Transcript Fundamental of regulation of genetic feedback loops

Prokaryotic Gene Regulation:
Lecture 5
Introduction
• The two types of transcription regulation
control in prokaryotic cells
• The lac operon an inducible regulatory
feedback system
• The tryp operon a repressible regulatory
feedback system.
• The role of bioinformatics analysis in such
systems
Gene Regulation
• All “genes” must have some way of regulating their expression (converting
the DNA to amino acids) in order to allow them to adopt appropriately to
the environment.
• In prokaryotic cells the process, owing to the simple nature of the genomic
material, is basically controlled “mainly” at the transcription level…
• Essentially the molecule “RNA polymerase” must bind to an “exposed“
part of DNA strand called a promoter; it must then move, in the 5’ to 3’
direction, transcribing the DNA of the “gene” to RNA.
• The transcription is controlled/ regulated at two stages:
– RNA polymerase binds to the DNA strand;
• RNA binds DNA transcription begins
• RNA is prevented from binding; DNA is not transcribed: gene not expressed.
– RNA polymerase moves in the 5’ to 3’ direction:
• If the movement if RNA polymerase is blocked transcription is stopped : gene not
expressed
• Otherwise transcription is completed and the gene is expressed.
Basic types transcriptional control
• Inducible: gene is expressed (DNA is
converted to RNA) only if the molecule
(inducer) is present.
• Repressible: if molecule is present gene
expression is turned off
• In both cases the molecule works via a
feedback loop (e.g. a thermostat)
Regulatory loops: transcription
• Feedback loop: product
of the gene expression
loops back
(directly/indirectly) and
alter the expression of
the same gene.
• The effect can be either :
– positive : “turns on”
transcription via an
inducer; e.g. [lactose ]
– Negative: “turns off”
transcription via a
repressor e.g. [tryptophan]
Gene
Gene product
Inducible Prokaryotic regulation
• Lactose, a complex sugar (glucose)
• In order for E. Coli to use (metabolise) the sugar a gene system referred to
as the “lac operon” must produce three enzyme(s) that allow lactose to be
utilised by the bacteria. For simplicity we will refer to the combined
system as: lactose dehydrogenise (a more detailed description of the
enzymes and their functions can be found in Klug p. 310).
• The function of this enzyme is to break convert lactose to glucose and
increase its absorption into the bacterial cell.
• Basically In order to ensure efficient functionality of the “lac operon”
system it must have the following properties:
– will not be expressed if there is no lactose
– will be expressed if there is lactose.
• Note a more complete description of the system, involving glucose, can be
found in the supplementary notes.
Function of Lac operon
Klug chapter 15
• The term operon is the common “gene regulatory
system” used in prokaryotic cells and generally a
number of genes are regulated as a one.
• In the E Coli Lac operon DNA there are:
–
–
–
–
–
1 repressor gene (
A promoter (where RNA polymerase binds)
A Cis–acting regulatory region (operator)
A leader region (not critical to expression)
3 structural genes: LacZ, LacY and LacA (refer to here as
lactose dehydrogenase)
Function of Lac operon
•
•
•
•
•
•
•
RNA polymerase binds to the promoter
region
The repressor gene produces a product “a
repressor protein”
This binds to the DNA at the operator region
and blocks RNA polymerase moving down
the DNA strand.
If lactose is present it alters the repressor
protein.
The alter repressor protein is unable to bind
to the DNA
RNA polymerase binds to the promoter
region and begins transcribing the 3
structural genes.
Repressor protein
RNA polymerase
When lactose levels drop to zero: what
happens?
Klug chapter 15
Glucose and the lac operon
• Lactose is metabolised into
glucose so what happens if
glucose is present.
• Catabolite-activation
protein (CAP): CAP must be
present to make RNA
polymerase binding
efficiently
• In the presence of glucose
the CAP is altered and
prevents RNA polymerase
binding to the promoter
region and so prevents
transcription.
Klug chapter 15
A repressible operon
• Tryptophan is an essential AA and
is normally made
(biosynthesised) by E Coli.
• If tryptophan is present externally
[in sufficient levels] then the
biosynthesis stops;
• The DNA strand has the same
elements as the lactose operon:
repressor gene, promoter,
operator, leader and genes
• In this system the repressor
protein is altered by tryptophan
and the modified repressor
protein now binds to the operator
region and blocks RNA
polymerase transcribing the
genes required to make
tryptophan.
Klug chapter 15
The tryptophan operon
•
•
•
•
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•
In addition in the presence of
tryptophan there is an additional
control mechanism called:
The attenuation regulatory
mechanism:
In the sequence prior to structural
genes is the attenuator region:
If tryptophan and its gene expression
is repressed they still found that
transcription was initiated… ; there
was “RNA” fragments of leader
[L]sequence
Thus altering the repressor protein is
not enough to prevent expression.
It seems that tryptophan also binds
to the attenuator [A]region and
prevents transcription beyond the
leader region.
Attenuator region
Leader region
Klug chapter 15
Importance of bioinformatics
• Bioinformatics can help with improving our understanding
of such regulation by:
– Finding potential gene regions and promoter regions since a
gene will be in close proximity to a promoter regions.
– The prokaryotic sequence normally has specific sequences
associated with it and so do genes [begin with AUG/ATG]. [This
will be covered in more detail in the lecture “finding
genes/promoters”
– Prokaryotic systems usually have polycistronic mRNA ; multiple
gene sequences in very close proximity (contiguous) or in some
cases the may even slightly overlap. (understanding
bioinformatics section 9.2)
– The gene sequence can be converted into amino acid sequence
via universal code
– Finally the promoter sequences and gene sequences can be
analysed using advanced computational techniques to help
understand or suggest how the “operon” works
Exam question
• Distinguish, using suitable examples, the
difference between inducible and repressible
transcription control in prokaryotic organisms.
(20 marks)
• Discuss, using examples, how analysis of DNA
sequences can help our understanding of
genes in prokaryotic cells. (10 marks)