Lac Operon
Allolactose
Converted from lactose by B-gal; has an allosteric affect on LacI protein preventing it from binding to lacO, preventing repression
What are the structural genes in the Lac operon? and what do they code for?
LacY-codes for lactose permease, LacZ- codes for beta galactosidase, LacA-Galactoside transaceytlase
What is the order of the Lac genes?
LacZ-LacY-LacA
What is the function of transacetylase?
Likely protects the cell from toxic products created by action β-gal
What happens when glucose is present?
Low cAMP. which means that CAP cannot bind to the activator binding site. This means that RNA polymerase cannot transcribe. Lac operon off
what are the two functions of B-galactosidase?
-Breaking lactose into glucose, and galactose. Galactose is converted to glucose, and glucose is metabolized by constituitutively produced enzymes. -Converts lactose to allolactose.
What are the two mechanisms regulate expression of the trp operon?
-Repressor-operator interaction -Transcription termination
What mutations affect the gene expression of an operon?
-mutations in the lac operator, and mutations in the LacI
What is a superrepressor?
A LacI mutant. It produces no lac enzymes. Repressor cannot bind to alloctose, so genes are not expressed
What is a constitutive gene?
A gene that is transcribed at a relatively constant level regardless of the cell environmental conditions.
What does it mean for a gene to be repressible?
A repressible gene is a gene whose transcription and expression are stopped when expression is no longer needed.
What is a repressor?
A repressor is a regulator molecule responsible for negative regulation of a gene.
What is inducer for the lac operon?
Allolactose
What happens in the presence of lactose?
Allolactose binds the repressor protein and transcription is initiated.
What is an activator?
An activator is a regulator molecule responsible for positive regulation of a gene.
The lac operon repressor protein can be described as what kind of protein?
An allosteric protein.
What is an inducer?
An inducer is a small molecule that joins with a regulatory protein to control transcription of the operon
What does the gene for LacZ encode for?
B-galactosidase
What does LacO do?
Binding site for repressor
What does the LacI product do?
Binds the operator
What is the nonsense mutations
Change of a codon into a stop codon. This affects the translation of downstream genes
Lactose is a ___________.
Disaccharide.
What happens when glucose is present?
It reduces cAMP levels leading to slow transcription of the lac gene
What happens when tryptophan is present in a trp operon?
It will bind to an aporerpressor protein. The active repssor binds the trp operator, and prevents transcription initiation. Repression reduces transcription of the trp operon about 70 fold
What scientists first figured out the Lac Operon? What else are they known for?
Jacob and Monod. They also first postulated the existence of mRNA.
What does mutation of LacO genes produce?
Lac Z genes were working but lac permease was inactive
Give an example of an inducible gene
Lac operon
What is the chemical break down of lactose?
Lactose----> Glucose + Galactose
When lactose is the sole carbon source, what occurs?
Levels of the three enzymes increase coordinately about 1,000 fold. -allolactose is the inducer molecule -The mRNA for the enzyme has a short half-life. When lactose is gone, Lac transcription stops
What is missense mutation and how does it affect?
Missense mutation is the change of a base pair, it affects the product
The lac operon is under what kind of regulatory conrol?
Negative (but is also inducible)
Describe negative regulation.
Negative regulation occurs when the regulatory molecule bind to the DNA and prevents the RNA polymerase from binding, thereby blocking transcription.
How does positive control work in lac operons?
No glucose present, only lactose. -Catabolite activator protein(CAP) binds cyclic AMP(cAMP) -CAP-cAMP complex is binds to CAP-site. -Binding CAP-cAMP complex recruits RNA polymerase to the promoter leading to transcription
LacI binding affinity with operators (O1, O2, O3)
O1>O2>O3, positive correlation with strength of repression
When are inducible genes expressed?
Only in the absense of a repressor and/or presence of an inducer
What does the repressor protein bind?
Operator
Within the leader mRNA are four regions that can form secondary structures by complementary base-pairing. Name the pairs, and what they can do.
Pair of 1 and 2 create a transcription pause signal pairing of sequence 3 and 4 causes termination signal pairing of 2 and 3 causes an antitermination signal, and transcription continues
What does the gene for Lac Y produce?
Permease
What is encoded by the lacY gene?
Permease
What is the permease function?
Permease transports lactose into the bacterium.
Describe positive regulation.
Positive regulation occurs when a regulatory molecule activates transcription. It binds the DNA and helps RNA polymerase bind to the promoter.
What does lac I do?
Produce repressors
What constitutes the regulatory region of the lac operon?
Promoter & Operator.
Where does the regulatory protein bind?
Promoter and operator
What is the function of RNA polymerase?
RNA pol binds the promoter to begin transcription.
If the repressor protein binds the operator, ________________________
RNA polymerase cannot initiate transcription.
Regulation of gene expression permits what?
Regulation of gene expression improves efficiency.
What are the products of the LacI?
Repressor protien
Which transcription regulators are trans-acting?
Repressor, Activator, TF
What is the molecule responsible for repressing gene expression of repressible genes?
Repressor. The repressor is often the end product of the biosynthetic pathway controled by the enzymes expressed by the gene.
What are regulatory elements?
Segments of DNA involved in regulation of structural gene transcription.
Give an example of a constitutive gene.
The lacI gene is constitutive. (It is the structural segment of the lac operon that is inducible)
What role do metabolites play in gene expression?
The presence or absence of metabolites can turn a gene on or off.
What is the operator region?
The region between the promoter and structural genes to which the repressor protein binds. It enables control of structural gene expression.
The repressor protein has ______ binding sites, the _____________ and ___________.
The repressor protein has 2 binding sites, the operator region and allolactose region.
What defines the behavior of regulation molecules?
Their binding.
What is so great about Monosaccharides?
They are useable; they are faster to metabolise than disaccharides.
How do prokaryotes regulate multiple genes in a Metabolic pathway?
They group the genes on one operon. These are a defined group of genes on the control of one promoter. This produces polycistronic mRNA.
In the presence of a glucose medium what level of lac gene products are produced?
Very low
Cooperative binding
When 2 DNA-binding proteins can bind to one another as well, it effectively increases their affinity for their binding sites
lacI protein
a homodimer, uses a helix-turn-helix structural motif to bind to DNA, so consensus binding sequence is palindrome
cAMP
a second messenger. When glucose is present the concentration of cAMP in the cell is low.
In presence of lactose and absence of glucose...
activated level of transcription (CAP interacts with RNApol)
What is the Lac Operon?
an operon that is required for the transport and metabolism of lactose. Consits of three adjacent structural genes. transcription of this operon does not occur in the presence of glucose.
In the absence of lactose, the repressor protein ________
binds the operator.
Lac Repressor protein
binds to the operator site and blocks transcription
Transcription of the lac operon is controlled by how many proteins, what kind of proteins, what are the proteins?
controlled by 2 regulatory proteins catabolite activator protein(cap) and the lac repressor protein
Explain allolactose
converted from lactose via beta-galacosidase. Binds to repressor causing a conformational change in the repressor. INDUCER
Core promoter and Sigma 32
core promoter contains functional binding sequences for both sigma-70 and -32 so rRNA expression continues normally during heat shock
Sigma factor cycle
cyclical addition and release of sigma factor from the RNA pol holoenzyme during each round of transcription
What happens to the polycistronic mRNA? What does this allow the bacteria to do?
is translated to produce beta-galactosidase, lactose permease, and Galactoside transaceytlase proteins. Allows the bacterium to transport lactose into the cell and metabolize it.
lacO
lac Operator, area where lacI protein (lac repressor, separate constitutively active promotor) can bind, blocking RNA polymerase from binding
What are the structural genes of the lac operon?
lacZ - beta-galactosidase (necessary to turn lactose into monosaccharides) lacY - lactose permease (necessary to get lactose into the cell) lacA - thiogalactoside transacetylase (not necessary for lactose metabolism)
The lac operon is involved in the metabolism of ___________.
lactose
Glucose and galactose are both______________
monosaccharides
Explain what happens when both glucose and lactose are present
not enough cAMP to bind to CAP. therefore cannot bind to the activator binding site. Without the activator RNA Polymerase can not transcribe. At first Lac operon is off. Once all glucose is used then Lac operon is on
When glucose and lactose both present at high concentrations...
operon undergoes basal transcription
In presence of glucose and absence of lactose...
repressed, no transcription
"Housekeeping" genes sigma factor versus Heat shock sigma factor
sigma-70; sigma-32. Compete with each other to bind to a free polymerase, which redirects transcription to different sets of genes
What happens if there is no lactose in the medium?
the lac repressor binds to the operator site blocking transcription. Lac operon off
What happens when there is no lactose in the cell?
the lac repressor protein is active and binds to the lac operator site. Blocking RNA polymerase from transcription.
CAP
to bind to the activator binding site and facilitate transcription of lactose CAP has to first attach to cyclic AMP (cAMP)
What happens when there is lactose in the cell?
when lactose enters the cell a small amount of it is converted into allolactose which binds to repressor causing a conformational change in the repressor and creating an inactive lac repressor. Thus preventing it from binding to the operator site. cAMP is present and able to bind to CAP which can bind to the binding site. Allowing RNA polymerase to bind to promoter and transcribes a polycistronic mRNA containing the Lacz, Lacy, and Laca genes.
When lactose is present, ezymes are required to metabolize it in order to produce allolactose. How does the cell get around this problem when the lac operon is repressed in the absence of lactose?
β-gal is made even in the absence of lactose because this enzyme is needed in case lactose becomes available. Transcription for the lac operon is not 100% inactive in the absence of lactose; enough transcription occurs to counter this conundrum.
What converts lactose to allolactose?
β-galactosidase
Thiogalactoside Transacetylase
•LacA gene •not involved in lactose metabolism per se, but detoxifies certain other sugars that can also enter cell via permease
Lac operon operator sequences
•O1, O2, O3; less than 500 bps apart from each other •O3 upstream of O1, O2 downstream •DNA bends when they bind forming a tetramer with lacI dimer which further stabilizes the lacI:DNA complex
cAMP
•a chemical derivative of ATP •concentration is inversely proportional to amount of glucose •2 molecules of cAMP bind to a CAP dimer, causing an alosteric change in conformation, positioning the 2 recognition helices so they can fit into the major grooves of a DNA helix
CAP
•catabolite activator protein •binds to lacO and cooperatively binds to CTD of RNA pol which helps to (i) recruit polymerase to the core promoter and (ii) hold polymerase on the promoter enough to initiate transcription •binding regulated by cAMP
ara operon
•encodes gene products needed for metabolism of sugar arabinose •unlike lac operon, induces ara transcription by binding to an activator protein, then allosteric change allowing binding adjacent to promoter recruiting RNA pol •presence of glucose represses ara transcription by inhibiting CAP activator protein
gal operon
•encodes gene products needed for metabolism of the sugar galactose •galactose induces gal operon transcription by binding to and inactivating a repressor protein •presence of glucose represses gal transcription by inhibiting the CAP activator protein
Sigma-32 and RBS
•heat shock temperatures melt the secondary structure of the mRNA which exposes the RBS usually blocked, increasing rate of translation
Lactose permease
•lacY gene •membrane transport protein essential for lactose to enter cytoplasm from extracellular fluid
Beta-Galactosidase
•lacZ gene •enzyme that cleaves lactose into glucose and galactose, and a tiny fraction into its isomer allolactose
Heat Shock
•proteins lose 3-D conformation at high temps, become non-functional; "denaturation" •E. Coli- temps >42 C induces a 15-fold increase in expression of "heat-shock" proteins, or molecular chaperones that help to fold both newly translated and misfolded proteins •sigma-32 protein rapidly increased in concentration during heat shock by postranscriptional regulation (translated more efficiently, degraded less rapidly)
What is the repressor gene of the lac operon?
LacI
How many regulatory elements does the lac operon have?
2
Even when all 3 operators are engaged by lacI protein dimers, there is still a very low level of expression from lac promoter that is essential because....
Allolactose, not lactose, is the chemical inducer of lac operon expression; its formation requires B-gal and lactose permease so they wouldn't be formed without some low level of expression
What is β-galactosidase?
Galactosides are a particular group of organic molecules. β-galactosidase is encoded by the lacZ gene. It is an enzyme that cleaves lactose in to monosacharides and converts lactose to allolacose.
How may gene expression be regulated?
Gene expression may be under positive or negative control.
Explain what happens when neither glucose or lactose are present
High concentration of cAMP and CAP is bound to the activator binding site. RNA polymerase can bind to the promoter but is blocked by the repressor (because there is no lactose to bind and alter shape) no transcription
What does it mean for a gene to be inducible?
Inducible genes are expressed only for specific conditions; they must be turned on. They often encode enzymes that catabolize a molecule.
Why is the lac operon important?
It allows for the digestion of lactose (if present) as a growth substrate if there is none of the preferred energy source glucose
What is lactose permease(M protein) used for?
It is required for transport of lactose across the cytoplasmic membrane
Give a general description of the lac operon.
The Lac Operon has structural genes, with an upstream regulatory region consisting of an operator (lacO) and a promoter (lacP)
What is the lacI gene?
The LacI gene encodes the repressor protein, which is a negative regulator of the transcription of the three structural lac genes.
Where does lac operons usually work?
The regulatory event typically occurs at a specific DNA sequence
What happens when Allolactose binds the repressor protein?
This binding causes a conformational change. The change alters the operator binding site and the repressor protein falls off the operator. RNA pol can now transcribe structural genes.
What molecule is responsible for turning on a gene that is currently off?
This molecule is called the inducer; it induces transcription of the inducible gene, activating gene expression.
What is encoded by the lacA gene?
Transacetylase
What does the gene for Lac A produce?
Transacetylase, no known
What does a mutation in the LacI gene do?
change the repressor protein's conformation, and prevent it from binding to the operator, results in expression of the operon