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
