Topic 8: Regulation Flashcards
Bacterial Transcription
- Involves RNA polymerase holoenzyme = core enzyme and sigma factor
- Sigma factors direct RNA polymerase to promoters, located behind the start site of transcription
- Different sigma factors can direct the core RNA polymerase to different genes as needed
- Once the RNA polymerase binds to the promoter it unwinds the DNA and transcription occurs
Bacterial Termination
Transcription stop in two ways
Rho-dependent:
- rho protein follows the RNA polymerase and removes it from the DNA when it reaches a termination sequence
Rho-independent:
- RNA hairpin loop forms when RNA is being made and the strand folds back on itself and catches the RNA polymerase behind it
- It stalls the RNA polymerase on a weak point (A-T bonds are weak as they are H-H bonds)
- At this weak point of dissociation, the RNA polymerase falls off the template DNA strand
- The terminator loop stops RNA polymerase when it doesn’t need to transcribe the rest of the gene
Differentiate between regulation of protein activity via covalent modifications or allosteric regulation
Covalent modifications:
- May also alter enzyme conformations (strongly affects enzymes)
- Some covalent modifications increase enzyme activity and some decrease the activity of enzymes
- Examples: phosphorylation, acetylation, methylation, glycosylation
- Attaching these or removing these molecules changes the conformation of the active site
Allosteric regulation:
- Involves allosteric proteins/enzymes
- activity inhibition or activation from the binding of an allosteric effector molecule
- Binding of a non-substrate molecule at a site away from the active site
- Changes conformation: By making the substrate bind better or causing the substrate to not bind as efficiently
-Many enzymes use allosteric regulation mechanisms
Differentiate between positive and negative control of transcription by describing regulatory proteins, effector molecules, and DNA binding sites
(Negative)
Negative Control of Transcription:
- May involve specific repression or induction in response to conditions
- Both are considered “negative control”
Repression: inhibits transcription in response to a signal
– Minority of enzymes are controlled by repression
– Typically affects anabolic (biosynthetic) enzymes
Induction: derepression of enzyme production in response to a signal
– Typically affects catabolic enzymes
– Enzymes synthesized only when substrates available
– No wasted energy
Effector molecules:
– Co-repressors change shape of repressor protein so it can bind to its operator
– Co-inducers binds to the repressor protein, changing its shape so it falls off the operator
Differentiate between positive and negative control of transcription by describing regulatory proteins, effector molecules, and DNA binding sites
(Positive)
Positive Control of Transcription:
- Allosteric regulator proteins activate binding of RNA polymerase to DNA
– activator proteins bind specifically to activator binding site of the promoter - Maltose catabolism
– Maltose activator protein only binds DNA in the presence of maltose - Positively controlled promoters only weakly bind RNA polymerase
– Activator protein recruits polymerase to promoter
– May cause DNA structural change
– May interact directly with polymerase
Activator site may be close to the promoter or up to hundreds of base pairs away
Explain the lac operon and how it is regulated by positive and negative control of transcription
- Multiple control elements on both the DNA and accessory proteins
- Inducible expression
- System is only turned on when needed (cheaper/easier to use glucose)
- Components allow use of lactose sugar
Negative control:
- repressor protein (LacI) binds to operator, blocking RNA polymerase and inhibiting transcription
- effector molecule (allolactose) induces transcription by inhibiting binding of the repressor to the operator
Positive control:
- Activator protein (cyclic AMP receptor protein; CRP) binds and increases transcription rates when effector molecule (cAMP) present (low glucose)
- When glucose goes down, cyclic AMP levels goes up in the cell
- Effector molecule induces conformational change in activator protein, which increases affinity for binding site, increasing RNA polymerase affinity for the Lac operon promoter
Describe how the tryptophan operon transcription is under negative control of transcription and attenuation;
Negative control of transcription:
- effector molecules can also inhibit transcription by binding to the repressor protein and enhancing its ability to bind to the operator (common for anabolic operons)
Negative control of attenuation:
- High levels of tryptophan causes a terminator loop to forms and stop transcription
- Ribosome comes in behind RNA polymerase and starts to make a leader peptide
- Ribosome will move quickly through the peptide as there is a lots of tryptophan around
Ribosome occupies region 2, so it will not stick to region 3
- Region 3+4 will make a hairpin loop
** high levels of tryptophan -> terminator loops forms and stops transcription of structural genes, tryptophan not made
Attenuation
- Interruption of transcription after initiation but before termination
- Attenuation cannot occur in eukaryotes
Explain how quorum sensing involves regulation of transcription within Allivibrio fischeri
Positive control of transcription:
- when grown to high density, the cells produce lots of N-acyl-homoserine lactose (AHL), which stimulates luminescence
- LuxI protein catalyzes AHL synthesis
- luxR, a regulator transcriptional activator, interacts with AHL when it reaches a high enough concentration
- binds the “lux box” DNA regulatory site (activator binding site)
- leads to transcription of luciferase protein genes and luxI, which creates positive feedback loop forming more AHL
No Quorum
- No light produced
- luxR = regulatory protein/ activator
- AHL = co-activator
Quorum
- positive feedback
- Light produced
- AHL diffuses out and comes pouring back in. When they come back in they start to bind to the other site on the luxR activator protein and change its conformation
- The AHL is a coactivator
- Transcription is efficient because you have the activator binding site with the luxR protein that is associated with its co-activator
- Transcription of lux genes and luciferase will occur with the help of ATP and O2
Describe the two elements of a two-component regulatory system;
- can use one protein as a sensor and another to control transcription
systems often involve: - a sensor kinase (e.g. HPK): detects the environmental stimulus
- a response regulator (RR): regulates transcription
Steps:
1. An external signal molecule binds the input domain of the sensor
2. The transmitter domain becomes phosphorylated using ATP
3. The sensor transfers the phosphate group to the response regulator (signal transduction)
4. The phosphorylated response regulator interacts with the DNA of the target gene and causes RNA polymerase to increase or decrease transcription
5. The response regulators have phosphatase activity and can dephosphorylate so it is a short-lived/temporary response
list the steps of an A. tumefaciens plant infection as they relate to two-component regulatory systems;
- vir genes found on the Ti plasmid are only expressed under conditions similar to a plant wound site
- virA/virG are required for expression of the other virulence genes
Chemotaxis in the presence of attractant or repellant
- A complex bacterial behaviour modulated by shifts in protein activity
- Chemotactic bacteria sense changes in chemical gradients over time
- Changes induce altered direction and duration of flagellar rotation, leading to directed movement over time
- Response to a signal
⚬ MCPs sense specific attractants/repellents in the environment
⚬ the methyl-accepting chemotaxis proteins bind to different combinations of chemicals
⚬ they need CheA in association with them to do the phosphorylation (CheA is the sensor kinase)
⚬ Depending on the signal, signal transduction with phosphorylation coming from ATP occurs - Control of flagella rotation
⚬ When MCP is bound to a repellent or there is no attractant bound, phosphorylated CheA transfers the phosphate group to CheY
⚬ When CheY is phosphorylated it interacts with the flagella motor proteins and changes their directions which causes a tumble
⚬ CheZ is always looking to dephosphorylate CheY and cause runs and no more tumbles - Adaptation
⚬ when methyl groups are added or removed to MCPs the sensitivity is altered
⚬ CheR always adds methyl groups
⚬ CheB removes methyl groups
*Video
Regulon and use of SOS response system and sigma factors
Regulons:
- Sets of genes that are coordinated together, responding to the same regulatory systems
- catabolite repression: shutdown of several systems that utilize various nutrients when glucose is present
-SOS response system: multigene system for wide-scale DNA repair in response to serious DNA damage
– Do not turn on the SOS response unless the chances of dying are very high
– two of the most important regulatory proteins for the SOS response regulon are recA and lexA
– When things aren’t okay then DNA is single-stranded and bound by RecA
– when RecA binds to the DNA it cleaves lexA so it can’t bind to operators, you get a transcription of genes to fix the DNA
– Detect DNA damage and fix it quick by cleaving lexA
– This process produces a lot of lexA
– As quickly as it starts it shuts off
