Flashcards in Ex2- Regulation of Gene Expression Deck (80):
What is Gene Expression?
The process by which the expression of genes is turned on and off at different times and under different conditions.
True or false? Microorganisms are able to control how much of each building block it makes.
True or false? Since biosynthesis of building blocks involves a series of enzymic reactions, the cells either turn on or off the enzyme synthesis or enzymic activity.
What is the most common/ easiest way to regulate gene expression? Furthermore, how is this able to control gene expression?
The most common way to regulate gene expression is through Transcription. This occurs because transcription regulator proteins bind to DNA through Helix-turn-Helix motifs. This binding can act as either a positive or negative and in some instances even both.
How does the negative binding of the transcription regulator proteins occur?
The repressor protein binds to the operator and prevents the binding of RNA pol to the promoter and thus inhibiting protein synthesis.
How does the positive binding of the transcription regulator proteins occur?
The activator protein is required for the initiation byDNA pol onto the DNA strand by increasing its affinity to the promotor region and thus enhancing protein synthesis.
What is Posttranslational regulation?
Any regulation that occurs after the gene has already been transcribed into mRNA with the most common being translational regulation.
What is the significance of Joshua Elderberry's work?
-had recognized and isolated a large number of lac- mutants.
• he had mapped the lac gene - quite accurately as it turned out.
-Developed procedures to isolate β-galactosidase and to measure its activity in vitro.
• Synthesized a number of different β-galactosides
What type of inducers are ß-galactosides? Also which is the most common in
Melvin Cohn identified ß-galactosides as inducers of enzyme activity but were not substrates for ß-galactosidase also known as Gratuitous inducers.
-with the most common being isopropyl-thio-ß-Dgalactsode (IPTG)
Can Enzymes adapt?
No the discovery of gratuitous inducers killed off any notion of enzyme adaptation and lead to a new concept known as induction.
What are the functions and names of the 3 loci responsible for lactose metabolism in E.coli?
-LacZ which coded for the ß-galactosidase;cleaves the lactose molecule to form glucose
-LacY which coded for the lactose premease;allowing lactose into the cell
-LacA which is coding for the transacetylase; unkown
What is the function of the lacI protein and hoe does this occur?
The function of the lacI protein is to act as a repressor protein . IN the absence of lactose binds to the lacO sequence located near the promoter and thus prevents the binding of the RNA polymerase blocking the transcription of the structural lac genes.
What happens when E.coli is in the presence of lactose?
In the presence of lactose the inducer (allolactose) binds to the repressor thus resulting in a conformational change that is no longer able to bind to the operator, and allowing RNA polymerase to bind to the lacP and transcribe the lacZ, lacY, and lacA genes.
What is the difference between a non inducible and constitutive mutation of the lac operon?
Non-inducible- mutations that seem to abolish expression of the genes.
Constitutive mutations are those that cause increased expression of the genes.
What is the most important characteristic in regards to the repressor protein of E.coli?
The most important property of the repressor is its characteristic pleiotropic specificity of action. Since the repressor is both highly specific and mutations of the I gene do not affect any other system, and it is pleiotropic since both the galactosidase and acetylase are affected simultaneously and quantitatively to the same extent, by such mutations.
True or False: Once the existence of a specific repressor is considered as established, the existence of an operator element is also established.
True ince the repressor is established the problem is not whether an operator exists, but where and how it intervenes in the system of information transfer.
What does it mean to say that the repressor protein is pleiotropic in the lac system?
It means that at the mutations in the lacI affect 3 unrelated genes
True or False?
The operator, its specific complimentarily configuration must be genetically determined; therefore it could not be affected by mutations which would alter or abolish its specific affinity for the repressor, without necessarily impairing its activity as initiator of information-transfer.
FALSE any mutation to the operator will cause the affinity of the operator to change and could thus lead to impairment or overexertion, and in other instances a complete loss of the RNA polymerase ability to transcribe the genes.
How are you able to isolate the lacl-mutant phenotype in E.coli?
The mutant alleles can be isolated by mutagenizing an F' lac factor, transforming it into a lac deletion strain of E. coli, and then plating it on X-gal plates in the absence of any inducer. Once blue colonies appear it would inmate the expression of ß-galactosidase, and will display constitutive expression of the operon since there is no functional repressor present to block it.
How many bp in length is the LacZ gene and what does it code for in the lac operon?
The lacZ gene is 3072 bp in length and codes for ß-galactosidase the largest peptide formed by E.coli
How many bp in length is the LacY gene and what does it code for in the lac operon?
The lac Y gene is 1251bp in length and codes for the active form of the enzyme known as ß-galactsidase permease
How many bp in length is the LacA gene and what does it code for in the lac operon?
LacA is 609bp in length and codes for tiogalactoside transacetylase
How many regulatory genes does the lac operon contain and which gene codes for the repressor protein and lactose repressor?
The lac operon has only one regulatory gene: lacI which codes for the regulatory protein, the lactose repressor.
True or false: the lac operon contains two promoters although the second promoters function is not clear.
How is the lac operon regulated and how does this mechanism work?
The lac operon is regulated by catabolite repression. When the catabolite activator protein (CAP), and the effector protein cAMP bind to the CAP-binding site located 61.5bp ahead of the promoter this change allows the RNA polymerase bind more effectively and begin transcription.
True or False: Mutations in the lacZ or lacY genes results in a loss of both activities.
False mutations in the lacZ or LacY genes result in a loss of either (but not both) activities as both of these mutations are recessive.
True or False: A recessive mutation in a linked gene, lacI, resulted in high levels of the lacZ and lacY even in the absence of an inducer, this is said to be a regulatory mutation.
True this is said to be a regulatory mutation because the mutation governs the expression of the structural genes.
True or False: Dominant mutations in the LacI(lacI S) resulted in an induced phenotype for both the lacZ and LacY. which act in cis.
False the Dominant mutations in the lacI( = LacIS) resulted in a non-inducing phenotype for both lacZ and LacY. Which act in TRANS.
What are the two different kinds of elements present in the lac operon's regulation?
The two elements that are present in the lac operons regulation are DNA binding sites and Proteins.
DNA binding sites include the LacP LacO and Pi sites. While the Proteins involves include: CAP, LacI, LacZ,LacA, and LacY.
How are you able to distinguish between DNA binding sites and mutant proteins?
Through the use of Pseudo-Diploid Genetics we are able to distinguish between a trans acting site ( protein) or a cis acting site (DNA binding site)
What is a Trans-acting site?
A trans-acting site is a diffusible protein that is able to act on another DNA sequence from afar.
What is a Cis-acting site?
A cis acting site is a DNA sequence that is not able to act from on another DNA sequence without being near another DNA site to function.
What does the Cis and Trans test tell us?
The cis test tells us that if the inducer is present it will then bind to the operator thus repressing the lacZ gene when necessary.
The Trans test tells us that in the presence of the inducer it is not able to bind to the operator and thus is always ensuring that lacZ is present.
How are we able to tell if there is a Cis dominant mutation in the lac operator?
When there is a cis Dominant mutation in the cis operator there is constitutive (high levels regardless of cell needs) Expression of both LAcZ and LacY genes.
How are we able to tell if there is a Cis dominant mutation in the lac promoter?
Cis Dominant mutations of the promoter result in the lack of expression of both the lacZ and lacY genes.
How do we interpret the results of the cis and trans test?
When conducting the cis and trans test any pair of elements that passes both the cis and the trans must be acing in trans and is therefore a coding region for a protein. An thus any pair that passes only the cis test must be acting in cis and is therefore a DNA binding site.
How is Catabolite Repression Regulation achieved?
This regulation is achieved in absence of glucose, in which the Catabolite activator protein turns on the lac Genes. In the presence of glucose, the cell will shut off the ability to use any other carbon source as it is the proffered source.
What is the role of Adenylate cyclase?
Adenylate Cyclase is an enzyme which synthesizes Cyclic AMP from ATP.
How does high and low levels of glucose affect Adenylate cyclase?
In the presence of high glucose the adenylate cyclase enzyme is inhibited and thus producing low levels of cAMP.
In the absence of glucose Adenylate cyclase is not reserved and thus cyclic levels of cAMP are high.
What is the role of cAMP in the lac operon?
When high levels of cAMP are present it forms a complex with the CAP protein, this formation then binds upstream at the lac operon. This binding is required in order to allow RNA polymerase to bind to the lac promoter and thus turn on transcription.
True or False: In the absence of cAMP and CAP binding there is no or very little transcription of the lac operon, even in in the presence of lactose.
True or False: CAP binding bends DNA, this bending results in a less efficient RNA polymerase binding.
False. Yes, the CAP binding does bend the DNA but this bending allows for the RNA polymerase to bind more efficiently and thus enhances transcription of the lac operon.
What is the arabinose operon used for?
The arabinose operon is responsible for the cells' ability to use arabinose as a carbon source.
What system is required for the positive regulation of the arabinose catabolic operon?
for the regulatory control of the arabinose operon the cell uses the cAMP/CAP regulatory system.
True or False: The arabinose operon uses both positive and negative control.
What are the 3 structural genes required to utilize the sugar arabinose?
What is the regulator protein used for both positive and negative control of the arabinose operon?
In the presence of arabinose AraC turns on transcription of the area operon by binding to the araI initiator site. (positive)
In the absence of arabinose araC protein undergoes a conformational change which thus allows it to bind to both araI and araO, creating a loop that inhibits transcription. In addition the ara operon is also subject to catabolite repression.
How many binding sites does araC have and which are they and in what order do they bind in?
araC has 3 binding sites which are as follows in the preceding order araI, araO2, and araO1
What are the 3 major levels of gene regulation in the area operon?
1) Autogenous Regulation
Under what 2 conditions is the ara operon expressed?
1) in the presence of the substrate arabinose. and
2) When the preferred carbon source glucose is low.
What occurs during the Autogenous Regulation level of gene regulation phase of the ara operon?
Regardless of the presence or absence of the substrate arabinose
What occurs during the repression level of gene regulation of the ara operon?
When glucose is present little cAMP is available thus there is no CAP binding protein that attaches to the binding site. Regardless of the presence or absence of arabinose 2 araC proteins bind to araO2 and araI resulting in the formation of a DNA loop. This loop does not allow for the activation of the araBAD promoter or the attachment of the RNA promoter.
What occurs in the activation level of gene regulation of the area operon?
When glucose is absent, sufficient camp is able to be produced forming a complex with CAP, thus resulting in the alleviation of the DNA loop. Although not effective unless the sugar arabinose is present.
A) If arabinose is also present, the AraCarab protein activates the ParaBAD promoter and the ara operon is expressed.
B) If arabinose is absent, even though cAMP/CAP is bound to the CRP binding site, AraC alone can not activate expression of ParaBAD. Thus, the ara operon is not expressed.
What 3 ways can the cell control the gene expression of the TRP Operon?
1)The enzyme catalyzing the first step in the pathway is inhibited by Trp (feedback control).
2)In the presence of Trp, a repressor protein binds to an operator upstream of the Trp operon and shuts off transcription
3)Attenuation. There is a 160 base pair region in the Trp mRNA that causes transcription to terminate prematurely if Trp is present.
What are global regulatory mechanisms?
Global regulatory mechanisms are the regulatory systems that simultaneously regulate numerous operons in response to major changes to the environment.
What is a regulon?
A single regulatory protein that controls a large number of operons.
What are catabolites?
small molecules resulting from the breakdown of larger molecules
What is catabolite repression?
It the mechanisms used for ensuring that the cell will preferentially use the best carbon source available.
What is Catabolite-sensitive operons?
It is one of the largest global regulatory systems in bacteria that are subject to catabolite repression regulation.
What is CAP?
CAP is also known as the cyclic AMP repressor protein. It the protein responsible for the regulation of carbon utilization.
True or False: Only a single promoter can be controlled by a single cAMP-CAP complex.
False. IN some cases several promoters are all controlled by a single cAMP-CAP complex bound to DNA.
How does the CAP protein work?
When the cAMP-CAP complex forms this causes the molecule to have greater affinity to the binding site, thus binding to DNA. As it bind the DNA molecule this causes the molecule to bend at a 90º angle , this bending in the DNA allows for RNA polymerase to bind tightly to DNA and thus transcription is initiated..
Where in the CAP protein is the activating region that interacts with RNA polymerase and other transcription factors?
The portion of the CAP protein that is known as the activation region include amino acids 156, 158, 159, and 162 which are involved n protein-protein interactions.
How many classes of CAP-activated promoters are there?
There are 3 classes of CAP-activated promoters which include class 1-3.
What are the characteristics of a Class I Cap activated promoter?
- only require CAP for activation.
-The CAP binding site is located 61.5bp upstream from the promoter
What are the characteristics of a Class II Cap activated promoter?
-Only require CAP for activation
- In these promoters the CAP binding region overlaps the promoter and is thus centered at 41.5 bp
What are the characteristics of a Class III Cap activated promoter?
-require that additional regulator proteins as well as the CAP protein be present for activation.
-typically the CAP binding site is located 90bp upstream but may vary depending on the additional regulator proteins required
Regulation of the virulence genes in pathogenic bacteria gives the organism the ability to what?
Regulation of the virulence genes in pathogenic bacteria allow them to adapt to their most and cause disease in eukaryotic hosts. Mutations of these genes affect the growth of the bacteria in the host but not in the environment.
When does the c. Diphtheria know it has entered the host and which prophage is responsible for the production of the diphthteria toxin?
The C. diphtheria harbor the prophage ß, when entering the bloodstream of the host it is presented with low iron concentrations which changes the conformation of the DtxR repressor protein allowing the transcription of the toxin.
What are the components and function of the Diphtheria A-B toxin?
Sub-unit A: is an enzyme that adds ADP-ribose to a modify histidine the host elongation factor EF-2 which blocks translation resulting in cellular death
Sub-unit B: bind to specific receptors helping the sub-unit A to enter the cell
What is the heat shock response and how does it work?
The heat shock response quickly increases the Hsps (stress response proteins or chaperones) in the cell after an increase of extreme temperature, the presence of ethanol or other organic solvents, or when the DNA has been severely damaged in the cell. Present in both bacteria and eukaryotes.
What are the 2 major regulons that are present in E.coli?
The 2 major regions in E.coli are as follows:
-Major regulon: Sigma -32 regulon that consists of about 30 genes
- Minor Regulon: Sigma E regulon consists of about 3 genes
True or False: Hsp are not always present
False. Hap are always present at low concentrations.
What are chaperones?
Chaperones are Hsp proteins that direct the folding of newly transcribed proteins. During heat shock they help refold the proteins to their active conformation or they target them for destruction. EXP: DnaK, DnaJ, GroE, GrpE.
Which protein is the cellular thermometer and what does it do?
DnaK protein that senses changes in theperature and induces transcription of the heat shock genes.
Which Hsp proteins act as proteases that degrade heat denatured proteins before they can poison the cell?
Lon and Clp
What occurs when the temperature rises and the heat shock proteins are activated?
-the major regulon: sigma 32 (encoded by rpoH) rises causing the rate of transcription to increase, mRNA is more stable and thus more is translated , while DnaK is used to refold the proteins and does not affect Sigma 32.
As the temperature goes down what happens to the activated proteins ?
As the the temperature time decreases DnaK binds to the sigma 32 this makes sigma 32 more susceptible to protease when DNA is too far denatured.
What happens to the salt concentration when the cell has been heated for some time?
The cellular salt conditions adjust to the temperature and increase. So that no more proteins are denatured. Allowing DnaK to bind to sigma -32 and exit heat shock.