Regulation of Gene Expressionin Prokaryotes I Flashcards
so far we know…
- DNA is arranged into genes.
- Genes provide for the storage of information.
- This information is expressed through the processes of:
Transcription
Translation
How is the expression of genes regulated such that we obtain a coordinated expression of the genetic material at the right time (and place) to obtain the desired effect?
- Very NB question
- Remember, not all genes are expressed at all times
Efficient expression of genetic information
is dependant on control mechanisms that promote or suppress gene activities.
How is the promotion/ suppression of gene expression achieved
In transcription:
The expression of genes relies on the presence of a cis-element termed a promoter and which is usually found upstream of the start codon of the gene.
a promoter
Determines when and what quantities a gene will be transcribed
Many of the early studies of gene expression where performed on bacteria and yeast
Why?
- They are easy to culture and have short generation times
- they can be easily mutated and pure mutated cultures can be
obtained for separate studies
Bacteria are ideal models for studies involving
- the induction of gene expression in response to changes in
environmental conditions - Bacteria regulate their gene expression in response to
environmental changes as well as a variety of non-environmentally
regulated cellular activities (such as cell division)
Adaptive hypothesis
Refers to the ability of organisms to adapt to their environment
E.G:
lactose in growth medium induces expression of enzymes specific for lactose metabolism
Various forms of gene expression
Constitutive, inducible, repressible systems
Which are under negative or positive control
Constitutive genes
- permanently expressed, regardless of the environmental conditions
- Call these housekeeping genes - expressed all the time- keep cell
alive
Inducible genes
- expressed in response to a particular condition
E.g: An inducer, such as lactose.
repressible systems
- genes may be repressed due to the presence of a particular
molecule - These molecules are often end products of a specific biosynthetic
pathway
positive control
- needs a substrate to directly stimulate transcription
Negative control
- occurs when a molecule turns off for transcription
Glucose
is the primary molecule used as a source of energy in all cellular metabolism
E. coli has the ability to grow on ______ as an alternative source of ______
lactose
carbon
Lactose is a disaccharide of
Galactose and glucose
Which enzyme digests lactose to produce galactose and glucose?
Beta-galactosidase
In the absence of lactose
- There are only a few copies of the Beta-galactosidase enzyme
present in the cell
In the presence of lactose
- Gene expression is induced and the number of enzymes available
rapidly increases to several thousand enzymes per cell - The enzymes = inducable
- lactose = inducer molecule
Lactose - the inducer
- technically, the inducer is allolactose - an isomer of lactose
- When lactose first enters the cell, a very small portion of it is
converted to allolactose by the Beta-galactosidase enzyme
operon
Prokaryotic genes with related functions are organised in groups and are expressed in a coordinated fashion
Watch slide 11 of prokGenRegnarrated1
The lac operon
- lac operon consists of 3 structural genes and
- 2 regulatory regions upstream (cis & trans action) - it is found 5’ to
the operon - The discovery of a regulatory gene and regulatory site which are
not part of the gene cluster - NB in understanding how the operon
was controlled - neither of these regions encode enzymes needed for lactose
metabolism = function of the 3 structural genes
Structural genes (related functions):
lacZ – b-galactosidase
lacY – b-galactoside permease
lacA – transacetylase
However, together, this entire gene cluster operates to provide a rapid response to the lactose status of the environment
Structural genes (related functions):
Genes and what they encode:
lacZ – b-galactosidase
> responsible for cleaving lactose into glucose and galactose
lacY – b-galactoside permease
> facilitates the entry of lactose into the cell
lacA – transacetylase
> May be involved in the removal of toxic products of lactose
metabolism (unclear)
Functions of these structural genes were identified through the study of lac minus mutants - first isolated and studied by
Joshua Lederberg
Lac z minus mutants - didn’t produce active beta-galactosidase
Lac Y minus mutants - unable to use lactose as an energy source
Polycistronic mRNA:
is an mRNA that encodes several proteins and is characteristic of many bacterial and chloroplast mRNAs.
Polycistronic mRNA: of the lac operon
Nederberg mapped all these genes
- order Z-Y-A
- they are transcribed as a single polycistronic mRNA = Co-ordinated
expression of all 3 genes
How does lactose regulate transcription of the 3 structural genes?
- Gene activity repressed in absence of lactose
> Lactose induces expression - Lactose analog - IPTG (Isopropylthiogalactoside)
(gratuitous inducer)
= showed that induction is not mediated through interactions with
enzymes
- Because ITPG cant interact with the Beta-galactoside enzyme - Constitutive mutations
- continuously turn the operon on, regardless if lactose was present
or not.
- mapping of the LacI mutation showed that the mutation was in a
sight near to, but distinct from the structural genes
= discovery of LacI gene = repressor gene (turns operon off)
- LacO^c (second constitutive mutant) due to mutation in the operator
region of the operon
In both of these mutations:
– lacI- and lacOc genes are produced continually and inducibility
cannot happen - enzyme regulatability is lost
Operon model – negative control
- lacI encodes an allosteric repressor molecule
= a molecule that combined with another molecule (lactose) induces
a conformational shift in the repressor and changes it chemical
activity - Repressor protein encoded by lacI binds to operator region
> Inhibits RNA polymerase binding (prevents transcription) - This represses the expression of the structural genes as the
promotor can’t read through towards the structural genes - Transcription thus only occurs when the repressor fails to bind to
the operator = under negative control - HOWEVER, when lactose is present, it can bind to the repressor
- This induces an allosteric conformational change - which changes
the binding site of the lac repressor and prevents it from binding to
the lac operator
Functioning of the lac operon
Listen to slide 16 and 17
Mutations in LacI and LacO^c
- Constitutive = continuous expression of lac operon
- The mutations interfere with molecular interactions between the lac
repressor and the operator region
Mutations in lacI
= LacI-
- Either cause a conformational change - prevents it from binding to
the operator region
- or causes the complete loss of the repressor
> In both cases, it loses its ability to bind to the operator region,
which results in the operon being continuously on
Mutations in lacOc
- Alters the nucleotide sequence of the operator region
= normal repressor protein can no longer recognize that sequence
and is unable to bind to the operator
Genetic proof for the operon model.
The operon hypothesis resulted in 3 major predictions which could be tested to determine the validity of the model
Hypothesis:
1. lacI produces a diffusible (trans-acting) product
2. lacO has no gene product - cis-acting element - involved in
regulating gene expression
> Also true for LacP - the promotor region
3. lacO must be adjacent to the structural genes in order to regulate
them, as they are a cis-element
> (also true for P)
How is this tested?
- bacteria may contain plasmids (in addition to their chromosome) which are independently replicated circular DNA molecules, that contain extra genes
> they have their own origins of replication = can replicate
independently of the bacterial chromosome
Listen to 20 - 28
