Flashcards in Gene Expression Deck (52):
What are the major modes of regulation in the cell and how do they differ?
1) Post translational regulation-controls the activity of preexisting enzymes/proteins. Very rapid process (seconds).
2) Regulate level of transcription and regulate translation - controls the amount of a enzyme/protein(s). Slower process (minutes)
DNA binding proteins have specific domains and typically bind to specific regions of DNA. Describe the 3 most common domains and where most DNA binding proteins bind on the DNA.
1) Helix-turn-helix domain: 1st helix is the recognition helix, 2nd helix is the stabilizing helix. Many different DNA-binding proteins contain this.
2) Zinc Finger Domain: Protein structure that binds a zinc ion. Typically 2 or 3 zinc fingers on proteins that use them for DNA binding.
3) Leucine Zipper Domain: Leucine residues are spaced every 7 amino acids. Does not interact directly with DNA. Nothing to do with DNA binding protein-protein interaction domain.
What are bacterial promoters comprised of and what is the difference in sequence in promoters that are naturally "on" and those that are naturally "off"?
Bacterial promoters are comprised of 2 short DNA sequences separated by a defined number of bases. -10 and -35
-Promoter sequences are optimized for RNA polymerase holoenzyme recommitment
-Have good matches to consensus binding sites
-Weak similarities to consensus binding sites
-Requires binding of transcription factors
-Expression based on conditions - "inducible"
What is the difference between repression, induction, and activation?
Repression: prevents the synthesis of an enzyme in response to a signal. Negative control.
Induction: Removal of repression by inducers. Negative control.
Activation: ambidextrous and can, simultaneously, contact more than one target site on RNA polymerase. utilizing signals. Positive control.
What is a promoter?
A region of DNA that initiates transcription of a particular gene.
What does a protein do?
Binds to the promoter to initiate production of mRNA transcript
What is an operon?
Set of genes transcribed under control of an operator gene. A segment of DNA containing adjacent genes including structural genes, operator genes, and a regulon.
What are operators?
A segment of DNA to which a transcription factor binds to regulate gene expression by repressing it. Protein that does this is a repressor.
Are all promoters either highly or weakly expressed or is there a gradient of expression for most promoters?
Gradient. Minimal expression has multiple repressors, slow rates, mutations in -10 to -35, most will be here....Maximum expression is depressed and 1 or more strong activators expressed at high rates.
What is the difference between an operon and a regulon?
Regulon: term for when a transcription factor has multiple genes/operons controlled by the same regulatory element. They are genes that are controlled by the same transcription factor.
Operons: Groups of adjacent genes and are coregulated.
What are master regulators?
Transcription factors that influence the expression of many genes.
From DNA to protein, where is gene expression regulation found?
Transcription, post-transcription, and translation
How is transcription controlled?
Helix-turn-helix, zinc fingers, leucine zippers in DNA-binding proteins or by RNA-based methods
What is the difference between repression, induction, and activation?
Repression - negative control
Induction - relief of negative control
Activation - positive control
How is the lac operon of E. coli controlled?
Positively by CRP/cAMP
Negatively by lacI
Induced by lactose
What is meant by global regulation?
Regulatory steps that effect many downstream genes
How do two-component systems work?
Phosphorylation cascade between conserved histidine in the sensor kinase and conserved aspartate in response regulator
What is quorum sensing and how is it controlled?
Cell density-dependent signaling; through AHL's binding to transcription factors and regulating expression.
What are the types of RNA-based genetic regulation
-Antisense and ncRNAs
Explain catabolite repression and how it contributes to the lac operon transcriptional control?
It is an example of global control. If glucose is present, everything else is off. Lac operon is under control of catabolite repression. It ensures the "best" carbon and energy source is used first. It prefers carbon source of glucose.
What happens during the regulation of the lac operon if there is a negative amount of Glucose and a negative amount of lactose?
What do bacteria do in response to various environmental signals?
They adapt to changes in their surroundings by using regulatory proteins to turn groups of genes on and off in response to various environmental signals.
What is the lactose operon?
An operon is a cluster of bacterial genes along with an adjacent promoter that controls the transcription of those genes. When the genes in an operon are transcribed, a single mRNA is produced for all the genes in that operon. This mRNA is said to be polycistronic because it carries the information for more than one type of protein.
What is the lac Operator?
The operator is a short region of DNA that lies partially within the promoter and that interacts with a regulatory protein that controls the transcription of the operon.
Provide an analogy of a promoter and an operon.
A promoter is like a door knob, in that the promoters of many operons are similar. An operator is like the key hold in a door knob, in that each door is locked by only a specific key, or a specific regulatory protein which is the product of a regulatory gene.
What is the lac Regulatory gene?
The regulatory gene lacI produces an mRNA that produces a Lac repressor protein, which can bind to the operator of the lac operon. Note that these genes are not necessarily close to the operons they affect. These genes produce proteins and is considered a repressor because it keeps RNA polymerase from transcribing the structural genes. This the Lac repressor inhibits transcription of the lac operon.
Explain the Lac Repressor Protein.
In the absence of lactose, the Lac repressor binds to the operator and keeps RNA polymerase from transcribing the lac genes. It would be energetically wasteful if lac genes were expressed when lactose was not present. This is negative regulation.
What is the effect of Lactose on the lac Operon?
When lactose is present, the lac genes are expressed because allolactose binds to the Lac repressor proteins and keeps it from binding to the lac operator.
What is allolactose and why is it important?
It is an isomer of lactose. Small amounts of allolactose are formed when lactose enters E-coli, for example. It binds to the allosteric site on the repressor protein causing a conformational change. As a result of this change the repressor can no longer bind to the operator region and falls off. RNA polymerase can then bind to the promoter and transcribe the lac genes.
Describe the lac Inducer: allolactose.
IT is called an inducer because it turns on, or induces the expression of, the lac genes. The presence of lactose ( and thus allolactose) determines whether or not the LAc repressor is bound to the operator.
What occurs if lactose is present?
Lac repressor cannot bind to operator. Lac mRNA is produced.
What occurs if no lactose is present?
Lac repressor binds to operator. No lac mRNA is produced.
What does the feedback control of the lac operon do?
When the enzymes encoded by the lac operon are produced they break down lactose and allolactose, eventually releasing the repressor to stop additional synthesis of lac mRNA. mRNA breaks down after a relatively short period of time.
What are the energy source preferences of E-coli?
Whenever glucose is present, E. coli metabolizes it before using alternative energy sources such as lactose, arabinose, galactose, and maltose.
What happens when both glucose and lactose are present? If glucose is exhausted?
The genes for lactose metabolism are transcribed at low levels. When both glucose and lactose are present, the genes for lactose metabolism are transcribed to a small extent. If glucose becomes exhausted, the RNA polymerase will start to transcribe the lac genes efficiently, allowing E. coli to metabolize lactose. Maximal transcription of the lac operon occurs only when glucose is absent and lactose is present. The action of cyclic AMP and a catabolite activator protein produce this effect.
What does the presence or absence of glucose on the lac operon affect?
It affects the concentration of cyclic AMP. The concentration of cyclic AMP in E. coli is inversely proportional to the concentration of glucose as the concentration of glucose decreases, the concentration of cyclic AMP increases.
What is the effect of lactose in the absence of glucose o the lac Operon?
In the presence of lactose and absence of glucose, cyclic AMP (cAMP) joins with a catabolite activator proteins that binds to the lac promoter and facilitates the transcription of the lac Operon. When the concentration of glucose is low, cAMP accumulates in the cell. The binding of cAMP and the catabolite activator protein to the lac promoter increase transcription by enhancing the binding of RNA polymerase to the lac promoter.
Is transcription controlled in Archaea?
Reliant on a single RNA polymerase for all transcription, and many of the known regulatory mechanisms employed for archaeal transcription mimic strategies also employed for eukaryotic and bacterial species. It has repressors that block RNA polymerase or TBP/TFB binding and activators. that recruit TBP directly.
What are two component systems?
They are phosphate based and used to transmit signals from outside the cell to inside the cell. Bacteria does it with 2 bacteria, but Eukaryotes have >2 because they have many more signals to respond to at the multicellular level. Bacteria do not expand into as many cell types.
What are the 2 components of signal transduction in bacteria?
Sensor kinase (H) and response regulator (D).
What is an example of a two component system?
Chemotaxis, or movement along a chemical gradient. This Che system regulates the direction of turning for the swimming apparatuses, the flagella. CheY is in charge of determining the spinning of the flagella. Unbound CheY means it will not spin.
What are the 3 stages of signaling?
1. Response to signal.
What are other two component systems?
What is quorum sensing in bacteria?
It means to assess the population in a local environment. G+ and G- bacteria do this differently. Production of small QS molecule accumulates in the extracellular milieu. Signal receptors receive the signal and translate it into new gene expression - many genes are induced/repressed in response to QS (e.g. motility, biofilm formation, light production, virulence factor expression). It is threshold dependent.
What types of transcriptional control exist?
Transcriptional control is often dictated by intracellular and intercellular signaling.
Two component systems
The stringent response
What is heat shock signal transduction in bacteria?
It is largely controlled by alternative sigma factors. Heat shock proteins counteract damage of denatured proteins and help cells recover from temperature stress. It also occurs in Archaea. Part of Sigma 70 family, but not sigma 70 itself. Highly structured with lots of stem loops. When heat increases, a highly structured RNA will begin to become more linear and the RNA will breakdown its circular groups to linear.
What types of RNA based regulation exist?
1. Antisense RNA
How does RNA based regulation work?
Regulatory RNA molecules exert their effects by base pairing with mRNA or DNA and blocking translation and/or targeting for degradation.
How do antisense RNAs and srRNAs work
Antisense RNAs are encoded on the opposite strand of DNA matching identically to their target(s). Each antisense/ncRNA can regulate one or multiple mRNAs and may function with or without the protein partner Hfq.
They can function positively or negatively
Positively: binding to RNA and preventing secondary structure from forming or exposing ribosome binding site.
Negatively: base pairing with the ribosome binding site or internal fragments to prevent translation initiation or elongation respectively.
What are riboswitches?
RNA domains within an mRNA molecule that can bind small molecules to control translation of mRNA. They are located at the 5' end of mRNA. Binding results from folding of RNA into a 3-D structure. Found in some bacteria, fungi, and plants.
What is attenuation?
Transcriptional control that functions by premature termination of mRNA synthesis. Control exerted after the initiation of transcription, but before its completion. mRNA stem-loop structure and synthesis of leader peptide are determining factors in attenuation.