Regulation of Gene Expression Flashcards
What is the lac operon?
Operon - A cluster of genes transcribed from a single promoter
The lac operon of E.coli contains genes involved in lactose metabolism only expressed when lactose is present and glucose is absent
E.coli first uses glucose and then when it is depleted lactose is used
This allows bacteria to adapt to adapt to its environment and not be debilitated by synthesising unnecessary enzymes
What is the composition of the lac operon?
The lac operon is composed of 3 genes; lacZ, Y and A:
lacZ = b-galactosidase
Cytoplasmic enzyme that breaks lactose into glucose + galactose
lacY = lactose permease
An integral membrane protein that transports lactose across cytoplasmic membrane
lacA = Transacetylase
May acetylate galactoside sugars (other than lactose), preventing them becoming substrates for β galactosidase
Also contains a promotor, operator and terminator - that are considered part of the transcriptional unit
What is significant about the 3 genes in the lac operon?
All three genes are co-transcribed to make a single mRNA
However, each gene is independently translated into protein from the same mRNA
They each have a different ribosome binding site
This type of mRNA is called a poly-cistronic or polygenic
Ensures each gene is expressed
What controls the elements and genes of the lac operon?
An adjacent gene LacI
This gene encodes a repressor that targets the lac operon’s operator
It has its own promotor and terminator = not part of the lac operon
It lies near the beginning of the LacZ gene
In the absence of inducer, lac repressor specifically binds to the operator to prevent the transcription of mRNA (it finds the operator by sliding along the DNA)
It can bind to 1/2 of 3 operators forming a loop in the DNA (CAP stabilises this loop)
When an inducer binds, the repressor dissociates from the operator, allowing transcription/translation of the lac enzymes
What are some structural features of the lac repressor?
- An N-terminal “headpiece” (containing a helix-turn-helix motif) that binds to DNA operators
- A linker - allows the headpiece to move freely
- Two domain core - binds inducers e.g. IPTG
- C-terminal a helix, needed for the quaternary structure
What are some inducers that bind to lac repressors?
Natural inducer - 1,6-allolactose an isomer of lactose
Synthetic inducer - isopropylthiogalactoside (IPTG), this resembles allolactose by isn’t degraded by b-galactosidease
They cause the repressor dimer to separate - it can no longer bind to the DNA = dissociation
What are the two control mechanisms of the lac operon?
Negative control - In the presence of glucose (repressor in use)
Positive control - In the absence of glucose (inducers and cAMP-CAP)
What is catabolic repression?
When there are large amounts of glucose (as E coli’s metabolic fuel choice)
Other catabolites aren’t fully expressed e.g. lactose, arabinose and galactose
It prevents wasteful duplication of energy-producing enzyme systems
Describe positive control of the catabolite-repressed operons?
In the absence of glucose
A decrease in glucose = increase in cAMP
Increased cAMP leads to relieved pressure on catabolic repression of lac operon
cAMP binds to CAP (catabolic activator protein)
cAMP-CAP binds upstream of the promotor in the major groove of DNA - forming a 90° bend of the DNA around the protein dimer
In the presence of cAMP-CAP RNA polymerase will initiate transcription more efficiently - as it increases the affinity of the RNA pol for the promotor
What is a regulon?
Regulon - all genes/operons that are regulated (whether positively or negatively) by the same regulatory protein
A change in a regulatory protein allows the co-ordinated regulation of multiple genes/operons, at different locations, in response to a common environmental signal
What is the trp operon in E coli?
5 genes encoding 3 enzymes of the shikimate pathway converting chorismite (an acid) to tryptophan
Has a single promotor, operator and terminator
The trp operon is controlled by the trpR - trp repressor
This binds trpL to form a complex that binds to the operator, reducing the rate of transcription
Tryptophan is a corepressor not an inducer
High levels of trp = trp bound to trpR to activate it
What is the second control mechanism of trp operon?
Other than the repressor mechanism the trp operon is controlled by attenuation
Attenuation regulates transcription termination - preventing the completion of transcription
The section between the operator and the first gene contains a leader peptide coding region and an attenuator sequence
The attenuator can form various hairpin structures
What is the outcome of the attenutation mechanism?
The 14 residue polypeptide leader sequence contains 2 trp residues
Abundance of trp = the ribosome doesn’t wait long for a trp-tRNA
A terminator (3.4) hairpin (AKA rho-independent transcriptional terminator)
is formed in the attenuation sequence
Making RNA polymerase detach and ending transcription
Low levels of trp = the ribosome will pause
The pausing causes an antitermination (2.3) hairpin to form in the attenuation sequence
This hairpin prevents formation of the terminator (3.4) and allows transcription to continue
What are riboswitches?
Metabolite-sensing RNAs
TPP (a coenzyme) has a riboswitch meaning depending on presence TPP it can bind to RNA
This is detected, as RNA changes its secondary structure upon binding
TPP masks the Shine-Dalgarno sequence (needed to initiate translation)
What is an example of a riboswitch target of antibiotics?
Pyrithiamine (antibiotic)
This is converted by the bacterial cell into pyrithiamine pyrophosphate - a TPP analog
This can’t function as a coezyme and therefore kills the cell
What are some factors of eukaryotic gene expression?
Chromatin structure (all in the chromatin section)
Transcriptional activators
PTM control mechanisms
Antibody diversity
What are some transcriptional activators in eukaryotes?
Enhancers are often Cis
Cis-acting regulatory elements: They are located on the same DNA molcule as the genes they control
Non-coding regions of DNA that regulate the expression of localised genes
Transcription factors are trans
Trans-acting factor: They encode diffusible factors that regulate the expression of genes at a distance - may be on different chromosomes from those that encode them
How can transcription factors stimulate transcription?
Some TF can cooperatively assemble with architectural proteins on an enhancer to form an enhanceosome
Enhanceosomes function as on/off switches, containing coacticators and corepressors that interact with proteins not DNA
TF contain a DNA-binding site and an activation domain
As each TF binds it increases initiation rate by factor 10 each time - they work together (synergise) to increase the rate
What is squelching?
Unbound transcription factors compete with DNA-bound transcription factors for their target sites
This reduces the rate at which the PIC is recruited to the associated promoter
This is why in the nucleus TF are bound to inhibitors unless actively engaged in transcriptional initiation
What is needed as well as transcriptional activators in initiation of eukaryotic transcription?
Mediators - they link transcriptional activators and RNAP II
It is a coactivator that binds to the CTD of RNAP II
It acts as an adaptor that bridges the DNA-bound transcriptional activators and RNAP II
= formation of stable PIC
What are enhancers?
Prevent interference by regulatory elements in neighbouring genes
They block enhancers from interacting with the promotor
Another type of insulator blocks the spreading of heterochromatin
How are transcription factors activated?
Signalled - e.g. by phosphorylation
Modification/ligand binding
Released by an inhibitor/repressor
Give an example of activation of a transcription factor?
JAK-STAT pathway
STAT - signal transducers and activators of transcription
- ligand binding induces cytokine receptor dimerisation
- The JAKs phosphorylate each other and associated receptors
- STATs bind and are phosphorylated
- Phosphorylated STATs dissociate and dimerise
- The STAT dimer moves to the nucleus where it functions as a TF
How are nuclear receptors activated?
Nuclear receptors are activated by hormones e.g. steroids and thyroid hormones
Nuclear receptors activate distinct but overlapping sets of genes
What are some eukaryotic posttranscriptional control mechanisms?
mRNA degredation
RNA interference
Roles of IncRNAs
Controlling translation initiation
Eukaryotic PTM control - describe mRNA degredation?
mRNA is degraded at different rates
Deadenylases - catalyses the removal of the poly(A) tail, so it can no longer interact with the PABP
Decapping enzyme - hydrolytically excises the m7G 5’ cap
mRNA can now be degraded by exonucleases - Xrn1
Eukaryotic PTM control - what is RNA interference?
RNA interference (RNAi) is RNA that interferes with gene expression They can bind and down-regulate gene expression
siRNA - small interference RNA
miRNA - microRNA
Eukaryotic PTM control - describe a mechanism of RNAi with siRNA?
- Trigger dsRNA is cleaved by Dicer (an RNase) into siRNA
- RNA-induced silencing complex (RISC) binds to the siRNA and separates its strands
- The siRNA binds to a complementary mRNA
- RISC (component called argonaute) cleaves the mRNA so it can’t be translated
Eukaryotic PTM control - describe a mechanism of RNAi with miRNA?
- Pri-miRNA is turned into Pre-miRNA using a microprocessor complex in the nucleus
- Pre-miRNA is cleaved by a Dicer in the cytosol to form miRNAs
- RISC binds to the passenger RNA to form guide RNA-RISC complex
- This can then bind to mRNA - silencing it and can lead to degradation
Eukaryotic PTM control - what are the roles of IncRNAs?
Long non-coding RNAs
They have many different roles in gene expression and effecting epigenetics
Examples:
Regulating the epigenetic differentiation of the skin
Regulate transcription by binding to the general transcription factors
Some bind to mRNAs to influence pre-mRNA
Eukaryotic PTM control - how can mRNA translation be controlled?
Active forms of proteins need to be readily available
eIF2 needs to be phosphorylated in order to form a tighter complex with eIF2B
