Lecture Notes

Week 2 Notes: Regulation of Bacterial Gene Expression- The Operon

Introduction: control of gene expression
 Trans-acting factors: genes code for product (protein/tRNA/rRNA) which functions on any copy of its target
DNA
o E.g. protein or RNA molecule that diffuses away from location of synthesis to act elsewhere (global)

 Cis-acting sequences: DNA site that affects only sequences of its own DNA or RNA molecule
o Does not code for protein
o E.g. promoters/operators/terminators: DNA sequence that only have local effects

 Structural gene: code for RNA/protein product that are non-regulators

 Regulator gene: code for regulators: product (protein) that controls expression of other genes (usually at
transcription) by binding to operators: cis-acting sequence sites on DNA
o Operators: Sites located upstream of target gene
 This interaction regulates target gene in positive (activator) or negative (repressor)
manner

Induction and repression
 Gene encoding for enzyme is regulated by concentration of its substrate or product
 Substrate absent: bacteria does not synthesise enzyme (to avoid wasting energy)
 Inducible regulation: gene regulated by presence of substrate (inducer)
 Repressible regulation: gene regulated by presence of product its enzyme pathway (co-repressor)

Negative control of gene expression
 Trans-acting repressor protein binds to cis-acting operator to prevent gene expression
 Repressor absent: gene is expressed
 Common in bacteria

Positive control of gene expression
 Trans-acting activator binds at cis-acting promoter to allow RNA pol to initiate transcription
 Absence of positive regulator: gene is inactive
 Common in eukaryotes

Regulatory circuits
 Negative inducible, negative repressible, positive inducible, positive repressible
 Unifying theme: regulatory proteins are trans acting factors that recognise cis acting elements just upstream
of target gene

The operon
 In bacteria, the operon is a functioning unit of genomic material containing a cluster of genes under the
control of a single regulatory signal or promoter
 Genes are transcribed together into a mRNA polycistronic strand
o All genes in the operon are either expressed together or not at all
 Genes coding for proteins that function in same pathway are located adjacent to each other and controlled as
a single unit

The lac operon
 Lac operon: protein products of operon enable bacteria to take up and metabolise B-galactoside sugars
 3 lac structural genes:
o lacZ encodes B-galactosidase (B-galactoside breakdown  glucose/galactose)
o lacY encodes B-galactoside permease (transports B-galactoside into cell)
o lacA encodes B-galactoside transacetlyase (transfers acetyl groups from acetyl-CoA to B-
galactosides)

, Week 2 Notes: Regulation of Bacterial Gene Expression- The Operon

 lac operon is inducible:
o B-galactoside = substrates of lac operon = inducer
o Addition of specific B-galactosides induces transcription of all 3 genes
o Lac mRNA is very unstable (restricts amount of protein made)
 Therefore induction can be rapidly reversed

 lac operon is negative inducible
o lacZYA operon transcription is controlled by lac repressor protein that binds to operator that
overlaps the promoter at the start of the cluster

lac repressor
 Tetramer (made of two dimers) of identical subunits
o Monomers form a dimer by making contacts b/w core sub-domains 1 and 2
o Dimers form a tetramer by interactions b/w the tetramerisation helices

 Coded by lacI gene, an independent transcription unit with its own promoter and terminator

 A single repressor subunit is divided into N-terminal DNA-binding domain, a hinge and the core of the protein
o DNA-binding domain: has 2 short alpha-helical regions that bind the major groove of the DNA
(helix-turn-helix motif)
o Inducer-binding site and regions responsible for multimerisation are located in the core

 Repressor is controlled by small inducer: allosteric control
o Repressor has two binding sites: for operator DNA & for inducer
 Inducer binding allosterically changes repressor conformation, and inactivates it into form
with lower operator affinity by changing properties of DNA binding site,
 Probably by changing hinge helices
 Inducer: allolactase, not the actual substrate of B-galactosidase
 Gratuitous inducer: resemble authentic inducers of transcription, but are not substrates for
the induces enzymes (e.g. IPTG)
 Lac repressor binding to operator is regulated by allosteric change in conformation
 Repressor binds to operator DNA, which is a palindromic sequence of 26 bp (dyad
symmetry)
 Each inverted repeat of operator binds to DNA-binding site of one repressor subunit
 Symmetry of operator matches symmetry of repressor

 Constitutive expression: state in which a gene is expressed continuously
 B-galactoside absence: lac operon is expressed at very low (basal) levels

Cis-acting constitutive mutations identify the operator
 Mutations
o In operator cause constitutive expression of lacZYA operon
o Are cis acting: Only affect genes on contiguous stretch of DNA
o In promoter prevent expression of lacZYA and are uninducible
 Cis-dominant: a site or mutation that affects the properties only if its own molecule of DNA, often indicating
that a site does not code for a diffusible product

Trans-acting mutations identify the regulator gene
 Mutations
o In lacI gene are trans acting and affect expression of all lacZYA clusters
o That eliminate lacI function cause constitutive expression and are recessive (lacI-)
o In the DNA-binding site of the repressor are constitutive b/c the repressor cannot bind the operator