AL-RAISALMILL M. ALAWI
OPERONS NEGATIVE CONTROL OF LAC OPERON
FINE CONTROL OF BACTERIAL TRANSCRIPT ● Negative control - implies that operon is turned on unless lac
THE LAC OPERON repressor intervenes to stop it.
● Lac Operon - first operon discovered which became the prime ● Off regulation - lac repressor; turns off the lac operon
example for operon concept: ○ When the repressor binds to the operator or as long as
● 1. Galactoside permease (lacY) - transports the lactose into cells there’s no lactose available, the operon is repressed ARTH
PARD
● 2. B - galactosidase (lacZ) - cuts lactose into galactose and ○ Repressor bound to operator – prevents RNA polymerase AND
glucose from binding to the promoter and transcribing the operon
● 3. Galactoside transacetylase (lacA) - unclear function in lactose ● lacl gene - product of a regulatory gene
● Metabolism they are transcribed to produce one messenger RNA ● Allosteric protein - an repressor; binds to one molecule to the REPR
known as polycistronic message - comes from a single promoter protein changes the shape of a remote site on the protein and GEN
(ciostEroanc)h cistron/ gene – has its own ribosome binding site alters its interaction with a second molecule
○ Each cistron – can be translated by separate ribosomes ○ Greek: allos = other + stereos = shape
that bind independently of each other ● Inducer - first molecule; binds to the repressor causing protein to
● E. coli cells growing on a medium containing sugars glucose and change to a conformation that favors dissociation from operator
lactose – cells exhaust the glucose and stop growing ● - an Allolactose; alternative form of lactose
○ growth resumes - after an hour NATURE OF INDUCER
○ During lag - cells have been turning on the lac operon and ● Inducer of lac operon - binds the repressor
beginning to accumulate the enzymes theyneed to ● Inducer of Allolactose - Alternative form of lactose
metabolize lactose ● When Beta - galactosidase cleaves lactose to galactose plus
● Diauxic - latin word auxilium meaning help glucose, it rearranges a small fraction of the lactose to allolactose
● Bacteria need an enzyme to transport the lactose into the cells REPR
○ Galactoside permease ●
DISCOVERY OF THE OPERON
○ Cells - need an enzyme to break the lactose down into 2
component sugars: Galactose & glucose FRANCOIS 1940s-1950s; studies the metabolism of lactose by E.
JACOB AND coli
JACQUES ●
CONTROL OF LAC OPERON MONOD
3 enzyme activities or genes were induced together by
Negative Control Positive Control galactosides
● like “brake” of car; needed ● like accelerator pedal
to released to be able to ● Activator - additional Constitutive mutants - needed no induction, genes are
move postive factor; responds to active all the time ●
● Brake - a protein called as low glucose levels by
lac repressor which keeps stimulating transcription of
which keeps the operon the lac operon MONOD, 1940 studying the inducibility of lactose metabolism in E.
turned off as long as ● High glucose levels -keep coli
THE M
lactose is absent the concentration of the
activator low, so BETA-GALACTO important feature of lactose metabolism ●
SIDASE
, AL-RAISALMILL M. ALAWI
POSITIVE CONTROL OF THE LAC OPERON ● Thomaz Steitz and colleagues - determined that CAP-cAMP TRYP
Positive control of the lac operon by a substance - sensing a lack of bends its target DNA by about 100 degress when it binds. Five g
glucose that reponds by activating the lac promoter ● Hen-Ming Wu and Donald Crothers - using electrophoresis synthe
concentration of a nucleotide (cyclic-AMP or cAMP) - rises as observed the DNA bend in the crystallography studies High o
concentration of glucose drops THE ARA OPERON Prese
CATABOLITE ACTIVATOR PROTEIN ● ara operon of E. coli - codes for enzymes required to metabolize NEGA
● cAMP added to E. coli - overcome the catabolite repression of lac the sugar arabinose , another catabolite- repressible operon ●
operon ● Two ara operators exists: ara01 & ara02
● Addition of cAMP - leads to activation of lac gene even with the ○ araC - regulated transcription of control gene of ara01 ●
presence of glucose ○ ara02 is located far upstream of the promoter it controls ●
● 2 parts of positive controller of the lac operon: (PBAD) between position 265 & 294
● 1. cAMP ● CAP-binding site - about 200 bp upstream yet can still stimulate ●
● 2. Protein factor such as: transcription
○ CAP or Catabolite Activator Protein ● AraC protein - another system of negative regulation ●
○ CRP or Cyclic-AMP Receptor Protein ARA OPERON REPRESSION LOOP MECH
○ crp - gene encoding this protein ● araO2 operator - controls transcription from a promoter 250 ●
○ Geoffrey Zubay and coworkers - showed that crude downstream ●
cell-free extract of E. coli would make B-galactosidase if it ● Robert Lobell and Robert Schleif found that if they inserted DNA
is supplied with cAMP fragments containing an integral number of double-helical turns ●
SIMULATION OF LAC OPERON (multiples of 10.5 bp) between the operator and the promoter, the
● CAP - cAMP complex - positively controls the activity of Beta- operator still functioned. However, if the inserts contained a
galactosidase nonintegral number of helical turns (e.g., 5 or 15 bp), the operator ●
○ CAP - binds cAMP tightly did not function
○ Mutant CAP - not bind cAMP tightly prepared ARA CONTROL PROTEIN DEFE
○ Compare activity and production of Beta - galactosidase ● araC - act as both positive and negative regulator 3 BINDING site: ●
using both ○ ara02 - far upstream site
○ complexes ○ ara01 - located between -106 & -144 ●
○ Low activity with mutant CAP – cAMP ○ aral is 2 half sites: aral1 - between -56 & -78 and aral2-
MECHANISM OF CAP ACTION -35 to -51 (each half site can bind to one monomer of AraC ●
● CAP-cAMP complex - binds to the lac promoter ARA CBAD OPERON
● Mutant whose lac gene is not stimulated by complex - had the ● ara operon - also known as araCBAD operon for its 4 genes:
mutation in the lac promoter ○ Three gene: asaB, A, and D - encodes the arabinose
● Mapping the DNA - showed that the activator-binding site lies just metabolizing enzymes
upstream of the promoter ○ transcribes rightward from the promoter araPBAD RIBOS
● Binding CAP and cAMP to the activation site- helps RNA ○ other gene, araC which encodes the control protein AraC ●
polymerase form an open promoter and transcribed leftward from the araPc promoter
RECRUITMENT AUTOREGULATION OF ARA C ●
● Formation of the closed promoter complex allows AraC to regulate its own synthesis
● Conversion of the closed promoter complex to the open promoter autoregulation - protein who controls its own synthesis
OPERONS NEGATIVE CONTROL OF LAC OPERON
FINE CONTROL OF BACTERIAL TRANSCRIPT ● Negative control - implies that operon is turned on unless lac
THE LAC OPERON repressor intervenes to stop it.
● Lac Operon - first operon discovered which became the prime ● Off regulation - lac repressor; turns off the lac operon
example for operon concept: ○ When the repressor binds to the operator or as long as
● 1. Galactoside permease (lacY) - transports the lactose into cells there’s no lactose available, the operon is repressed ARTH
PARD
● 2. B - galactosidase (lacZ) - cuts lactose into galactose and ○ Repressor bound to operator – prevents RNA polymerase AND
glucose from binding to the promoter and transcribing the operon
● 3. Galactoside transacetylase (lacA) - unclear function in lactose ● lacl gene - product of a regulatory gene
● Metabolism they are transcribed to produce one messenger RNA ● Allosteric protein - an repressor; binds to one molecule to the REPR
known as polycistronic message - comes from a single promoter protein changes the shape of a remote site on the protein and GEN
(ciostEroanc)h cistron/ gene – has its own ribosome binding site alters its interaction with a second molecule
○ Each cistron – can be translated by separate ribosomes ○ Greek: allos = other + stereos = shape
that bind independently of each other ● Inducer - first molecule; binds to the repressor causing protein to
● E. coli cells growing on a medium containing sugars glucose and change to a conformation that favors dissociation from operator
lactose – cells exhaust the glucose and stop growing ● - an Allolactose; alternative form of lactose
○ growth resumes - after an hour NATURE OF INDUCER
○ During lag - cells have been turning on the lac operon and ● Inducer of lac operon - binds the repressor
beginning to accumulate the enzymes theyneed to ● Inducer of Allolactose - Alternative form of lactose
metabolize lactose ● When Beta - galactosidase cleaves lactose to galactose plus
● Diauxic - latin word auxilium meaning help glucose, it rearranges a small fraction of the lactose to allolactose
● Bacteria need an enzyme to transport the lactose into the cells REPR
○ Galactoside permease ●
DISCOVERY OF THE OPERON
○ Cells - need an enzyme to break the lactose down into 2
component sugars: Galactose & glucose FRANCOIS 1940s-1950s; studies the metabolism of lactose by E.
JACOB AND coli
JACQUES ●
CONTROL OF LAC OPERON MONOD
3 enzyme activities or genes were induced together by
Negative Control Positive Control galactosides
● like “brake” of car; needed ● like accelerator pedal
to released to be able to ● Activator - additional Constitutive mutants - needed no induction, genes are
move postive factor; responds to active all the time ●
● Brake - a protein called as low glucose levels by
lac repressor which keeps stimulating transcription of
which keeps the operon the lac operon MONOD, 1940 studying the inducibility of lactose metabolism in E.
turned off as long as ● High glucose levels -keep coli
THE M
lactose is absent the concentration of the
activator low, so BETA-GALACTO important feature of lactose metabolism ●
SIDASE
, AL-RAISALMILL M. ALAWI
POSITIVE CONTROL OF THE LAC OPERON ● Thomaz Steitz and colleagues - determined that CAP-cAMP TRYP
Positive control of the lac operon by a substance - sensing a lack of bends its target DNA by about 100 degress when it binds. Five g
glucose that reponds by activating the lac promoter ● Hen-Ming Wu and Donald Crothers - using electrophoresis synthe
concentration of a nucleotide (cyclic-AMP or cAMP) - rises as observed the DNA bend in the crystallography studies High o
concentration of glucose drops THE ARA OPERON Prese
CATABOLITE ACTIVATOR PROTEIN ● ara operon of E. coli - codes for enzymes required to metabolize NEGA
● cAMP added to E. coli - overcome the catabolite repression of lac the sugar arabinose , another catabolite- repressible operon ●
operon ● Two ara operators exists: ara01 & ara02
● Addition of cAMP - leads to activation of lac gene even with the ○ araC - regulated transcription of control gene of ara01 ●
presence of glucose ○ ara02 is located far upstream of the promoter it controls ●
● 2 parts of positive controller of the lac operon: (PBAD) between position 265 & 294
● 1. cAMP ● CAP-binding site - about 200 bp upstream yet can still stimulate ●
● 2. Protein factor such as: transcription
○ CAP or Catabolite Activator Protein ● AraC protein - another system of negative regulation ●
○ CRP or Cyclic-AMP Receptor Protein ARA OPERON REPRESSION LOOP MECH
○ crp - gene encoding this protein ● araO2 operator - controls transcription from a promoter 250 ●
○ Geoffrey Zubay and coworkers - showed that crude downstream ●
cell-free extract of E. coli would make B-galactosidase if it ● Robert Lobell and Robert Schleif found that if they inserted DNA
is supplied with cAMP fragments containing an integral number of double-helical turns ●
SIMULATION OF LAC OPERON (multiples of 10.5 bp) between the operator and the promoter, the
● CAP - cAMP complex - positively controls the activity of Beta- operator still functioned. However, if the inserts contained a
galactosidase nonintegral number of helical turns (e.g., 5 or 15 bp), the operator ●
○ CAP - binds cAMP tightly did not function
○ Mutant CAP - not bind cAMP tightly prepared ARA CONTROL PROTEIN DEFE
○ Compare activity and production of Beta - galactosidase ● araC - act as both positive and negative regulator 3 BINDING site: ●
using both ○ ara02 - far upstream site
○ complexes ○ ara01 - located between -106 & -144 ●
○ Low activity with mutant CAP – cAMP ○ aral is 2 half sites: aral1 - between -56 & -78 and aral2-
MECHANISM OF CAP ACTION -35 to -51 (each half site can bind to one monomer of AraC ●
● CAP-cAMP complex - binds to the lac promoter ARA CBAD OPERON
● Mutant whose lac gene is not stimulated by complex - had the ● ara operon - also known as araCBAD operon for its 4 genes:
mutation in the lac promoter ○ Three gene: asaB, A, and D - encodes the arabinose
● Mapping the DNA - showed that the activator-binding site lies just metabolizing enzymes
upstream of the promoter ○ transcribes rightward from the promoter araPBAD RIBOS
● Binding CAP and cAMP to the activation site- helps RNA ○ other gene, araC which encodes the control protein AraC ●
polymerase form an open promoter and transcribed leftward from the araPc promoter
RECRUITMENT AUTOREGULATION OF ARA C ●
● Formation of the closed promoter complex allows AraC to regulate its own synthesis
● Conversion of the closed promoter complex to the open promoter autoregulation - protein who controls its own synthesis
