? Transcriptional Regulation in Bacteria
? Thanks to the relative ease of doing genetics with bacteria,
transcriptional regulation in bacteria is better understood than
that in other organisms and has served as a framework for
understanding transcriptional regulation in eukaryotic organisms,
There are important differences between the mechanisms of
transcriptional regulation in bacteria and higher organisms,many
of which relate to the presence of a nuclear membrane,
Nevertheless,many of the strategies used are similar throughout
the biological world,and many general principles have been
uncovered through studies of bacterial transcriptional regulation.
(A) The two general types of
transcriptional regulation,In
negative regulation,a repressor binds
to an operator and turns the operon
off,In positive regulation,an
activator protein binds upstream of
the promoter and turns the operon on,
(B) Graph showing the usual
locations of activator sites relative to
operators,Activator sites are usually
farther upstream,Each datum point
indicates the middle of the known
region on the DNA where a
regulatory protein binds,
? The Bacterial Operon
? The concept of an operon is central to hypotheses about
bacterial transcriptional regulation,Bacterial genes are often
arranged so that more than one gene can be transcribed into the
same polycistronic mRNA,In such cases,the genes are said to
be cotranscribed,Cotranscription of more than one gene into a
polycistronic mRNA seems to be unique to bacteria and their
phages and affects the types of translational initiation regions
(TIR) used on the mRNA,In eukaryotes,generally the first
AUG codon in an mRNA is used to initiate translation so that
only one polypeptide can be encoded by each mRNA,
However,bacterial TIRs are much more complex,Shine-
Dalgarno and other sequences help define the TIR,Because of
its distinct structure,a bacterial TIR will be recognized
wherever it appears in the mRNA,and more than one TIR can
be recognized in the same mRNA.
? A bacterial operon is the region on the DNA that includes
genes cotranscribed into the same mRNA plus all of the
adjacent cis-acting sequences required for transcription of
these genes,including the genes' promoter as well as
operators and other sequences involved in regulating the
transcription of the genes,Because the genes of an operon
are all transcribed from the same promoter and use the same
regulatory sequences,all the genes of an operon can be
transcriptionally regulated simultaneously.
? Repressors and Activators
? Before discussing our examples of transcriptional regulation in
bacteria,we give a brief overview of the types of regulation
known to occur and define some of the terms that describe the
factors involved,The transcription of bacterial operons is
regulated by the products of regulatory genes,which are often
proteins called repressors or activators,These regulatory
proteins bind close to the operon's promoter and regulate
transcription from the promoter,Sometimes,a regulatory protein
can play dual roles and can also perform an enzymatic reaction
in the pathway encoded by the operon,Because they bind to
DNA,repressors and activators often have the helix-turn-helix
motif shared by many DNA binding proteins,
? Repressors bind to sites called operators and turn off the
promoter,thereby preventing transcription of the genes of the
operon,Activators,in contrast,bind to activator sites and turn
on the promoter,thereby facilitating transcription of the operon
genes.
? Negative and Positive Regulation
? Another important concept is the distinction between negative and
positive regulation by regulatory proteins,If a regulatory protein
in its active state turns off the expression of the operon,the operon
is said to be negatively regulated by the regulatory protein,If the
regulatory protein in its active state turns on the operon,the
operon is positively regulated by the regulatory protein,An
operon regulated by a repressor is therefore negatively regulated,
because the presence of the active repressor prevents transcription
of that operon,In contrast,an operon regulated by an activator is
positively regulated,because in its active state the activator
protein turns on transcription of the operon under its control.
? Activator and repressor proteins usually bind to different regions
of the DNA,
? Activators usually bind upstream of the -35 sequence of the
promoter,where they can make contact with the RNA
polymerase bound to the promoter,Repressors often bind to
the promoter region itself,or at least very close to it,and
thereby block access by RNA polymerase to the promoter.
? Some regulatory proteins can be both repressors and activators,
depending upon the situation,The λ repressor is an example,It
represses transcription from the the pL and pR promoters but
activates the pRM promoter,The binding site on the DNA for
the regulatory protein often changes when the protein shifts
from being an activator to a repressor,
? Inducers and Corepressors
? Whether a regulatory protein is active sometimes depends on
whether it is bound to a small molecule,Small molecules that
bind to proteins and change their properties are called effectors,
An effector that binds to a repressor or activator and thereby
initiates transcription of an operon is called the inducer of the
operon,In contrast,an effector that binds to a repressor and
causes it to block transcription is called a corepressor.
? The activity of regulatory proteins is not necessarily altered
only by binding to small molecule effectors,Some repressors
and activators are covalently altered under some conditions,
for example,by methylation or phosphorylation.
? Genetic Evidence for Negative and Positive
Regulation
? Negatively and positively regulated operons behave very differently
in genetic tests,One difference is in the effect of mutations that
inactivate the regulatory gene for the operon,If an operon is
negatively regulated,a mutation that inactivates the regulatory gene
will allow transcription of the operon genes,even in the absence of
inducer,If the regulation is positive,mutations that inactivate the
regulatory gene will prevent transcription of the genes of the operon,
even in the presence of the inducer,A mutant in which the genes of
an operon are always transcribed,even in the absence of inducer,is
called a constitutive mutant,Constitutive mutations are much more
common with negatively than with positively regulated operons
because any mutation that inactivates the repressor will result in the
constitutive phenotype,With positively regulated operons,a
constitutive phenotype can be caused only by changes that do not
inactivate the activator protein but alter it so that it can activate
transcription without binding to the inducer,Such changes tend to be
rare.
? Complementation tests reveal another difference between
negatively and postively regulated operons,Constitutive
mutations of a negatively regulated operon are often recessive
to the wild type,This is because any normal repressor protein
in the cell encoded by a wild-type copy of the gene will bind to
the operator and block transcription,even if the repressor
encoded by the mutant copy of the gene in the same cell is
inactive,In contrast,constitutive mutations in a solely
positively regulated operon should be dominant to the wild
type,A mutant activator protein that is active without inducer
bound might activate transcription even in the presence of a
wild-type activator protein.
? Negative Regulation:The E,coli lac Operon
? The classic example of negative regulation is regulation of the E,
coli lac operon,which encodes the enzymes responsible for the
utilization of the sugar lactose,The experiments of Francois
Jacob and Jacques Monod and their collaborators on the
regulation of the E,coli lac genes are excellent examples of the
genetic analysis of a biological phenomenon in bacteria,
Although these experiments were performed in the late 1950s,
only shortly after the discovery of the structure of DNA and the
existence of mRNA,they still stand as the framework with
which all other studies of gene regulation are compared.
? GENETICS OF THE lac OPERON
? When Jacob and Monod began their classic work,it was
known that the enzymes of lactose metabolism are inducible in
that they are expressed only when the sugar lactose is present
in the medium,If no lactose is present,the enzymes are not
made,From the standpoint of the cell,this is a sensible
strategy,since there is 'no point in making the enzymes for
lactose utilization unless there is lactose available for use as a
carbon and energy source.
? To understand the regulation of the lactose genes,Jacob and
Monod first isolated many mutations affecting lactose
metabolism and regulation,which fell into two fundamentally
different groups,Some mutants were unable to grow with
lactose as the sole carbon and energy source and so were Lac-,
Other mutants made the lactose-metabolizing enzymes
whether or not lactose was present in the medium and so were
constitutive mutants.
? To analyze the regulation of the lac genes,Jacob and Monod needed
to know which of the mutations affected trans-acting gene
products—either protein or RNA—involved in the regulation and
how many different genes these mutations represented,They also
wished to know if any of the mutations were cis acting (affecting
sites on the DNA involved in regulation).
? To answer these questions,they needed to perform complementation
tests,which require that the organisms be diploid,with two copies of
the genes being tested,Bacteria are normally haploid,with only one
copy of each of their genes,but are "partial diploids" for any genes
carried on an introduced prime factor,Recall that a prime factor is a
plasmid into which some of the bacterial chromosomal genes have
been inserted,By introducing prime factors carrying various mutated
lac genes into cells with different mutations in the chromosomal lac
genes,Jacob and Monod performed complementation tests on each
of their lac mutations,Their methods depended upon the type of
mutation being tested.
? COMPLEMENTATION TESTS WITH lac
MUTATIONS
? Whether a particular lac mutation is dominant or recessive was
determined by introducing an F factor carrying the wild-type
lac region into a strain with the lac mutation in the
chromosome,If the partial diploid bacteria are Lac- and can
multiply to form colonies on minimal plates with lactose as the
sole carbon and energy source,the lac mutation is recessive,If
the partial diploid cells are Lac- and cannot form colonies on
lactose minimal plates,the lac mutation is dominant,Jacob
and Monod discovered that most lac mutations are recessive to
the wild type and so presumably inactivate genes whose
products are required for lactose utilization.
? The question of how many genes are represented by recessive
lac mutations could be answered by performing pairwise
complementation tests between different lac mutations,Prime
factors carrying the lac region with one lac mutation were
introduced into a mutant strain with another lac mutation in
the chromosome,In this kind of experiment,if the partial
diploid cells are Lac+,the two recessive mutations can
complement each other and are members of different
complementation groups or genes,If the partial diploid cells
are Lac-,the two mutations cannot complement each other and
are members of the same complementation group or gene,
Jacob and Monod found that most of the lac mutations sorted
into two different complementation groups,which they named
lacZ and lacY We now know of another gene,lacA,which was
not discovered in their original selections because its product
is not required for growth on lactose.
Complementation of two recessive mutations,One mutation is in the
chromosome,and the other is in a prime factor,If the two mutations
complement each other,the cells will be Lac+ and will grow with
lactose as the sole carbon and energy source,The mutations will not
complement if they are in the same gene or if one affects a
regulatory site or is polar,
? cis-Acting lac Mutations
? Not all lac mutations affect diffusible gene products and can
be complemented,Immediately adjacent to the lacZ mutations
are other lac mutations that are much rarer and have radically
different properties,These mutations cannot be complemented
to allow the expression of the lac genes on the same DNA,
even in the presence of good copies of the lac genes,
Recessive mutations that cannot be complemented are cis
acting and presumably affect a site on DNA rather than a
diffusible gene product like RNA or protein.
? To show that a lac mutation is cis-acting,i.e.,affects only the
expression of genes on the same DNA where it occurs,we
could introduce an F' factor containing the potential cis-acting
lac mutation into cells containing either a lacZ or a lacY
mutation in the chromosome,Trans-acting gene products
encoded by the F' factor lacZ or lacY genes would complement
the chromosomal lacY or lacZ mutations,respectively,
However,if the resulting phenotypes are Lac-,the lac mutation
in the F' factor must prevent expression of both LacZ and
LacY proteins from the F factor,The mutation in the F factor
is therefore cis-acting.
? One type of the cis-acting lac mutations affects lacp and is a
promoter mutation that prevents transcription of the lacZ and
lacY genes by changing the binding site for RNA polymerase
on the DNA,Another type of cis-acting mutation is a polar
mutation in lacZ that prevents the transcription of the
downstream lacY gene.
The lacp mutations cannot be complemented and are cis acting,A
lacp mutation in the prime factor will prevent expression of any of
the other lac genes on the prime factor,so that a lac mutation in the
chromosome will not be complemented,Partial diploid cells will be
Lac-.
? Regulation by Attenuation of Transcription
? In the above examples,the transcription of an operon is
regulated through the initiation of RNA synthesis at the
promoter of the operon,However,this is not the only known
means of regulating operon transcription,Another mechanism
is the attenuation of transcription,Unlike repressors and
activators,which turn on or off transcription from the
promoter,the attenuation mechanism works by terminating
transcription—which begins normally—before the RNA
polymerase reaches the first structural gene of the operon,The
classic examples of regulation by attenuation are the his and
trp operons of E,coli,Closely related mechanisms regulate
such E,coli biosynthetic operons as leucine (leu),
phenylalanine (phe),threonine (thr),and isoleucine-valine (ilv)
and the Bacillus subtilis tRNA synthetase genes,
? Genetic Evidence for Attenuation
? the trp operon is negatively regulated by the TrpR repressor
protein,However,early genetic evidence suggested that this is
not the only type of regulation for trp,If the trp operon were
regulated solely by the TrpR repressor,the levels of the trp
operon enzymes in a trpR mutant would be the same in the
absence and the presence of tryptophan,However,even in a
trpR null mutant,the expression of these enzymes is higher in
the absence of tryptophan than in its presence,indicating that
the trp operon is subject to another regulatory system.
? Early evidence suggested that tRNATrp plays a role in the
regulation of the trp operon in the absence of TrpR (see
Landick and Yanofsky and Morse and Morse,Suggested
Reading),Mutations in the trytophanyl-tRNA synthetase (the
enzyme responsible for transferring tryptophan to tRNATrp)
and mutations in the structural gene for the tRNATrp,as well as
mutations in genes whose products are responsible for
modifying the tRNATrp increase the expression of the operon,
All these mutations presumably lower the amount of
aminoacyl-ated-tRNATrp in the cell,suggesting that this other
regulatory mechanism is not sensing the amount of free
tryptophan in the cell but,rather,the amount bound to the
tRNATrp.
? Other evidence suggested that the region targeted by this other
type of regulation is not the promoter but a region downstream
of the promoter called the leader region,or trpL,Deletions in
this region,which lies between the promoter and trpE,the first
gene of the operon,eliminate the regulation,Double mutants
with both a deletion mutation of the leader region and a trpR
mutation are completely constitutive for expression of the trp
operon,Deletions of the leader region are also cis acting and
affect only the expression of the trp operon on the same DNA,
Later evidence indicated that transcription terminated in this
leader region in the presence of tryptophan because of an
excess of aminoacylated-tRNATrp,Because the regulation
seemed to be able to stop,or attenuate,transcription that had
already initiated at the promoter,it was called attenuation of
transcription,in agreement with an analogous type of
regulation already discovered for the his operon.
? MODEL FOR REGULATION OF THE
trp OPERON BY ATTENUATION
? A current model for regulation of the trp operon by
attenuation,According to this model,the percentage of
the tRNAlrp that is aminoacylated (i.e.,has tryptophan
attached) determines which of several alternative
secondary-structure hairpins will form in the leader
RNA,Four RNA regions can form three different
hairpins,The secondary structure of an RNA,or a
hairpin,results from comple mentary pairing between
the bases in RNA transcribed from inverted repeated
sequences.
? Whether or not transcription termination occurs depends on
whether the attenuation mechanism senses relatively low or high
levels of tryptophan,The trpL region,which contains two
adjacent trp codons,provides the signal,The trp codons are there
to allow the ribosome to test the water before the RNA
polymerase is allowed to plunge into the structural genes of the
operon,If levels of tryptophan are low,the levels of
tryptophanyl-tRNATrP (tRNATrP with tryptophan attached) will
also be low,When a ribosome encounters one of the trp codons,
it will temporarily stall,unable to insert the amino acid,This
stalled ribosome in the trpL region therefore communicates that
the tryptophan concentration is low and that transcription should
continue.
? How the hairpins operate in attenuation,Four different regions in
the trpL leader RNA—regions 1,2,3,and 4—can form three
different hairpins,1:2,2:3,and 3:4,The formation of hairpin 3:4
causes RNA polymerase to terminate transcription because this
hairpin is part of a factorindependent transcription termination
signal.
? Whether hairpin 3:4 forms is determined by the dynamic relationship
between ribosomal translation of the trp codons in the trpL region and
the progress of RNA polymerase through the trpL region,After RNA
polymerase initiates tran scription at the promoter,it moves through
the trpL region to a site located just after region 2,where it pauses,
The hairpin formed by mRNA regions 1 and 2 is an important part of
the signal to pause,The pause is short,probably less than 1 s,but it
ensures that a ribosome has time to load on the mRNA before the
RNA polymerase proceeds to region 3,The moving ribosome may
help release the paused RNA polymerase by colliding with it.
? The process of ribosome translation through the trp codons of trpL
then determines whether hairpin 3:4 will form,causing termination,or
2 will pair instead with 3,preventing formation of the 3:4 hairpin,
Region 3 will pair with region 2 if the ribosome stalls at the trp
codons because of low tryptophan concentrations,If the ribosome
does not stall at the trp codons,it will continue until it reaches the
UGA stop codon at the end of trpL,By remaining at the stop codon
while region 4 is synthesized,the ribosome will prevent hairpin 2:3
from forming,Therefore hairpin 3:4 can form and terminate
transcription.
? GENETIC EVIDENCE FOR THE MODEL
? No model is satisfactory unless it is supported by
experimental evidence,The existence and in vivo functioning
of hairpin 2:3 were supported by the phenotypes produced by
mutation trpL75,This mutation,which changes one of the
nucleotides and prevents pairing of two of the bases holding
the hairpin together,should destabilize the hairpin,In the
trpL75 mutant,transcription terminates in the trpL region,
even in the absence of tryptophan,consistent with the model
that formation of hairpin 2:3 normally prevents formation of
hairpin 3:4.
? That translation of the leader peptide from the trpL region is
essential to the regulation is supported by the phenotypes of
mutation trpL29,which changes the AUG start codon of the
leader peptide to AUA,preventing initiation of translation,In
trpL29 mutants,termination also occurs even in the absence of
tryptophan,The model also explains this observation as long as
we can assume that the RNA polymerase paused at hairpin
coding sequence 1:2 will eventually move on,even without a
translating ribosome to nudge it,and will eventually transcribe
the 3:4 region,Without a ribosome stalled at the trp codons,
however,hairpin 1:2 will persist,preventing the formation of
hairpin 2:3,If hairpin 2:3 does not form,hairpin 3:4 will form
and transcription will terminate.
? One final prediction of the model is that stopping translation at
other codons in trpL should also relieve attenuation,The codon
immediately downstream of the second tryptophan codon in the
trpL region is for arginine,Starving the cells for arginine also
prevents attenuation of the trp operon,fulfilling this prediction
of the model.
Structure and relevant features of the leader
region of the trp operon involved in regulation
by attentuation,TT (in blue) indicates the two
trp codons in the leader region,
Induction and Repression
? The regulation of β-galactosidase synthesis has been intensively studied and serves
as a primary example of how gene expression is controlled,This enzyme catalyzes
the hydrolysis of the sugar lactose to glucose and galactose,When E,coli grows
with lactose as its carbon source,each cell contains about 3,000 β-galactosidase
molecules,but has less than three molecules in the absence of lactose,The enzyme
β-galactosidase is an inducible enzyme -that is,its level rises in the presence of a
small molecule called an inducer (in this case the lactose derivative allolactose).
? The genes for enzymes involved in the biosynthesis of amino acids and other
substances often respond differently from genes coding for catabolic enzymes,An
amino acid present in the surroundings may inhibit the formation of the enzymes
responsible for its biosynthesis,This makes good sense because the microorganism
will not need the biosynthetic enzymes for a particular substance if it is already
available,Enzymes whose amount is reduced by the presence of an end product are
repressible enzymes,and metabolites causing a decrease in the concentrations of
repressible enzymes are corepressors,Generally,repressible enzymes are necessary
for synthesis and always are present unless the end product of their pathway is
available,Inducible enzymes,in contrast,are required only when their substrate is
available; they are missing in the absence of the inducer.
Gene Induction
The regulator gene,Reg.,synthesizes an active repressor that binds to the
operator,o,and blocks RNA polymerase binding to the promoter,p,unless the
inducer inactivates it,In the presence of the inducer,the repressor protein is
inactive and transcription occurs.
Gene Repression
The regulator gene,Reg.,synthesizes an inactive repressor protein that must be
activated by corepressor binding before it can bind to the operate,o,and block
transcription,In the absence of the corepressor,the repressor is inactivate and
transcription occurs.
Negative Control
A controlling factor can either inhibit or activate transcription,
Although the responses to the presence of metabolites are
different,both induction and repression are forms of negative
control,mRNA synthesis proceeds rapidly in the absence of
the active controlling factor.
Positive Control
The preceding section shows that operons can be under
control,resulting in induction and repression,In contrast,
some operons function only in the presence of a controlling
factor － that is,they are under positive operon control.
Positive Control of the Lac operon
When cyclic AMP is absent or present at a low level,the CAP protein remains inactive
and does not bind to the promoter,In this situation RNA polymerase also does not
bind to the promoter and transcribe the operon’s genes.
Negative regulation of the trp operon by the TrpR repressor.
Another element of control,called attenuation,has been recognized in
several operons,The word attenuation means,to lessen in amount”,
We described regulating transcription at initiation; that is,repressors
block the synthesis of RNA whereas activator proteins encourage
synthesis,
In transcription attenuation the control occurs after initiation of RNA
synthesis but before its completion,That is,the number of completed
transcripts from a gene or an operon is reduced,even though the number
of initialed transcripts is not,Most of the first examples of attenuation
involved regulating genes controlling the biosynthesis of certain amino
acids in gram-negative Bacteria,The first such system to be described
was the tryptophan operon in Escherichia coli.
The Tryptophan Operon Leader,
(a) Organization and base pairing of the traptophan operon leader region,The
promoter and operator are to the left of the segment diagrammed,and the first
structural gene (trpE) begins to the left of the attenuator,
(b and c) The stretches of DNA marked off as 1 through 4 can base pair with
each other to form hairpin loops,segment 2 with 1,and segment 3 with 2 or 4.
Attenuation
Control
The control of
tryptophan operon
function by
attenuation.
Mutations
Conditional mutations are those that are expressed
only under certain environmental conditions.
Biochemical mutations are those causing a change in
the biochemistry of the cell,Since these mutations
often inactivate a biosynthetic pathway,they
frequently make a microorganism unable to grow on a
medium lacking an adequate supply of the pathway’s
end product,The mutant cannot grow on minimal
medium and requires nutrient supplements,Such
mutants are called auxotrophs,whereas microbial
strains that can grow on minimal medium are
prototrophs.
Mutations occur in one of two ways,
(1) Spontaneous mutations arise occasionally in
all cells and develop in the absence of any
added agent.
(2) Induced mutations,on the other hand,are
the result of exposure of the organism to some
physical or chemical agent called a mutagen.
Transition and Transversion Mutations,Errors in replication due to base
tautomerization.
(a) Normally AT and GC pairs are formed when keto groups participate in
hydrogen bonds,In contrast,enol tautomers produce AC and GT base pairs.
Spontaneous
Mutations.
(b) Mutation as a consequences of tautomerization during DNA
replication,The temporary enolization of guanine leads to the
formation of an AT base pair in the mutant,and a GC to AT
transition mutation occurs,The process requires two replication
cycles,The mutation only occurs if the abnormal first-generation
GT base pair is missed by repair mechanisms.
Additions and Deletions,A
hypothesis mechanism for
the generation of additions
and deletions during
replication,The direction of
replication is indicated by the
large arrow,In each case
there is strand slippage
resulting in the formation of a
small loop that is stabilized
by the hydrogen bonding in
the repetitive sequence,the
AT stretch in this example,
DNA synthesis proceeds to
the right in this figures,
(a) If the new strand slips,an
addition of one T results,
(b) Slippage of the parental
strand yields a deletion (in
this case,a loss of two Ts).
Mutagenesis by the Base
Analog 5-Bromouracil
(a) Base pairing of the normal
keto form of 5-BU is shown in
the top illustration,The enol
form of 5-BU (bottom
illustration) base pairs with
guanine rather than with
adenine as might be expected
for a thymine analog.
(b) If the keto form of 5-BU is
incorporated in place of
thymine,its occasional
tautomerization to the enol
form (BUe) will produce an AT
to GC transition mutation.
Methyl-
Nitrosoguanideine
Mutagenesis.
Mutagenesis by
methyl-
nitrosoguanidine due
to the methylation of
guanine.
Thymine Dimer,Thymine dimers are formed by ultraviolet
radiation,The enzyme photolyase cleaves the two colored bonds
during photoreactivation.
Frameshift Mutation,A frameshift mutation resulting from insertion of
a GC base pair,The reading frameshift produces a different peptide
after the addtion.
Replication Plating
The use of replica plating in
isolating a lysine auxotroph,
Mutants are generated by treating a
culture with a mutagen,The culture
containing wild type and
auxotrophs is plated on complete
medium,After the colonies have
developed,a piece of sterile
velveteen is pressed on the plate
surface to pick up bacteria from
each colony,Then the velvet is
pressed to the surface of other
plates and organisms are
transferred to the same position as
on the master plate,After
determining the location of Lys-
colonies growing on the replica with
complete medium,the auxotrophs
can be isolated and cultured.
Mutant Selection
the production and
direct selection of
auxotroph revertants will
be selected after
treatment of a lysine
auxotroph culture
because the agar
contains minimal
medium that will not
support auxotroph
growth.
The Ames
Test for
Mutagenicity
DNA Repair
Excision Repair is a general repair system that
corrects damage that causes distortions in the double
helix,A repair endonuclease or uvrABC endonuclease
removes the damaged bases along with some bases on
either side of the lesion,The resulting single-stranded
gap,about 12 nucleotides long,is filled by DNA
polymerase Ⅰ,and DNA ligase joins the fragments.
This system can remove thymine dimers and repair
almost any other injury that produces a detectable
distortion in DNA.
Excision Repair,Excision repair of a thymine dimer that has distorted the double helix,
The repair endonuclease or uvrABC endonuclease is coded for by the uvrA,B,and C
genes.
Removal of Lesions
Thymine dimers and alkylated bases often are directly
repaired,Photoreactivation is the repair of thymine
dimers by splitting them apart into separate thymines
with the help of visible light in a photochemical
reaction catalyzed by the enzyme photolyase,Because
this repair mechanism does not remove and replace
nucleotides,it is error free.
Photoreactivation,Shown is a schematic of how the photolyases enzyme with the help
of species-specific wavelengths of light enzymatically cleaves pyrimidine dimers and
thus restores the integrity of the DNA,(A) A DNA sequence (B) That sequence
following exposure to UV (approximately 254 nm),The triangle represents the
pyrimidine dimer that was formed,(c) A molecule of photolyase recognizes the dimer,
binds to it,and sits there until it is activated by specific wavelengths of light (D),Once
activated,the dimer is cleaved and the DNA sequence is restored (E).
Postreplication Repair
Despite the accuracy of DNA polymerase action and continual
proofreading,errors still are made during DNA replication,
Remaining mismatched bases and other errors are usually
detected and repaired by the mismatch repair system in
E.coli,The mismatch correction enzyme scans the newly
replicated DNA for mismatched pairs and removes a stretch
of newly synthesized DNA around the mismatch,A DNA
polymerase then replaces the excised nucleotides,and the
resulting nick is sealed with a ligase,Postreplication repair is
a type of excision repair.
An E.coli
retrieval system
uses a normal
strand of DNA
to replace the
gap left in a
newly
synthesized
strand opposite
a site of
unrepaired
damage.
Recombination Repair
In recombination repair,damaged DNA for which there is no
remaining template is restored,This situation arises if both
bases of a pair are missing or damaged,or if there is a gap
opposite a lesion,In this type of repair the recA protein cuts
a piece of template DNA from a sister molecule and puts it
into the gap of uses it in to replace a damaged strand.
The recA protein also participates in a type of inducible repair
known as SOS repair,In this instance the DNA damage is so
great that synthesis stops completely,leaving many large gaps,
RecA will bind to the gaps and initiate strand exchange.
The SOS Repair process,In the absence of damages,repair genes are expressed in E.coli at low levels due to
binding of the lexA repressor protein at their operators (o),When the recA protein binds to a damaged
region- for example,a thymine dimer created by UV radiation- it destroys lexA and the repair genes are
expressed more actively,The uvr genes code for the repair endonuclease or uvrABC endonuclease
? Thanks to the relative ease of doing genetics with bacteria,
transcriptional regulation in bacteria is better understood than
that in other organisms and has served as a framework for
understanding transcriptional regulation in eukaryotic organisms,
There are important differences between the mechanisms of
transcriptional regulation in bacteria and higher organisms,many
of which relate to the presence of a nuclear membrane,
Nevertheless,many of the strategies used are similar throughout
the biological world,and many general principles have been
uncovered through studies of bacterial transcriptional regulation.
(A) The two general types of
transcriptional regulation,In
negative regulation,a repressor binds
to an operator and turns the operon
off,In positive regulation,an
activator protein binds upstream of
the promoter and turns the operon on,
(B) Graph showing the usual
locations of activator sites relative to
operators,Activator sites are usually
farther upstream,Each datum point
indicates the middle of the known
region on the DNA where a
regulatory protein binds,
? The Bacterial Operon
? The concept of an operon is central to hypotheses about
bacterial transcriptional regulation,Bacterial genes are often
arranged so that more than one gene can be transcribed into the
same polycistronic mRNA,In such cases,the genes are said to
be cotranscribed,Cotranscription of more than one gene into a
polycistronic mRNA seems to be unique to bacteria and their
phages and affects the types of translational initiation regions
(TIR) used on the mRNA,In eukaryotes,generally the first
AUG codon in an mRNA is used to initiate translation so that
only one polypeptide can be encoded by each mRNA,
However,bacterial TIRs are much more complex,Shine-
Dalgarno and other sequences help define the TIR,Because of
its distinct structure,a bacterial TIR will be recognized
wherever it appears in the mRNA,and more than one TIR can
be recognized in the same mRNA.
? A bacterial operon is the region on the DNA that includes
genes cotranscribed into the same mRNA plus all of the
adjacent cis-acting sequences required for transcription of
these genes,including the genes' promoter as well as
operators and other sequences involved in regulating the
transcription of the genes,Because the genes of an operon
are all transcribed from the same promoter and use the same
regulatory sequences,all the genes of an operon can be
transcriptionally regulated simultaneously.
? Repressors and Activators
? Before discussing our examples of transcriptional regulation in
bacteria,we give a brief overview of the types of regulation
known to occur and define some of the terms that describe the
factors involved,The transcription of bacterial operons is
regulated by the products of regulatory genes,which are often
proteins called repressors or activators,These regulatory
proteins bind close to the operon's promoter and regulate
transcription from the promoter,Sometimes,a regulatory protein
can play dual roles and can also perform an enzymatic reaction
in the pathway encoded by the operon,Because they bind to
DNA,repressors and activators often have the helix-turn-helix
motif shared by many DNA binding proteins,
? Repressors bind to sites called operators and turn off the
promoter,thereby preventing transcription of the genes of the
operon,Activators,in contrast,bind to activator sites and turn
on the promoter,thereby facilitating transcription of the operon
genes.
? Negative and Positive Regulation
? Another important concept is the distinction between negative and
positive regulation by regulatory proteins,If a regulatory protein
in its active state turns off the expression of the operon,the operon
is said to be negatively regulated by the regulatory protein,If the
regulatory protein in its active state turns on the operon,the
operon is positively regulated by the regulatory protein,An
operon regulated by a repressor is therefore negatively regulated,
because the presence of the active repressor prevents transcription
of that operon,In contrast,an operon regulated by an activator is
positively regulated,because in its active state the activator
protein turns on transcription of the operon under its control.
? Activator and repressor proteins usually bind to different regions
of the DNA,
? Activators usually bind upstream of the -35 sequence of the
promoter,where they can make contact with the RNA
polymerase bound to the promoter,Repressors often bind to
the promoter region itself,or at least very close to it,and
thereby block access by RNA polymerase to the promoter.
? Some regulatory proteins can be both repressors and activators,
depending upon the situation,The λ repressor is an example,It
represses transcription from the the pL and pR promoters but
activates the pRM promoter,The binding site on the DNA for
the regulatory protein often changes when the protein shifts
from being an activator to a repressor,
? Inducers and Corepressors
? Whether a regulatory protein is active sometimes depends on
whether it is bound to a small molecule,Small molecules that
bind to proteins and change their properties are called effectors,
An effector that binds to a repressor or activator and thereby
initiates transcription of an operon is called the inducer of the
operon,In contrast,an effector that binds to a repressor and
causes it to block transcription is called a corepressor.
? The activity of regulatory proteins is not necessarily altered
only by binding to small molecule effectors,Some repressors
and activators are covalently altered under some conditions,
for example,by methylation or phosphorylation.
? Genetic Evidence for Negative and Positive
Regulation
? Negatively and positively regulated operons behave very differently
in genetic tests,One difference is in the effect of mutations that
inactivate the regulatory gene for the operon,If an operon is
negatively regulated,a mutation that inactivates the regulatory gene
will allow transcription of the operon genes,even in the absence of
inducer,If the regulation is positive,mutations that inactivate the
regulatory gene will prevent transcription of the genes of the operon,
even in the presence of the inducer,A mutant in which the genes of
an operon are always transcribed,even in the absence of inducer,is
called a constitutive mutant,Constitutive mutations are much more
common with negatively than with positively regulated operons
because any mutation that inactivates the repressor will result in the
constitutive phenotype,With positively regulated operons,a
constitutive phenotype can be caused only by changes that do not
inactivate the activator protein but alter it so that it can activate
transcription without binding to the inducer,Such changes tend to be
rare.
? Complementation tests reveal another difference between
negatively and postively regulated operons,Constitutive
mutations of a negatively regulated operon are often recessive
to the wild type,This is because any normal repressor protein
in the cell encoded by a wild-type copy of the gene will bind to
the operator and block transcription,even if the repressor
encoded by the mutant copy of the gene in the same cell is
inactive,In contrast,constitutive mutations in a solely
positively regulated operon should be dominant to the wild
type,A mutant activator protein that is active without inducer
bound might activate transcription even in the presence of a
wild-type activator protein.
? Negative Regulation:The E,coli lac Operon
? The classic example of negative regulation is regulation of the E,
coli lac operon,which encodes the enzymes responsible for the
utilization of the sugar lactose,The experiments of Francois
Jacob and Jacques Monod and their collaborators on the
regulation of the E,coli lac genes are excellent examples of the
genetic analysis of a biological phenomenon in bacteria,
Although these experiments were performed in the late 1950s,
only shortly after the discovery of the structure of DNA and the
existence of mRNA,they still stand as the framework with
which all other studies of gene regulation are compared.
? GENETICS OF THE lac OPERON
? When Jacob and Monod began their classic work,it was
known that the enzymes of lactose metabolism are inducible in
that they are expressed only when the sugar lactose is present
in the medium,If no lactose is present,the enzymes are not
made,From the standpoint of the cell,this is a sensible
strategy,since there is 'no point in making the enzymes for
lactose utilization unless there is lactose available for use as a
carbon and energy source.
? To understand the regulation of the lactose genes,Jacob and
Monod first isolated many mutations affecting lactose
metabolism and regulation,which fell into two fundamentally
different groups,Some mutants were unable to grow with
lactose as the sole carbon and energy source and so were Lac-,
Other mutants made the lactose-metabolizing enzymes
whether or not lactose was present in the medium and so were
constitutive mutants.
? To analyze the regulation of the lac genes,Jacob and Monod needed
to know which of the mutations affected trans-acting gene
products—either protein or RNA—involved in the regulation and
how many different genes these mutations represented,They also
wished to know if any of the mutations were cis acting (affecting
sites on the DNA involved in regulation).
? To answer these questions,they needed to perform complementation
tests,which require that the organisms be diploid,with two copies of
the genes being tested,Bacteria are normally haploid,with only one
copy of each of their genes,but are "partial diploids" for any genes
carried on an introduced prime factor,Recall that a prime factor is a
plasmid into which some of the bacterial chromosomal genes have
been inserted,By introducing prime factors carrying various mutated
lac genes into cells with different mutations in the chromosomal lac
genes,Jacob and Monod performed complementation tests on each
of their lac mutations,Their methods depended upon the type of
mutation being tested.
? COMPLEMENTATION TESTS WITH lac
MUTATIONS
? Whether a particular lac mutation is dominant or recessive was
determined by introducing an F factor carrying the wild-type
lac region into a strain with the lac mutation in the
chromosome,If the partial diploid bacteria are Lac- and can
multiply to form colonies on minimal plates with lactose as the
sole carbon and energy source,the lac mutation is recessive,If
the partial diploid cells are Lac- and cannot form colonies on
lactose minimal plates,the lac mutation is dominant,Jacob
and Monod discovered that most lac mutations are recessive to
the wild type and so presumably inactivate genes whose
products are required for lactose utilization.
? The question of how many genes are represented by recessive
lac mutations could be answered by performing pairwise
complementation tests between different lac mutations,Prime
factors carrying the lac region with one lac mutation were
introduced into a mutant strain with another lac mutation in
the chromosome,In this kind of experiment,if the partial
diploid cells are Lac+,the two recessive mutations can
complement each other and are members of different
complementation groups or genes,If the partial diploid cells
are Lac-,the two mutations cannot complement each other and
are members of the same complementation group or gene,
Jacob and Monod found that most of the lac mutations sorted
into two different complementation groups,which they named
lacZ and lacY We now know of another gene,lacA,which was
not discovered in their original selections because its product
is not required for growth on lactose.
Complementation of two recessive mutations,One mutation is in the
chromosome,and the other is in a prime factor,If the two mutations
complement each other,the cells will be Lac+ and will grow with
lactose as the sole carbon and energy source,The mutations will not
complement if they are in the same gene or if one affects a
regulatory site or is polar,
? cis-Acting lac Mutations
? Not all lac mutations affect diffusible gene products and can
be complemented,Immediately adjacent to the lacZ mutations
are other lac mutations that are much rarer and have radically
different properties,These mutations cannot be complemented
to allow the expression of the lac genes on the same DNA,
even in the presence of good copies of the lac genes,
Recessive mutations that cannot be complemented are cis
acting and presumably affect a site on DNA rather than a
diffusible gene product like RNA or protein.
? To show that a lac mutation is cis-acting,i.e.,affects only the
expression of genes on the same DNA where it occurs,we
could introduce an F' factor containing the potential cis-acting
lac mutation into cells containing either a lacZ or a lacY
mutation in the chromosome,Trans-acting gene products
encoded by the F' factor lacZ or lacY genes would complement
the chromosomal lacY or lacZ mutations,respectively,
However,if the resulting phenotypes are Lac-,the lac mutation
in the F' factor must prevent expression of both LacZ and
LacY proteins from the F factor,The mutation in the F factor
is therefore cis-acting.
? One type of the cis-acting lac mutations affects lacp and is a
promoter mutation that prevents transcription of the lacZ and
lacY genes by changing the binding site for RNA polymerase
on the DNA,Another type of cis-acting mutation is a polar
mutation in lacZ that prevents the transcription of the
downstream lacY gene.
The lacp mutations cannot be complemented and are cis acting,A
lacp mutation in the prime factor will prevent expression of any of
the other lac genes on the prime factor,so that a lac mutation in the
chromosome will not be complemented,Partial diploid cells will be
Lac-.
? Regulation by Attenuation of Transcription
? In the above examples,the transcription of an operon is
regulated through the initiation of RNA synthesis at the
promoter of the operon,However,this is not the only known
means of regulating operon transcription,Another mechanism
is the attenuation of transcription,Unlike repressors and
activators,which turn on or off transcription from the
promoter,the attenuation mechanism works by terminating
transcription—which begins normally—before the RNA
polymerase reaches the first structural gene of the operon,The
classic examples of regulation by attenuation are the his and
trp operons of E,coli,Closely related mechanisms regulate
such E,coli biosynthetic operons as leucine (leu),
phenylalanine (phe),threonine (thr),and isoleucine-valine (ilv)
and the Bacillus subtilis tRNA synthetase genes,
? Genetic Evidence for Attenuation
? the trp operon is negatively regulated by the TrpR repressor
protein,However,early genetic evidence suggested that this is
not the only type of regulation for trp,If the trp operon were
regulated solely by the TrpR repressor,the levels of the trp
operon enzymes in a trpR mutant would be the same in the
absence and the presence of tryptophan,However,even in a
trpR null mutant,the expression of these enzymes is higher in
the absence of tryptophan than in its presence,indicating that
the trp operon is subject to another regulatory system.
? Early evidence suggested that tRNATrp plays a role in the
regulation of the trp operon in the absence of TrpR (see
Landick and Yanofsky and Morse and Morse,Suggested
Reading),Mutations in the trytophanyl-tRNA synthetase (the
enzyme responsible for transferring tryptophan to tRNATrp)
and mutations in the structural gene for the tRNATrp,as well as
mutations in genes whose products are responsible for
modifying the tRNATrp increase the expression of the operon,
All these mutations presumably lower the amount of
aminoacyl-ated-tRNATrp in the cell,suggesting that this other
regulatory mechanism is not sensing the amount of free
tryptophan in the cell but,rather,the amount bound to the
tRNATrp.
? Other evidence suggested that the region targeted by this other
type of regulation is not the promoter but a region downstream
of the promoter called the leader region,or trpL,Deletions in
this region,which lies between the promoter and trpE,the first
gene of the operon,eliminate the regulation,Double mutants
with both a deletion mutation of the leader region and a trpR
mutation are completely constitutive for expression of the trp
operon,Deletions of the leader region are also cis acting and
affect only the expression of the trp operon on the same DNA,
Later evidence indicated that transcription terminated in this
leader region in the presence of tryptophan because of an
excess of aminoacylated-tRNATrp,Because the regulation
seemed to be able to stop,or attenuate,transcription that had
already initiated at the promoter,it was called attenuation of
transcription,in agreement with an analogous type of
regulation already discovered for the his operon.
? MODEL FOR REGULATION OF THE
trp OPERON BY ATTENUATION
? A current model for regulation of the trp operon by
attenuation,According to this model,the percentage of
the tRNAlrp that is aminoacylated (i.e.,has tryptophan
attached) determines which of several alternative
secondary-structure hairpins will form in the leader
RNA,Four RNA regions can form three different
hairpins,The secondary structure of an RNA,or a
hairpin,results from comple mentary pairing between
the bases in RNA transcribed from inverted repeated
sequences.
? Whether or not transcription termination occurs depends on
whether the attenuation mechanism senses relatively low or high
levels of tryptophan,The trpL region,which contains two
adjacent trp codons,provides the signal,The trp codons are there
to allow the ribosome to test the water before the RNA
polymerase is allowed to plunge into the structural genes of the
operon,If levels of tryptophan are low,the levels of
tryptophanyl-tRNATrP (tRNATrP with tryptophan attached) will
also be low,When a ribosome encounters one of the trp codons,
it will temporarily stall,unable to insert the amino acid,This
stalled ribosome in the trpL region therefore communicates that
the tryptophan concentration is low and that transcription should
continue.
? How the hairpins operate in attenuation,Four different regions in
the trpL leader RNA—regions 1,2,3,and 4—can form three
different hairpins,1:2,2:3,and 3:4,The formation of hairpin 3:4
causes RNA polymerase to terminate transcription because this
hairpin is part of a factorindependent transcription termination
signal.
? Whether hairpin 3:4 forms is determined by the dynamic relationship
between ribosomal translation of the trp codons in the trpL region and
the progress of RNA polymerase through the trpL region,After RNA
polymerase initiates tran scription at the promoter,it moves through
the trpL region to a site located just after region 2,where it pauses,
The hairpin formed by mRNA regions 1 and 2 is an important part of
the signal to pause,The pause is short,probably less than 1 s,but it
ensures that a ribosome has time to load on the mRNA before the
RNA polymerase proceeds to region 3,The moving ribosome may
help release the paused RNA polymerase by colliding with it.
? The process of ribosome translation through the trp codons of trpL
then determines whether hairpin 3:4 will form,causing termination,or
2 will pair instead with 3,preventing formation of the 3:4 hairpin,
Region 3 will pair with region 2 if the ribosome stalls at the trp
codons because of low tryptophan concentrations,If the ribosome
does not stall at the trp codons,it will continue until it reaches the
UGA stop codon at the end of trpL,By remaining at the stop codon
while region 4 is synthesized,the ribosome will prevent hairpin 2:3
from forming,Therefore hairpin 3:4 can form and terminate
transcription.
? GENETIC EVIDENCE FOR THE MODEL
? No model is satisfactory unless it is supported by
experimental evidence,The existence and in vivo functioning
of hairpin 2:3 were supported by the phenotypes produced by
mutation trpL75,This mutation,which changes one of the
nucleotides and prevents pairing of two of the bases holding
the hairpin together,should destabilize the hairpin,In the
trpL75 mutant,transcription terminates in the trpL region,
even in the absence of tryptophan,consistent with the model
that formation of hairpin 2:3 normally prevents formation of
hairpin 3:4.
? That translation of the leader peptide from the trpL region is
essential to the regulation is supported by the phenotypes of
mutation trpL29,which changes the AUG start codon of the
leader peptide to AUA,preventing initiation of translation,In
trpL29 mutants,termination also occurs even in the absence of
tryptophan,The model also explains this observation as long as
we can assume that the RNA polymerase paused at hairpin
coding sequence 1:2 will eventually move on,even without a
translating ribosome to nudge it,and will eventually transcribe
the 3:4 region,Without a ribosome stalled at the trp codons,
however,hairpin 1:2 will persist,preventing the formation of
hairpin 2:3,If hairpin 2:3 does not form,hairpin 3:4 will form
and transcription will terminate.
? One final prediction of the model is that stopping translation at
other codons in trpL should also relieve attenuation,The codon
immediately downstream of the second tryptophan codon in the
trpL region is for arginine,Starving the cells for arginine also
prevents attenuation of the trp operon,fulfilling this prediction
of the model.
Structure and relevant features of the leader
region of the trp operon involved in regulation
by attentuation,TT (in blue) indicates the two
trp codons in the leader region,
Induction and Repression
? The regulation of β-galactosidase synthesis has been intensively studied and serves
as a primary example of how gene expression is controlled,This enzyme catalyzes
the hydrolysis of the sugar lactose to glucose and galactose,When E,coli grows
with lactose as its carbon source,each cell contains about 3,000 β-galactosidase
molecules,but has less than three molecules in the absence of lactose,The enzyme
β-galactosidase is an inducible enzyme -that is,its level rises in the presence of a
small molecule called an inducer (in this case the lactose derivative allolactose).
? The genes for enzymes involved in the biosynthesis of amino acids and other
substances often respond differently from genes coding for catabolic enzymes,An
amino acid present in the surroundings may inhibit the formation of the enzymes
responsible for its biosynthesis,This makes good sense because the microorganism
will not need the biosynthetic enzymes for a particular substance if it is already
available,Enzymes whose amount is reduced by the presence of an end product are
repressible enzymes,and metabolites causing a decrease in the concentrations of
repressible enzymes are corepressors,Generally,repressible enzymes are necessary
for synthesis and always are present unless the end product of their pathway is
available,Inducible enzymes,in contrast,are required only when their substrate is
available; they are missing in the absence of the inducer.
Gene Induction
The regulator gene,Reg.,synthesizes an active repressor that binds to the
operator,o,and blocks RNA polymerase binding to the promoter,p,unless the
inducer inactivates it,In the presence of the inducer,the repressor protein is
inactive and transcription occurs.
Gene Repression
The regulator gene,Reg.,synthesizes an inactive repressor protein that must be
activated by corepressor binding before it can bind to the operate,o,and block
transcription,In the absence of the corepressor,the repressor is inactivate and
transcription occurs.
Negative Control
A controlling factor can either inhibit or activate transcription,
Although the responses to the presence of metabolites are
different,both induction and repression are forms of negative
control,mRNA synthesis proceeds rapidly in the absence of
the active controlling factor.
Positive Control
The preceding section shows that operons can be under
control,resulting in induction and repression,In contrast,
some operons function only in the presence of a controlling
factor － that is,they are under positive operon control.
Positive Control of the Lac operon
When cyclic AMP is absent or present at a low level,the CAP protein remains inactive
and does not bind to the promoter,In this situation RNA polymerase also does not
bind to the promoter and transcribe the operon’s genes.
Negative regulation of the trp operon by the TrpR repressor.
Another element of control,called attenuation,has been recognized in
several operons,The word attenuation means,to lessen in amount”,
We described regulating transcription at initiation; that is,repressors
block the synthesis of RNA whereas activator proteins encourage
synthesis,
In transcription attenuation the control occurs after initiation of RNA
synthesis but before its completion,That is,the number of completed
transcripts from a gene or an operon is reduced,even though the number
of initialed transcripts is not,Most of the first examples of attenuation
involved regulating genes controlling the biosynthesis of certain amino
acids in gram-negative Bacteria,The first such system to be described
was the tryptophan operon in Escherichia coli.
The Tryptophan Operon Leader,
(a) Organization and base pairing of the traptophan operon leader region,The
promoter and operator are to the left of the segment diagrammed,and the first
structural gene (trpE) begins to the left of the attenuator,
(b and c) The stretches of DNA marked off as 1 through 4 can base pair with
each other to form hairpin loops,segment 2 with 1,and segment 3 with 2 or 4.
Attenuation
Control
The control of
tryptophan operon
function by
attenuation.
Mutations
Conditional mutations are those that are expressed
only under certain environmental conditions.
Biochemical mutations are those causing a change in
the biochemistry of the cell,Since these mutations
often inactivate a biosynthetic pathway,they
frequently make a microorganism unable to grow on a
medium lacking an adequate supply of the pathway’s
end product,The mutant cannot grow on minimal
medium and requires nutrient supplements,Such
mutants are called auxotrophs,whereas microbial
strains that can grow on minimal medium are
prototrophs.
Mutations occur in one of two ways,
(1) Spontaneous mutations arise occasionally in
all cells and develop in the absence of any
added agent.
(2) Induced mutations,on the other hand,are
the result of exposure of the organism to some
physical or chemical agent called a mutagen.
Transition and Transversion Mutations,Errors in replication due to base
tautomerization.
(a) Normally AT and GC pairs are formed when keto groups participate in
hydrogen bonds,In contrast,enol tautomers produce AC and GT base pairs.
Spontaneous
Mutations.
(b) Mutation as a consequences of tautomerization during DNA
replication,The temporary enolization of guanine leads to the
formation of an AT base pair in the mutant,and a GC to AT
transition mutation occurs,The process requires two replication
cycles,The mutation only occurs if the abnormal first-generation
GT base pair is missed by repair mechanisms.
Additions and Deletions,A
hypothesis mechanism for
the generation of additions
and deletions during
replication,The direction of
replication is indicated by the
large arrow,In each case
there is strand slippage
resulting in the formation of a
small loop that is stabilized
by the hydrogen bonding in
the repetitive sequence,the
AT stretch in this example,
DNA synthesis proceeds to
the right in this figures,
(a) If the new strand slips,an
addition of one T results,
(b) Slippage of the parental
strand yields a deletion (in
this case,a loss of two Ts).
Mutagenesis by the Base
Analog 5-Bromouracil
(a) Base pairing of the normal
keto form of 5-BU is shown in
the top illustration,The enol
form of 5-BU (bottom
illustration) base pairs with
guanine rather than with
adenine as might be expected
for a thymine analog.
(b) If the keto form of 5-BU is
incorporated in place of
thymine,its occasional
tautomerization to the enol
form (BUe) will produce an AT
to GC transition mutation.
Methyl-
Nitrosoguanideine
Mutagenesis.
Mutagenesis by
methyl-
nitrosoguanidine due
to the methylation of
guanine.
Thymine Dimer,Thymine dimers are formed by ultraviolet
radiation,The enzyme photolyase cleaves the two colored bonds
during photoreactivation.
Frameshift Mutation,A frameshift mutation resulting from insertion of
a GC base pair,The reading frameshift produces a different peptide
after the addtion.
Replication Plating
The use of replica plating in
isolating a lysine auxotroph,
Mutants are generated by treating a
culture with a mutagen,The culture
containing wild type and
auxotrophs is plated on complete
medium,After the colonies have
developed,a piece of sterile
velveteen is pressed on the plate
surface to pick up bacteria from
each colony,Then the velvet is
pressed to the surface of other
plates and organisms are
transferred to the same position as
on the master plate,After
determining the location of Lys-
colonies growing on the replica with
complete medium,the auxotrophs
can be isolated and cultured.
Mutant Selection
the production and
direct selection of
auxotroph revertants will
be selected after
treatment of a lysine
auxotroph culture
because the agar
contains minimal
medium that will not
support auxotroph
growth.
The Ames
Test for
Mutagenicity
DNA Repair
Excision Repair is a general repair system that
corrects damage that causes distortions in the double
helix,A repair endonuclease or uvrABC endonuclease
removes the damaged bases along with some bases on
either side of the lesion,The resulting single-stranded
gap,about 12 nucleotides long,is filled by DNA
polymerase Ⅰ,and DNA ligase joins the fragments.
This system can remove thymine dimers and repair
almost any other injury that produces a detectable
distortion in DNA.
Excision Repair,Excision repair of a thymine dimer that has distorted the double helix,
The repair endonuclease or uvrABC endonuclease is coded for by the uvrA,B,and C
genes.
Removal of Lesions
Thymine dimers and alkylated bases often are directly
repaired,Photoreactivation is the repair of thymine
dimers by splitting them apart into separate thymines
with the help of visible light in a photochemical
reaction catalyzed by the enzyme photolyase,Because
this repair mechanism does not remove and replace
nucleotides,it is error free.
Photoreactivation,Shown is a schematic of how the photolyases enzyme with the help
of species-specific wavelengths of light enzymatically cleaves pyrimidine dimers and
thus restores the integrity of the DNA,(A) A DNA sequence (B) That sequence
following exposure to UV (approximately 254 nm),The triangle represents the
pyrimidine dimer that was formed,(c) A molecule of photolyase recognizes the dimer,
binds to it,and sits there until it is activated by specific wavelengths of light (D),Once
activated,the dimer is cleaved and the DNA sequence is restored (E).
Postreplication Repair
Despite the accuracy of DNA polymerase action and continual
proofreading,errors still are made during DNA replication,
Remaining mismatched bases and other errors are usually
detected and repaired by the mismatch repair system in
E.coli,The mismatch correction enzyme scans the newly
replicated DNA for mismatched pairs and removes a stretch
of newly synthesized DNA around the mismatch,A DNA
polymerase then replaces the excised nucleotides,and the
resulting nick is sealed with a ligase,Postreplication repair is
a type of excision repair.
An E.coli
retrieval system
uses a normal
strand of DNA
to replace the
gap left in a
newly
synthesized
strand opposite
a site of
unrepaired
damage.
Recombination Repair
In recombination repair,damaged DNA for which there is no
remaining template is restored,This situation arises if both
bases of a pair are missing or damaged,or if there is a gap
opposite a lesion,In this type of repair the recA protein cuts
a piece of template DNA from a sister molecule and puts it
into the gap of uses it in to replace a damaged strand.
The recA protein also participates in a type of inducible repair
known as SOS repair,In this instance the DNA damage is so
great that synthesis stops completely,leaving many large gaps,
RecA will bind to the gaps and initiate strand exchange.
The SOS Repair process,In the absence of damages,repair genes are expressed in E.coli at low levels due to
binding of the lexA repressor protein at their operators (o),When the recA protein binds to a damaged
region- for example,a thymine dimer created by UV radiation- it destroys lexA and the repair genes are
expressed more actively,The uvr genes code for the repair endonuclease or uvrABC endonuclease
