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Presented By Dr. Nitin M. Kamble PhD scholar BTY
Gene regulation in prokaryotes
O In the absence of regulatory proteins O Promoter O Rna polymerase (rho70) O -10, -35, UP element O Consensus
O Regulators-DNA binding proteins O Positive/activator- help RNA polymerase
bind DNA by recruitment and cooperative binding O Negative/repressors-block binding O Eg: lac genes in E. coli
Activator works by allostery .
O Eg: in glnA promoter. activator MerR interact with promoter DNA . activator NtrC interact with RNA polymerase bound in closed complex O In merT promoter.
DNA looping NtrC activator binding site 150 bp upstream of promoter .
.Structural gene clusters are coordinately controlled O Genes coding for proteins that function in the same pathway may be located adjacent to one another. O Controled as asingle unit that is transcribed into a polycistronic mRNA O Eg: bacterial structural genes. protein responsible for transportation small molecules in the cell. metabolic pathways.
.Lac operon (6000bp) O Francois Jacob and Jacques Monod O Includes cis acting regulator elements and O O O O O protein coding structural genes. Polycistronic message lacZ-b-galectosidase (500 kd) lacY-lactose permease (30 kd membrane bound) lacA.thiogalectoside transacetylase The lac genes are controlled by negative regulation : they are transcribed unless turned off by regulator protein.
O lac genotype O Mutation in lacZ and lacY gene O Cell cant utilize B galactoside O Mutation in lacA gene have no effect .
lac repressor.coded by lacI constitutively expressed Has its own promoter and terminator It is tetramer of identical subunits (38kd each).Lac genes are controlled by a lac repressor O Transcription of lacZYA gene is controlled by a repressor O O O O O O protein. . operator that overlap the promoter at the start of the cluster. Mutation in lacI causes structural genes to remain in their expressed condition. overlapping right end of promoter. Binding site. Operator extends from -5 upstream of mRNA startpoint to +21 within transcription unit.
O CAP & Lac repressor have opposing effects on RNA polymerase binding to lac Lac operator ² A) 21bp sequence promoter symmetry with twofold B) recognized by 2 subunits of Lac repressor C) lac operator overlap the promoter D) physically prevents RNA polymerase binding CAP (Catabolite activator protein or cAMP receptor protein) A) RNA pol binds poorely in absence of CAP B) promoter -35 sequence not optimal and lacks UP element C) CAP binds 60 bp upstream and helps in cooperative binding of RNA pol .
CAP has separate activating and DNA binding surfaces .
Ensuring low level of by b galactosidase. O Allolactose+lac repressor--. O Lactose---enter cell³allolactose by b galactosidase³control lac repressor O Expression of lac genes is leaky. O High Glucose level----lower intracellular cAMP³which needed by Cap to bind DNA. .The activities of lac repressor and CAP are controlled allosreically by their signals.conformational changes³inhibit operator binding.
Combinatorial control O Signal integartion : low lavel of glucose and presence of lactose O Gal genes-galctose presence and low level of glucose O Repressor coded by galR gene mediates effect of inducer galactose O Whereas activator is CAP .
within 2-3 minutes. the lac operon is expressed only at a very low(basal) level. In the absence of b galactosides .lac operon can be induced O The synthesis of enzymes in response to the O O O O O O appearance of specific substrate is called as induction. Bacteria and yeast Small molecules that induces an operon are identical with or related to the substrate for its enzyme B-galcotoside are the substrate for the enzymes coded by lacZYA. . as a result . induction can be rapidly reversed half life is 3 minutes. Addition of specific b-galctosides induces transcription of all the three genes of the operon. The lac mRNA is extremely unstable.
Repression Rapid response to changes in nutrient supply. Provides ability to metabolize new substrate.coli synthesizes tryptophan by tryptophan synthetase if however tryptophan is provided in medium the production of enzyme is immediately stoped. . Provide ability to shut off endogenous synthesis of compounds that suddenly apperas in the medium Eg: E.
Repressor is inactivated by an allosteric interaction in which binding at its site changes the properties of DNA binding site.Repressor is controlled by small molecule inducer O An inducer functions by converting the repressor O O O O O protein into a form with lower operator affinity. For lac system natural inducer is byproduct of lacZ enzyme . B 1-6 allolactose. The ability to act inducer or corepressor is highly specific. Repressor has two binding sites . Dual property of repressor. it can prevent transcription and can recognize inducer. . one for the operator and another for inducer.
.Gratuitous inducer O Molecule that induces enzyme synthesis but are not metabolized. O Isopropylthiogalctoside (IPTG) not recognized by b.galctosidase but it is very efficient inducer of lac genes.
cis-acting constitutive mutation identify the operator The continued expression of gene that do not respond to regulation is called constitutive gene expression and mutant as constitutive mutants. Mutation in the operator causes consitutive expression of all three structural genes. . Oc mutation are cis-acting and affect only those genes on the contiguous stretch of DNA.
Mutation in the DNA binding site of repressor are constitutive because the repressor cannot binds to operator.Trans-acting mutations identify the regulator gene. . Mutation that eliminates lacI function causes constitutive expression and are recessive. Mutation in promoter are uninducible and cis acting. O Mutation in the lacI gene are trans-acting and O O O O affect expression of all lacZYA clusters in the bacterium. Mutation in the DNA binding site of inducer are uninducible.
NtrC and MerR : transcriptional activators that work by allostery rather than recruitment O NtrC control glnA gene. . open complex wheras MerR changes promoter DNA. O RNA polymerase is bound to promoter in stable closed complex. O The NtrC induces stable changes in polymerase . involved in Nitrogen metabolism O MerR control merT gene . encoding enzyme that makes cell resistant to toxic effect of mercury.
which phosphorylate it by NtrB exposing DNA binding domain of activator. . O Four Binding site 150 bp upstream of promoter and bind as a dimer in cooperative manner O Interact with 54 RNA polymerase O NtrC has ATPase activity which provides enegy to induce conformational changes.NtrC has ATPase activity and works from DNA sites far from the gene O Separate activating and binding domain O Bind DNA under low nitrogen level.
.Convert RNA polymerase from closed unstable to open stable complex.
MerR activates transcription by twisting promoter DNA Distance between -10 and -35 is 19 bp instead of 15 to 17 bp. Recognized by 70 polymerase Binding of Hg to MerR changes conformation .
Gal repressor involved in galactose metabolism.Some repressor hold RNA polymerase at the promoter. repressor bind DNA besides promoter and interact with RNA polymerase inhibiting transition from closed to open complex. When bind adjacent to weak promoter PA3. In absence of galactose. When bind adjacent to strong promoter PA2c. . P4 protein from phage 29 growing on B. repress polymerase as overall binding energy provided by polymerase binding to promoter and extra enegy provided by activator so strong that inhibit escape of polymerase . activate polymerase as extra binding energy helps recruit polymerase. subtilis. O MerR hold promoter DNA in a conformation incompatible O O O O O with transcription initiation.
AraC and control of araBAD operon by activation The magnitude of induction of the araBAD promoter by arabinose is very large so often used in expression vectors. .
phosphoribosyl anthranilate transferase trpC. O trpB-tryptophan synthatase B O trpA.Gene regulation at steps after transcription initiation 5 genes in trp operon] trpE-anthranilate synthetase trpD.phosphoribosyl anthranilate isomerase-indol glycerol phosphate synthatase.tryptophan synthatase a O O O O .
O Tryptophan limitating O Expression of all 5 genes O When tryptophan in excess bind to repressor causing conformational changes in it O Trp repressor bind to operator and inhibit trnascription O Attenuation.when little tryptophan is available premature termination of transcript before trpE. .
O High level of tryptophan transcription stops at 139 bp .
O Transcription termination at trp attenuator .
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