Lac Operon Flashcards
What is abortive initiation?
the repetitive synthesis and release of short nascent RNAs by RNAP
What is scrunching of DNA?
the change in conformation during initiation of transcription
What is ChIP-seq used for?
to study protein-DNA interactions on a genome-wide scale
What is a regulator gene?
a gene that encodes for a product that controls the expression of other genes
What is an operon?
a unit of bacterial gene expression and regulation, consisting of a promoter, operator and structural genes
What does a cis-acting site affect?
the activity only of sequences on its own molecule of DNA (or RNA) i.e. the site does not code for protein
What does a trans-acting product suggest?
it is a diffusible protein or RNA
What happens in negative control?
a repressor protein binds to an operator to prevent a gene from being expressed
What happens in positive control?
a TF (activator) is required to bind at the promoter in order to enable RNA polymerase to initiate transcription
What does LacI gene have?
its own promoter and terminator
What is transcription of the LacZYA operon controlled by?
the lac repressor that binds to an operator that overlaps the promoter at the start of the cluster (PO)
What is the polycistronic transcription unit of the LacZYA operon made up of?
- promoter
- lacZ, Y and A
- terminator
What does β galactosidase do?
break down and hydrolyse lactose into glucose and galactose
What does permease do?
help transport the beta galactosidase into the cells
How big is the lac operon operator?
the first 26 bp of each transcription unit
Where does the lacZ gene start on the transcription unit?
base 39
What is the lac repressor?
a tetramer of identical subunits coded by the lacI gene
What is the lac repressor controlled by?
an inducer
What does the lac inducer do?
convert the lac repressor to a form with lower operator affinity to allow RNAP to initiate transcription
What are the 2 binding sites on the lac repressor?
- one for the operator DNA
- one for the inducer
What does lacZ encode for?
β galactosidase
What is IPTG?
a gratuitous inducer i.e. a molecular mimic of allolactose
What does IPTG do?
trigger transcription of the lac operon and induce protein expression where the gene is under the control of the lac operator
What are the 3 components of a single repressor subunit?
- N-terminal DNA-binding domain
- a hinge
- the core of the protein
What does the DNA-binding domain of the repressor subunit contain?
two short α-helical regions (helix-turn-helix) that bind the major groove of DNA
What do all HTH DNA binding proteins do?
bind DNA as dimers that are 3.4 nm apart
What do the amino acid side chains of the C-terminal helix of the HTH do?
recognise the specific DNA sequence to which the protein binds
What is the lac operator?
a palindromic sequence of 26 bp consisting of adjacent inverted repeats
What is a palindrome?
a DNA sequence that reads the same on each strand of DNA when the strand is read in the 5’ to 3’ direction
What does each inverted repeat of the lac operator do?
bind to the DNA-binding site of one repressor subunit
What does inducer binding to the repressor do?
cause a change in repressor conformation that reduces its affinity for DNA and releases it from the operator
Give examples of DNA motifs recognised by bacterial regulator proteins
- lac repressor
- catabolite activator protein (CAP)
- lambda repressor
What do mutations in the cis-dominant operator cause?
constitutive expression of all 3 lac structural genes because the operator is unable to be bound with repressor protein i.e. RNAP has unrestrained access to the promoter
What can the cis-dominant operator control?
only adjacent lac genes
What do mutations that inactivate the lacI gene cause?
the operon to be constitutively expressed because the mutant repressor protein cannot bind to the operator
What is meant by the lacI- gene being repressive?
induction of a normal lacI+ gene can restore control even in the presence of a defective lacI- gene
What do mutations in the inducer-binding site of the repressor do?
prevent lac operon transcription which causes uninducibility
What explains the dominant negative behaviour of the lacI-d mutation?
mutant subunits can combine with normal subunits and interfere with their function; it only requires 1 subunit of the multimer to be lacI-d to block repressor function
What does full repression of the lac operon require?
the repressor to bind to an additional operator downstream (O2) or upstream (O3), as well as to the primary operator at the lacZ promoter (O1)
What is catabolite repression?
the ability of glucose to prevent the expression of a number of genes
What does cAMP do?
convert inactive CAP to a form that binds the promoter and assists RNAP in initiating transcription
What activates a dimer of CAP?
a single molecule of cAMP
What does CAP do?
activate the α subunit of RNAP by interacting with the CTD
What is cAMP controlled by?
the level of glucose in the cell; a low glucose level allows cAMP to be made
What happens to the lac operon in the presence of both glucose and lactose?
- β-galactosidase is not needed
- lac operon is off
- glucose causes low levels of cAMP, and CAP fails to bind to its site in the lac operon
What happens to the lac operon in the presence of only glucose?
- β-galactosidase is not needed
- lac operon is off
- lac repressor bind to its operator and CAP fails to bind
What happens to the lac operon in the presence of only lactose?
- β-galactosidase is needed
- CAP binds and turns the lac operon on
What happens to the lac operon in the absence of both glucose and lactose?
- β-galactosidase is not needed
- lac operon is off because Lac repressor bound to its operator prevents CAP from turning the lac operon on
What are the 2 agar plates used to examine the lac+ phenotype?
- one has nutrient agar containing IPTG and X-gal, which is cleaved by beta-galactosidase to produce a blue reaction product
- the other is a control and contains nutrients and X-gal only
What is blue-white screening used for?
the detection of successful ligation in vector-based gene cloning
