotides to specify 20 different amino acids, the nucleotide
sequence is interpreted in codons, groups of three nucleotides.
 Properties of the genetic code

1. The genetic code is redundant. There are 64 possible codons but only 20
amino acids.
2. The genetic code is degenerate. A single amino acid may have more then
one triplet code
3. Non overlapping – a set of three adjacent bases are treated as a complete
group. Each group is called a codon.
4. No punctuation. There are no spaces or commas separating neighboring
codons.
5. There is a start codon corresponding to the amino acid methionine. When
translation begins the first amino acid is always methionine. After
translation this amino acid is removed as part of editing the protein.
6. There are three non coding stop or nonsense codons. These tell the
machinery of translation that the end of the protein has been reached.
7. The code is almost universal. The same genetic code is used by almost all
organisms.
 Ribosomes
 A cell's protein synthesis takes place in
organelles called ribosomes. Ribosomes are
composed of proteins and rRNAs. The major
role of the ribosome is to catalyse coupling of
amino acids into protein according to the
sequence specified by the mRNA.
Ribosome Whole Small Subunit Large Subunit
Source Ribosome
Prokariotes 70S 30S 50S
16S RNA 23S & 5S RNAs
21 proteins 31 proteins

Eukariotes 80S 40S 60S

18S RNA 28S, 5.8S, & 5S RNAs
33 proteins 49 proteins
 Messenger RNA
 A prokaryotic mRNA molecule encoding a single polypeptide has 5
prime non-coding leader sequence.
The rest of the mRNA contains a coding sequence that starts with an
AUG start codon and ends with a stop codon (UAA, UAG or UGA) and a
3 prime non-coding trailer sequence.
A eukaryotic mRNA molecule has, in addition to the above, a 5’cap and a
3’ poly(A) tail.
One important difference is that eukaryotic mRNAs lack a ribosome
binding site (SD site)
 Translation may be divided into three distinct steps:

 1. Activation of Amino Acids - linkage of the appropriate amino

acid to each tRNA.
 2. Initiation, results in the formation of an initiation complex in
which the ribosome is bound to the specific initiation (start) site
on the mRNA.
 3. Elongation, consists of joining amino acids to the growing
polypeptide chain according to the sequence specified by the
message.
 4. The termination - the ready-made protein is released from the
ribosome.
 5. Post-translational processing of protein.
 1. Activation of Amino Acids

 Activation of amino acids occurs in a two step process catalyzed by

aminoacyl-tRNA Synthetases, which catalyze linkage of the
appropriate amino acid to each tRNA. There are at least 20
different aminoacyl-tRNA synthetases.
 Activation of amino acids requires energy in the form of ATP. First
the enzyme attaches the amino acid to the α-phosphate of ATP with
the concomitant release of pyrophosphate. This is termed an
aminoacyl-adenylate intermediate. In the second step the enzyme
catalyzes transfer of the amino acid to either the 2'– or 3'–OH of the
ribose portion of the 3'-terminal adenosine residue of the tRNA
generating the activated aminoacyl-tRNA.
 The 2-step reaction is summarized below:
 amino acid + ATP ------ aminoacyl-AMP + PPi
 aminoacyl-AMP + tRNA ------ aminoacyl-tRNA + AMP
1. Activation of Amino Acids
 Aminoacyl-tRNA Synthetase
 There is generally a different Aminoacyl-tRNA
Synthetase (aaRS) for each amino acid. Accurate
translation of the genetic code depends on attachment
of each amino acid to an appropriate tRNA. Each aaRS
recognizes its particular amino acid and tRNAs coding
for that amino acid.
 Initiation of Translation

 The initiation of translation requires recognit- formation of a 70S

translation initiation complex (in prokaryotes) in which the ribosome is
bound to the specific initiation (start) site on the mRNA .
 Ion of an AUG codon, that in prokaryotes is located adjacent to a Shine-
Delgarno element in the mRNA. The Shine-Delgarno element is
recognized by complimentary sequences in the small subunit rRNA (16S).
 Initiation of Translation

Initiation of translation requires several specific steps:

 A ribosome must dissociate into its' 30S and 50S subunits.
 Three initiation factors (IF1, IF2, IF3) and GTP bind to the small ribosomal subunit.
The initiation factors bind to the 30S ribosomal subunit assure antiassociation to
the 50S subunit.
 The ribosome accepts a mRNA molecule, binding initially to the Shine-Delgarno
sequence at the 5'-end.
 The initiator aminoacyl tRNA (fmet-tRNAfmet) is attached to the 30S subunit. fmet-
tRNAfmet molecule is attached with the appropriate anti-codon at the starting point
– AUG codon.
 The large ribosomal subunit 50S joins the preinitiation complex to form the 70S
initiation complex. The resulting 70S initiation complex has fMet-tRNAfMet residing
in the ribosome's P site.
 The IF-1, IF-2 and IF-3 are released.
 The energy needed to stimulate the formation of the 70S initiation complex comes
from the hydrolysis of the GTP in GDP+Pi
 Elongation of Translation
 In prokaryotes, three elongation factors are required for
translation: EF-Tu, EF-Ts, and EF-G.
 EF-Tu (elongation factor thermo unstable) mediates the
entry of the aminoacyl tRNA into a free site of the ribosome.
 EF-Ts serves as the guanine nucleotide exchange factor for
EF-Tu, catalyzing the release of GDP from EF-Tu.
 EF-G catalyzes the translocation of the tRNA and mRNA
down the ribosome at the end of each round of polypeptide
elongation.
 Chain elongation during protein synthesis requires the
presence of a peptidyl tRNA or, in the first elongation cycle,
an fMet-tRNAfMet at the peptidyl (P) site.
 Elongation of Translation
 Elongation begins with the binding of the second aminoacyl tRNA at
the ribosomal aminoacyl (A) site. The tRNA is escorted to the A site by
the elongation factor EF-Tu, which also carries two bound GTPs. As the
tRNA binds, the GTPs are hydrolyzed and EF-Tu is released. EF-Ts
helps recycle the EF-Tu.
 A peptide bond is formed between the carboxyl group of the terminal
amino acid (or fMet in the first cycle) at the P site and the amino group
of the newly arrived amino acid at the A site.
This reaction is catalyzed by the peptidyl transferase .
 Translocation - after EF-G-GTP binds to the ribosome and GTP is
hydrolyzed, the tRNA carrying the elongated polypeptide translocates
from the A site to the P site. The discharged tRNA moves from the P
site to the E (exit) site and leaves the ribosome. Consequently, the next
mRNA codon is moved into the A site, which is open for the next
aminoacyl tRNA.
These events are repeated for each additional amino acid.
 Termination of Translation
 Termination of protein synthesis depends on
release factors that recognize the three stop
codons.
 When a stop codon (UAG, UAA, or UGA) arrives
at the A site, it is recognized and bound by a
protein release factor. This protein causes the
polypeptide to be transferred to a molecule of
water to cause its release from the tRNA and the
dissociation of the other components of the
elongation complex.
 
 This is a molecule of messenger RNA.
It was made in the nucleus by
transcription from a DNA molecule.

codon

AUGGGCUUAAAG CAGUGCACGUU

mRNA molecule
 A ribosome on the rough
endoplasmic reticulum attaches to
the mRNA molecule.

ribosome

AUGGGCUUAAAG CAGUGCACGUU
 Amino acid

tRNA molecule

A transfer RNA molecule arrives.

It brings an amino acid to the first three bases

(codon) on the mRNA.

anticodon The three unpaired bases (anticodon) on the

tRNA link up with the codon.

UAC
AUGGGCUUAAAG CAGUGCACGUU
 Another tRNA molecule comes into place,
bringing a second amino acid.

Its anticodon links up with the second codon on

the mRNA.
CC
G
UAC
AUGGGCUUAAAG CAGUGCACGUU
 Peptide bond

A peptide bond forms between the two amino

acids.

UAC CCG
AUGGGCUUAAAG CAGUGCACGUU
 The first tRNA molecule releases its amino acid and
moves off into the cytoplasm.

A C
U
CCG
AUGGGCUUAAAG CAGUGCACGUU
 The ribosome moves along the mRNA to the next
codon.

CCG
AUGGGCUUAAAG CAGUGCACGUU
 Another tRNA molecule brings the
next amino acid into place.

AA
U
CCG
AUGGGCUUAAAG CAGUGCACGUU
 A peptide bond joins the second and third
amino acids to form a polypeptide chain.

CCG CCG
AUGGGCUUAAAG CAGUGCACGUU
The process continues.

The polypeptide chain gets longer.

This continues until a termination (stop)

codon is reached.

The polypeptide is then complete. AC

G
GUC
AUGGGCUUAAAG CAGUGCACGUU
 5.Post-translational processing of protein

 Protein synthesis establishes the primary structurefor a

protein. Additional processing is required to convert it to
it’s biologically active form.
 This may include:
 Folding - proteins have to be folded into the proper three
dimensional conformation to work properly. Protein folding
result from interaction of side chains. Proteins called chaperones
act as catalysts to guide this process.
 Biochemical modifications
 Proteolytic cleavage
 Amino acid modification: phosphorilation, hydroxylation, etc.
 attachment of other groups (carbohydrates, prosthetic groups)
  
 Principles of Gene Regulation
 Genes for products that are required at all
times, such as those for the enzymes of central
metabolic path- ways, are expressed at a more
or less constant level in virtually every cell of a
species or organism. Unvarying expression of
a gene is called constitutive gene expression.
 For other inducible gene products , cellular
levels rise and fall in response to molecular
signals; this is regulated gene expression.
 Jacob and Monod model
 The model for regulation of the lactose (lac) operon includes the genes
for β-galactosidase (Z) and galactoside permease (Y), the operon
includes a gene for thiogalactoside transacetylase (A), whose
participate in the breakdown of lactose.
 Jacob and Monod found the molecule was later shown to be a
protein, now called the Lac repressor. Repression is not absolute.
 When cells are provided with lactose, the lac operon is induced. An
inducer molecule binds to a specific site on the repressor causing a
conformational change in the repressor that results in its dissociation
from the operator
 The inducer in this system is lactose. Lactose entering the E. coli cell
is converted to lactose in a reaction catalyzed by the few copies of β-
galactosidase in the cell. Lactose then binds to the Lac repressor. After
the repressor dissociates, the Lac operon genes are expressed and the
concentration of β-galactosidase increases by a factor of 1,000  
The lac Operon Is Subject to Positive Regulation

 The operator-repressor-inducer interactions provide an

intuitively satisfying model for an on/off switch in the
regulation of gene expression.
 Another major environmental factor affecting the
expression of the lac genes is the presence or absence of
glucose. Glucose is the preferred cellular energy source
because of its central place in cellular metabolism.
 Hence, expressing the genes required to metabolize
sugars such as lactose, galactose, and arabinose would
be wasteful if glucose were abundant.
 What happens to the expression of the lac operon if both
glucose and lactose are present?
 The repressive effect of glucose is mediated by cAMP and a
protein called catabolite gene activator protein,
abbreviated CAP.
 When glucose is absent, CAP binds to a specific site near
the lac promoter and stimulates RNA transcription 50-fold.
 CAP is therefore a positive regulatory element responsive to
glucose levels,
 the Lac repressor is a negative regulatory element responsive to
lactose. The two act in concert; CAP has little effect on the system
when the Lac repressor is blocking transcription, and dissociation
of the repressor from the operator has little effect unless CAP is
present to facilitate transcription. Stimulation by CAP is
necessary because the wild-type lac promoter is a relatively weak
promoter ;
 ■ In addition to repression by the Lac
repressor, the E. coli lac operon undergoes
positive regulation by the cAMP receptor
protein (CRP). When [glucose] is low, [cAMP]
is high and CRP-cAMP binds to a specific site
on the DNA, stimulating transcription of the
lac operon and production of lactose-
metabolizing enzymes.
FIGURE 28–18 Combined effects of glucose and lactose on
expression of the lac operon. (a) High levels of transcription
take place only when glucose concentrations are low (so
cAMP levels are high and CRP-cAMP is bound) and lactose
concentrations are high (so the Lac repressor is not bound).
(b) Without bound activator (CRP-cAMP), the lac promoter is
poorly transcribed even when lactose concentrations are high
and the Lac repressor is not bound.
 Regulation of Gene Expression in
Eukaryotes
■ In eukaryotes, positive regulation is more common than negative
regulation, and transcription is accompanied by large changes in
chromatin structure.
■ Large complexes of proteins are generally required to regulate
transcriptional activity. The effects of DNA-binding transactivators
on Pol II are mediated by coactivator protein complexes such as
TFIID or mediator.
■ Hormones affect the regulation of gene expression in one of two
ways. Steroid hormones interact directly with intracellular receptors
that are DNA-binding regulatory proteins; binding of the hormone
has either positive or negative effects on the transcription of genes
targeted by the hormone. Nonsteroid hormones bind to cell-surface
receptors, triggering a signaling pathway that can lead to
phosphorylation of a regulatory protein, affecting its activity.
■ Development of a multicellular organism presents
the most complex regulatory challenge. The fate of
cells in the early embryo is determined by
establishment of anterior-posterior and dorsal-
ventral gradients of proteins that act as
transcriptional transactivators or translational
repressors, regulating the genes required for the
development of structures appropriate to a particular
part of the organism. Sets of regulatory genes
operate in temporal and spatial succession,
transforming given areas of an egg cell into
predictable structures in the adult organism.
Protein Synthesis Is Inhibited by Many Antibiotics and Toxins

 One of the best-understood inhibitory antibiotics

is puromycin. Puromycin has a structure very
similar to the 3' end of an aminoacyl-tRNA (Fig.
26-34). It binds to the A site and participates in all
elongation steps up to and including peptide
bond formation, producing a peptidyl puromycin.
However, puromycin will not bind to the P site,
nor does it engage in translocation. It dissociates
from the ribosome shortly after it is linked to the
carboxyl terminus of the peptide, prematurely
terminating synthesis of the polypeptide.
Puromycin action
Puromycin resembles the
aminoacyl end of a charged tRNA
and can bind to the ribosomal A
site, where it can participate in
peptide bond formation (a).
The product of this reaction,
instead of being translocated to the
P site, dissociates from the
ribosome, causing premature chain
termination. 
 Antibiotics
 Tetracyclines inhibit protein synthesis in bacteria by
blocking the A site on the ribosome, inhibiting binding of
aminoacyl-tRNAs.
  Chloramphenicol inhibits protein synthesis by bacterial
(and mitochondrial and chloroplast) ribosomes by blocking
peptidyl transfer but does not affect cytosolic protein
synthesis in eukaryotes.
 Conversely, cycloheximide blocks the peptidyl transferase
of 80S eukaryotic ribosomes but not that of 70S bacterial
(and mitochondrial and chloroplast) ribosomes.
 Streptomycin, a basic trisaccharide, causes misreading of
the genetic code in bacteria at relatively low concentrations
and inhibits initiation at higher concentrations.
 Toxins
 Several other inhibitors of protein synthesis are
notable because of their toxicity to humans and
other mammals. Diphtheria toxin (Mr 65,000)
catalyzes the ADP-ribosylation of a
diphthamide (a modified histidine) residue on
eukaryotic elongation factor eEF2, thereby
inactivating it.
  Ricin, an extremely toxic protein of the castor
bean, inactivates the 60S subunit of eukaryotic
ribosomes.