Genetics Chpt 16
Name six different levels at which gene expression might be controlled
(1) Alteration or modification of the gene structure at the DNA level (2) Transcriptional regulation (3) Regulation at the level of mRNA processing (4) Regulation of mRNA stability (5) Regulation of translation (6) Regulation by posttranslational modification of the synthesized protein
What is the lac operon and how does it control the metabolism of lactose?
-3 genes, lacZ, lacY and lacA -lacZ gene codes for the enzyme β- galactosidase, breaks the disaccharide lactose into galactose and glucose, and converts lactose into allolactose -lacY gene codes for lactose permease, necessary for the passage of lactose through the E. coli cell membrane -lacA gene codes for the enzyme thiogalactoside transacetylase whose function in lactose metabolism has not yet been determined. -lacI gene codes for the repressor, which binds at the operator region and inhibits transcription of the lac operon by preventing RNA polymerase from successfully initiating transcription.
Some mutations in the trp 5' UTR region increase termination by the attenuator. Where might these mutations occur and how might they affect the attenuator?
-Any mutations that disrupt the formation of the antiterminator (2&3) would increase termination. -Such as a deletion in region 2 or 3 -Any mutation that doesn't allow the ribosome to migrate on the trpOperon -Or prevents ribosome from stalling, by eliminating the tryptophan codons that come before region 1
How catabolite repression allow a bacterial cell to use glucose instead of other sugars?
-Because glucose represses the transcription of other sugars, only enzymes involved in the metabolism of glucose will be used. -Operons that have catabolic repression are under the positive control of CAP. -CAP forms a complex with cAMP. -Glucose decreases the levels of cAMP, so CAP isn't activated
Mutation prevents CAP from binding to the promoter in the lac operon. Whats the effect on transcription?
-CAP binding to the promoter stimulates RNA poly to bind to the promoter. This increases transcription levels. -RNA poly will bind poorly if CAP is absent, causing low levels of transcription
Lactose present Glucose present Condition 1 Yes No Condition 2 No Yes Condition 3 Yes Yes Condition 4 No No Which would the lac operon produce the greatest amount of β-galactosidase? The least?
-Condition 1: lac operon requires the presence of lactose and the absence of glucose for the maximum amount of B to be made. Lactose (allolactose) binds to the lac repressor, making it not want to bind to the promoter. It steals it away (defender), leaving it open for RNA poly to jump in and bind at the operator. Without glucose, lots of cAMP is synthesized and complexes with CAP. This complex is like an energy drink for RNA poly and it makes it transcribe really fast. -Condition 2: least amount would be made. No lactose to keep the repressor away so it binds to the promoter. Transcription is inhibited by the repressor.
What would be the effect of a drug that altered the structure of allolactose so that it was unable to bind to the regulator protein?
-If allolactose can't bind to the regulator protein, then it can't defend from the lac repressor. So transcription would be repressed.
What is catabolite repression?
-Its when the presence of glucose represses the transcription of genes involved in the metabolism of sugars
An operon, blob operon, produces enzymes that convert compound A into compound B. Its controlled by a regulatory gene S. Enzymes are only synthesized in the absence of B. If S is mutated and enzymes are synthesized in the presence and absence of B.... Does S produce a repressor or activator? Is it inducible or repressible?
-Most likely codes for a repressor since enzymes are synthesized in the absence of B -The blob operon is probably a repressible because when B is there, enzymes aren't synthesized (it is repressed) and when B is gone they can be synthesized
Positive control in a repressible operon
-Positive control needs an activator to turn it on -Repressible operons need to be repressed, its already on so it needs an activator and then if the activator is active, its good *Active activator
What is the difference between positive and negative control?
-Positive transcriptional control. Requires an activator protein to stimulate transcription. -Negative transcriptional control. Requires a repressor protein to inhibit transcription. -An inducible operon isn't normally transcribed, it must be turned on (induced) by an inducer molecule. This molecule either stimulates activator protein in a positive operon or stimulates repressor protein in a negative operon -A repressible operon is normally transcribed, it must be turned off (inhibited) by the repressor becoming active in a negative operon or the activator being turned off in a positive operon
A mutant strain of E. coli produces β-galactosidase in the presence and the absence of lactose. Where in the operon might the mutation in this strain occur?
-Probably occurs in the operator. -It may cause the lac repressor unable to bind to the operator so transcription is NEVER inhibited -Or the lac repressor itself was mutated so it can't bind to the operator
What are riboswitches? How do they control gene expression?
-Regulatory sequences in RNA molecules -Regulatory molecules bind at the riboswitches and may result in repression or induction by affecting the formation of secondary structures -Some binding results in termination prematurely of the mRNA molecules, preventing translation initiation
What is antisense RNA? How does it control gene expression?
-Small RNA molecules that are complementary to other DNA or RNA sequences -Form RNA protein complexes -Control gene expression by binding to the 5' UTR region of an mRNA molecules and blocking the attachment of the ribosome, stopping translation -Or they cleave mRNA to stop translation
What is attenuation?
-The termination of transcription prior to the structural genes of an operon -Its the result of the formation of a termination hairpin structure or attenuator in mRNA
Regulatory genes
Encode proteins that regulate transcription/translation
Structural genes
Encode proteins used in cell metabolism or structure
Why is gene regulation important in bacterial cells?
Gene regulation allows for biochemical and internal flexibility while maintaining energy efficiency by the bacterial cells
Deletions in region 2 of the mRNA 5' UTR, would result in what?
If 2 is deleted, then 2 and 3 can't bind and so attenuation will occur
A mutation at the operator site prevents the regulator protein from binding. What happens if the: - Regulator protein is a repressor in a repressible operon - Regulator protein is a repressor in an inducible operon
If the protein is a repressor in a repressible operon, the operon is always on. If the repressor was able to bind then there will be no transcription. BUT this mutation causes it to be unable to bind. So transcription occurs all the time. If the protein is a repressor in an inducible operon, then that protein is normally bound to the operon, blocking transcription, forcing it to be induced. If that protein isn't present, then transcription would always occur.
A mutation that prevents the binding of the ribosome to the 5′ end of the mRNA 5′ UTR would result in what?
If the ribosome doesn't bind, then region 1 is free to bind with region 2, and region 3 will bind to region 4. This creates a termination in transcription (attenuation) because it forms a hairpin structure with a string of U bases following it. Normally, a hairpin structure is formed but there is only pairing between regions 2 and 3. So the U string that follows 4 isn't directly attached to the hairpin structure and termination does not occur.
In Streptococcus pneumoniae the ability to carry out transformation requires from 105-124 genes (com regulon). This is activated in response to CSP. When enough CSP accumulates, it attaches to a receptor protein that stimulates the transcription of genes within the com regulon and ultimately results in transformation. Is it: -Negative inducible -Negative repressible -Positive inducible -Positive repressible
It is Positive Inducible. Increased levels induce transcription. CSP stimulates the activity of the receptor, doesn't inhibit it. If it was negative inducible, CSP would be repressing the receptor activity. If it was negative repressible, the expression would be produced until it was expressed. If it was positive repressible, the expression would be produced until it was expressed.
Negative control in an inducible operon (negative inducible operon)
Negative = repressor, inducible = must be made active *Active repressor -Binds to the operator -Blocks the binding of RNA poly, prevents transcription -For transcription to occur, something must prevent the repressor from binding
Negative control in a repressible operon
Negative = repressor, repressible = transcription always happens *Inactive repressor (not actively repressing) -Transcription occurs normally, must be turned off -Repressor here is inactive since transcription is occurring -Binding of substrate makes the repressor active, it represses, turns of transcription
Positive control in an inducible operon
Positive = activator, inducible = must be made active *Inactive activator (activator, has to be inactive so it can be induced) -Transcription is normally off -Inactive activator doesn't normally bind because it's inactive -A substrate makes it active, binds and ribosome binds to induce transcription
Deletions in region 3 or 4 of the mRNA 5' UTR, would result in what?
Region 3 wouldn't be able to pair with region 4 causing the attenuation factor to be created, so transcription would continue
Regulatory elements
Sequences that aren't transcribed, but are involved in regulation of other sequences
Which strand of DNA (upper or lower) is the template strand?
The bottom strand It is 3' to 5' and the template strand is read left to right, 3' to 5'.
A mutation that creates a stop codon early in region 1 of the mRNA 5' UTR, would result in what?
The ribosome would stop, or fall off, then the first two would bind and the second two would bind and attenuation would occur because 3 and 4 form the attenuator
Deletion of the string of adenine nucleotides that follows region 4 in the 5' UTR, would result in what?
The string of adenine nucleotides are REQUIRED for the attenuation hairpin to function as a terminator. So if they are deleted transcription would continue
A mutation that changes the tryptophan codons in region 1 of the mRNA 5'UTR into codons for alanine, would result in what?
The tryptophan codons are needed in region 1, because this makes the ribosome pause so 2 and 3 can bind. If they now code for alanine, the ribosome wouldn't pause, 1 & 2 would bind and 3 & 4 would bind causing attenuation
At which level of gene regulation does attenuation occur?
Transcription
