Gene Regulation
level of gene expression can vary under different conditions. Benefit is the encoded proteins are produced only when they are required therefor the cell avoids wasting valuable energy making proteins it does not need.
Unregulated Genes/ Constitutive Genes
have constant levels of expression in all conditions over time. Encode proteins needed for survival.
Processes regulated at the genetic level
Metabolism- some proteins function in the metabolism of small molecules. For example, some enzymes are needed for a bacterium to metabolize certain sugars.
Response to environmental factors- Some proteins help a bacterium to survive environmental stress l
transcriptional regulation
at the rate of RNA synthesis can be increased or decrease, not necessarily turned off or on.
involved the actions of regulatory proteins that can bind to the dna and affect the rate of transcription of one or more nearby genes.
2 Types of regulatory Prote
Repressor
a regulatory protein that binds to the dna and INIBITS transcription. Negative Control
Activator
regulatory protein that increases the rate of transcription. Positive Control
Small Effector molecules
do not bind directly to dna to alter transcription - Effectors exert its effects by binding to an activator or repressor. The binding of the effector molecule causes a conformational change in the regulatory protein influencing whether or not the protein
Inducer / INDUCIBLE GENES
small effector molecule that causes transcription to increase. An inducer can do this with two ways
1. bind to a repressor protein and prevent it from binding to the dna
2. bind to an activator protein and cause it to bind to the dna.
Both cases - transcr
the presence of a small effector molecule may INHIBIT transcription.
two ways
1. CORE REPRESSOR - a small molecule that binds to a repressor protein causing the protein to bind to the dna
2. INHIBITOR - binds to an activator protein and prevents it from binding to the dna
both compressor/inhibitor act to REDUCE the rate of
***A repressor is a _ that _ transcription
regulatory protein , inhibits
*** Which of the following conditions would cause transcription to be activated?
a repressor plus an inducer
Regulation of the lac operon
found in e.coli
involves genes that play a role in the utilization of lactose which is a sugar found in milk, the regulation of these genes involves a repressor protein and activator protein.
The phenomenon of enzyme adaptation is due to the synthesis of cellular proteins
francois Jacob and Jacques Monod
Enzyme Adaptation - the observation that a particular enzyme appears within a living cell only after the cell has been exposed to the substrate for that enzyme. When a bacterium is not exposed to a particular substance it
lactose metabolism in Ecoli
1. the expose of bacterial cells ot lactose increased levels of lactose-utilizing enzymes by 1000- to 10000-fold
2. anti body and labeling techniques revealed that the increase in the activity of these enzymes was due to increased synthesis of the enzymes
Observations concluded that
enzyme adaptation is due to the synthesis of specific cellular proteins in response to lactose in the environment.
lac operon encodes proteins involved in lactose metabolism
in a bacteria it is common for a few genes to be arranged together in an OPERON - two or more genes under the transcriptional control of a single promotor
operon encodes a POLYCISTRONIC RNA - contains the sequences of two or more genes
Why do operons occur in bacteria?
one biological advantage of an operon is that it allows a bacterium to coordinately regulate a group of genes that are involved with a common functional goal, the expression of the genes occurs as a single unit.
for transcription to take place an operon i
Fig 16.3
two units are present
unit 1: lac operon contains a CAP site, promotoer (lacP), operator site (lacO), three structural genes (lacZ, Y, and A) and a terminator.
lacZ encodes the enzyme beta-galactosidase - an enzyme that cleaves lactose into galactose and
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lacA encodes galactoside transacetylase, an enzyme that covalently modifies lactose and lactose analogs.
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the cap site and operator site are short dna segments that function in gene regulation.
CAP SITE - a dna sequence recognized by an activator protein called the catabolite activator protein
OPERATOR SITE - a sequence of bases that provide a binding site fo
second transcriptional unit involved in genetic regulation is the lac-i gene which is NOT apart of the lac operon
the lac I gene, expressed at low levels, has its own promoter (i pomoter)
lac i gene encodes the LAC REPRESSOR, a protein that is important for the regulation of the lac operon. The lac repressor functions as a HOMOTETRAMER - a protein composed of 4 ident
lac operon is regulated by a repressor protein
Transcription can occur in more than one way
This way is inducible and under negative control. this regulation involves the lac repressor protein which binds to the sequence of nucleotides found within the lac operator site. Once bound, the lac repressor
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the ability of the lac repressor to bind to the operator site depends on whether or not allolactose is bound to it. Each of the repressor proteins four subunits has a single binding site for allolactose, the inducer.
How does a small molecule like allolactose exert its effects?
when allolactose binds to the repressor, a conformational change occurs in the lac repressor protein that prevents it form binding to the operator site. Under these conditions, RNA polymerase is now free to transcribe the operon.
In this process, it would
Allosteric poteins
have at least two binding sites, the effector molecule binds to the proteins ALLOSTERIC SITE, which is a site other than the proteins active site.
Rare mutation
in the lac-i gene that alter the regulation of the lac operon reveals that the lac repressor is composed of a protein domain that binds to the dna and another domain that contains the allolactose-binding site
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lac-i negative mutations that result in the contitutive expression of the lac operon aka it is expressed in teh presence and absence of lactose
this may result in an inability to synthesize any repressor protein or they may produce a repressor protein tha
lac-iS mutations have the opposite effect
the lac operon cannot be induced even in the presence of lactose. These mutations are typically located in the domain that binds allolactose. The mutation usually results in a lac repressor protein that cannot bind the allolactose. If the lac repressor is
the regulation of the lac operon allows bacterium to respond to environmental change
in the absence of lactose, no inducer is available to bind to the lac repressor. The lac repressor binds to the operator site and inhibits transcription. When bacterium is exposed to the lactose (a very small amount) can be transported into the cytoplasm
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the cytoplasmic level of allolactose gradually rises and allolactose binds to the lac repressor. The binding of allolactose promites a conformational change that prevents the repressor from binding to the lac operator site therefor allowing transcription
Understanding how the induction process is shut off in a lactose-depleted environment
consider the interaction between allolactose and lac repressor
The lac repressor has a measurable affinity for allolactose. the binding of allolactose to the lac repressor is reversible. The likeliood that allolactose will bind to the repressor depends on
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Now the lac repressor is unlikely to be bound to allolactose. When the allolactose is released the lac repressor returns to the conformation that binds to the operator site. The binding of the ressor shuts down the lac operon when lactose is depleted from
lac-i gene encodes a diffusible repressor protein
Paradee identitfied a few rare mutant strains of bacteria that had abnormal lactose adaptation.
lac-i negative resulted in the constitutive expression of the lac operon even in the abscence of lactose.
Bacterial Conjugation
mattings between recipient cells termed F- and donor cells (Hfr) transferred a portion of the bacterial chromosome.
Sometimes an F factor also carries genes that were originally found within bacterial chromosome. These types of F factors are called F' fac
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production of merozygotes was instrumental in allowing Paradee to elucidate the function of the lac-i gene
2 key points
1. two lac-i genes in a merozygote may be different alleles
ie. lac i gene on the chromosome may be lac i negative allele that causes c
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16.7
lac-i mutant strain was already known to constitutively express the lac operon and compared it to the corresponding merozygote. The merozygote had a lac-i negative mutant gene on the chromosome and a normal lac i gene on an F' factor. These two strai
interpret the data
Yellow = absence or presence of lactose
the presence of lactose was not needed to induce the operon due to a defective lac i gene.
merozygote strain - absence of lactose the lac operon were repressed, even the operon on the bacterial chromosome.
Why? beca
Interaction between regulatory protein and dna sequences illustrated in this experiment have leg to the definition of two genetic terms.
TRANS EFFECT - a form of genetic regulation that can occur even though two dna segmenta are not physically adjacent. the action of the lac repressor on the lac operon is a trans effect
TRANS ACTING FACTOR - a regulatory protein like the lac repressor
CIS
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a loss of function mutation in a gene encoding a repressor protein has the same effect as a mutation in an operator site that cannot bind a repressor protein. Both cases the genes of the lac operon are constitutivel expressed. in a merozygote the results
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a mutation in a transacting factor can be complemented by the introduction of a second gene with normal function.
a mutation in a cis acting element is not affected by the intro of another cis acting element with a normal funciton into the cell
lac operon is also regulated by an activator protein
lac operon can also be transcriptionally regulated in a second way, CATABOLITE REPRESSION. This is influenced by the presence of glucose which is a catabolite - a substance that is broken down inside the cell
the presence of flucose leads to repression of
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Using ttwo sugars by a bacterium = DIAUXIC GROWTH
glucose is not the small effector that binds to a genetic regulatory molecule, CYCLIC AMP (cAMP) which is produced from ATP via an enzyme is. The transport of glucose into the cell stimulates a signaling p
CAP
determines whether the lac operon is expressed in the presence or absence of lactose/glu.
when lac is only present, cAMO levels are high.
cAMP bind to CAP and then CAP binds to the CAP site and stimulates the ability of rna polymerase to begin transcripti
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the effect of glu called CATABOLITE REPRESSION involves the action of an inducer cAMP and an activator protein CAP, not a repressor.
lac operon has 3 operator sites for the lac repressor
O1 is the operator site slightly downstream from the promoter
O2 is father down in the lacZ coding sequence.
O3 upstream from the promoter
If O2+3 are missing, repression is dramatically reduced even thwne O1 is present.
When O1 is missing, even in the pr
16.9
Repressor must bind to O1,2,3to cause full repression. The lac repressor can bind to O1+2 or O1+3. Never O2+3. If either O2 or O3 were missing, maximal repression is not acheived because it is less likely for the repressor to bind when only two operator s
***What is an operon?
a group of genes under the control of a single promoter
*** The binding of _ to the lac repressor causes the lac repressor to _ o the operator site thereby _ transcription
glu, not bind, increase
*** on its chromosome, an eocli cell is lac i negative, lac z, y, and a. It has an F' factor that is lac i,z,y,a positive. What is the expected level of expression of the lac operon genes (lac Z,Y,A) in the absence of lactose?
neither expressed
*** When an e coli is exposed to glu how does this affect the regulation of the lac operon via CAP?
cAMP does not bind to CAP and transcription is decreased
Regulation of the trp operon
encodes enzymes involved with the snthesis of the amino acid tryptophan. Regulated by a repressor protein and ATTENUATION - where transcription begins but is stopped prematurely.
the trp operon is regulated by a repressor protein
contains 6 genes
trp L E D C B A
EDCBA genes encode enzymes involved in tryptophan biosynthesis
L plays a regulatory role
R not a part of the trp operon encodes the TRP REPRESSOR protein. when levels within cell is low the trp repressor cannot bind to the
trp operon is also regulated by Attenuation
can occur in bacteria because the processes of transcription and translation are coupled. Bacterial ribosomes quickly attach to the 5' end of the mRNa soon after its synthesis begins via RNA polymerase. During attenuation, transcription actually begins bu
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terp opeorn downstream from the operator site is imporant during attenuation, the first gene in the trp operon is the L which encodes a peptide containing 14 a called the LEADER PEPTIDE. two features are key in the attenuation mechanism
1. twp tryptophan
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Region 2 is complementary to 1 and 3. Region 3 is complementary to 2 and 4. The three stem loops are possible
12, 23, 34
ie. if region 2 forms a stem loop with region 1 it cannot at the same time form a stemloop with region 3. then region 3 cannot form a
conditions that favor the formation of the 3-4 stem loop
rely on the translation of the L gene.
3 scenarios are possible
1. Region 1 rapidly hydrogen bonds to region 2 and 3 is left to hydrogen bond to region 4 therefor the terminator stem loop forms and the transcription is terminated just past the L gene at t
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2. coupled transcription and translation occur under conditions in which the tryptophan concentration is low, then the cells cannot make a sufficient amount of charged trna. the ribosomes pause at the trp codons in the L mrna because it is waiting for cha
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3. coupled transcription and translation occur under conditions in which a sufficient amount of tryptophan is present in the cell. In this case, translation of the L mrna progresses to its stop codon where the ribosome pauses. The pausing at the stop codo
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attenuation is a mechanism to regulat transcription that is found in several other operons involved with aa biosynthesis. the mrna that encode the leader peptides are rich in odons for the particular aa that is synthesizes by the enzymes encoded by the pa
Translational and Post translational regulation
Post translational regulation refers to the functional control of proteins that are already present in the cell rather than regulation of transcription or translation. Can either activate or inibit the function of a protein. Compared with transcription co
repressor proteins and antisense rna can inhibit translation
for some bacterial genes, mrna is regulated by the binding of proteins that influence the ability of ribosomes to translate the mrna into polypeptide.
TRANSLATIONAL REGULATORY PROTEIN recognizes a seq within the mrna. TRANSLATIONAL REPRESSORS - proteins a
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2.