BIOL253: Genetics L14: Regulation of Transcription

Why do cells need to regulate transcription?

• Prokaryotes and eukaryotes have many genes that encode different proteins.
• However, although a cell might require lots of some proteins, they might only require a little of others
(refer back to sigma factors in lecture 9).
• They also only need to produce some proteins at particular times (e.g. during development) or (in
multicellular organisms) some cells only ever need to produce a subset of the proteins encoded by the
genome.

Transcription Factors
• Proteins that regulate transcription.
• Contain a DNA binding domain and a transactivating domain. (Transactivating domain particularly
found in eukaryotes)
• DNA binding domain recognises a particular DNA sequence.
• Transactivating contain interacts with other proteins – helps to assemble RNA polymerase complex
onto transcriptional start site.
• It has been estimated that 10% of the genes in the human genome code for transcription factors
(control expression of all other enzymes and proteins within the cell).

Examples of DNA binding domains:

• Helix-turn-helix motif. Found in all types of organisms. Found in lambda repressor, protein produced by
bacteriophage, phage lambda. Contain 2 alpha helices (blue and red), helices orientated in different
angles to one another. Heli-turn-helix proteins often bind as dimers. Critical role in binding to DNA.
• Zine finger domain – very common in mammals and eukaryotes. Contains alpha helix and beta pleats.
Orientated by binding of zinc atom to 2 cystines and 2 histidine’s. 3 zinc ions as have 3 consecutive
zinc fingers, multiples often seen in zinc finger domains. Combining zinc fingers in different ways can
get different recognition of different DNA sequences.
• Coiled coil domains – leucine zipper and helix-loop-helix motifs. 2 extended alpha helices coiled around
each other. Alpha helical bits that interact with the DNA is disordered (not alpha helix when not bound
to DNA)

Regulatory Mechanisms – Prokaryotes

• RNA polymerase holoenzyme (RNA polymerase and sigma factor) can transcribe
any gene with a functional promoter (DNA sequences that allows a sigma actor to
bind and recruit the RNA polymerase to transcriptional start site (promoter strength
is one level of regulation – see last lecture)
• Targeted gene regulation is used most:
- At transcription initiation (most often)
- At elongation or termination
- Regulation from the transcribed RNA itself
- Regulatory proteins that decrease transcription are called repressors
(negative regulatory mechanisms) – inhibit binding of RNA polymerase.
Occludes site RNA polymerase would bind to.
• Proteins that increase transcription are activators.
• The regulatory DNA sequences they bind to are called operators

Architectural DNA binding protein – lead DNA to fold back on itself, so regulatory protein
can interact with RNA polymerase even though binding site is far from where the RNA
polymerase would bind.
BIOL253: Genetics L14: Regulation of Transcription
Very close to promoter = proximal regulatory sequences.

Regulatory Mechanisms – Eukaryotes

Regulatory sequences located further from transcriptional start site (promoter) = distal regulatory sequences.
• Distal (1000s bp) regulatory sequences more frequently used than in bacteria – called enhancers or
enhancer elements. Can be up- or downstream of the gene.
• Regulatory sequences in eukaryotes – can either be upstream or downstream of transcriptional start
site (prokaryotes only upstream)
• Regulatory sequences frequently bind several regulatory proteins – allows expression to be tuned.
• Relies on the fact that DNA can fold back on itself – transcription factor bound to enhancer element can
still interact with RNA polymerase.
• Combinatorial gene regulation – where combine several different regulatory sequences all in one area,
all within one enhancer for example. Regulatory sequences combine several different transcription
factors – allows expression to be tuned to requirements of the cell. Determine how much mRNA is
going to be produced.

Control of gene expression in bacteria

• Constitutive or ‘Housekeeping’ genes – always expressed ‘constitutively expressed’
• Encode proteins cell always needs e.g. for glucose metabolism
• Regulated genes – expression required only under certain circumstances e.g. changes in temperature
(heat shock) or availability of nutrients.
• Expression is regulated at the level of transcription. (in eukaryotes – post transcriptional regulation)

Example: Role of RecA in the control of the SOS response

LexA protein represses expression of SOS genes in the absence of
damage. Found upstream of various genes that are required for
response to genotoxic damage. In absence of DNA damage LexA
represses the expression of these genes – so not going to effort of
producing those proteins and enzymes when not needed- when RecA
inactive. When replication fork stalls in response to DNA damage – it
exposes regions of the DNA that are single stranded. RecA can bind
to regions of that ssDNA to make a nuclear protein filament. Binding
of RecA- able to direct cleavage of Lex A repressor – inactivated –
cant bind to DNA. Transcription of SOS genes – to repair DNA
damage.
Two of the genes are produced – produce more RecA to respond to
more DNA damage. Another transcription target of the SOS genes is
D-IN one – can bind to RecA- inhibit it and prevent it from cleaving
LeXA – LexA can increase and bind to its operator sequence and
SOS gene transcription is shut down.
(Lecture 8)

Operons
•  gens that are collocated in the E.coli genome. The co-located genes encode proteins that work
together. Expression of genes which encode proteins that work together (e.g. to metabolise lactose or
to synthesise tryptophan) is coordinated by organisation of genes into operons.
• In an operon genes adjacent to each other are transcribed together into one polycistronic mRNA –
(codes more than one protein) which is then translated into separate proteins encoded by each gene in
the operon. (often seen in prokaryotes – not as often seen in eukaryotes)
BIOL253: Genetics L14: Regulation of Transcription
Utilisation of Lactose: regulation of the E.coli lac operon
• E. coli uses glucose as energy source. Express proteins for glucose
metabolism constitutively (housekeeping genes)
• Genes involved in metabolising other sugars e.g., lactose – regulated in sugar
specific way. Presence of sugar stimulate expression of genes that metabolise
that sugar. So bacteria not wasting resources producing enzymes that are
useless in the absence of the sugar target.
• Lactose is a disaccharide, can be used as an alternative energy and carbon
source for bacteria. (but bacteria prefers glucose)
• When lactose is added as the sole carbon source:
• Rapid increase in lac operon mRNA transcript (transcript is unstable- short
lived, once remove lactose, degraded rapidly)
• Rapid synthesis of enzymes required to metabolise lactose – beta galactosidase and permease – both
involved in metabolising lactose.

Regulation of lactose metabolism in E.coli

b-galactosidase (enzyme) converts lactose (disaccharide) to d-galactose
and d-glucose (monosaccharides) or to allolactose:
Permease encodes a protein that facilitates entry of lactose into the cell.
Isomerization reaction – lactose by the action of beta-galactosidase. Can
isomerise lactose into allolactose – important role in regulating lac operon.
b-Galactoside transacetylase transfers an acetyl group from acetyl-CoA to
eliminate toxic thiogalactosides from the cell

The genes are expressed via an operon

Promoter region (green), lac operator DNA sequence, co-located genes that can be transcribed to produce one
polycistronic mRNA. LacZ – codes for beta galactosidase enzyme, Lac Y encodes permease, Lac A that
encodes transacetylase (adds acetyl groups from acetyl CoA to various sugar – not sure why in lac operon)
The operon (lacZ, lacY and lacA) is transcribed to produce a single mRNA that is translated to produce three
proteins:
b-galactosidase – lacZ
Permease - lacY
Transacetylase – lacA
The lacI gene (not part of the operon)is situated immediately upstream of the operon – it has its own promoter
and is constitutively expressed (housekeeping gene). Protein produced is the lac repressor protein and is
alwaus produced by the cell. It plays an important role in regulating transcription of the lac operon…

The Lac repressor (Lac I)

When there is no lactose present, LacI binds to lacO (the lac operator). No
transcription occurs as RNA Pol can’t bind:

When lactose is present, allolactose binds to LacI, altering its shape (conformational change) – cant bind to
DNA, promoter sequence available for RNA pol to bind, this allows transcription to occur (derepression):
(need allolactose that is produced by the product of the LacZ gene – for the
gene to be expressed – can occur as oppression by lac I isn’t perfect – get
leeky expression – in absence of lactose – get very small amounts of mRNA
produced – produces just enough beta galatosidase to ensure when lactose
does become available, that it can be converted to allolactose and quicly get
expression of genes.

What happens when glucose is present?

Bacteria prefers glucose – better energy source. Prefer to use glucose to
lactose.
• Presence of glucose inactivates adenylate cyclase and dramatically
reduces levels of cAMP. (adenylate cyclase converts ATP to cAMP)
BIOL253: Genetics L14: Regulation of Transcription
• cAMP is therefore an indicator of glucose levels:
• cAMP high when glucose low
• cAMP low when glucose high

Catabolite activator protein (CAP)

Involved in positive regulation of the lac operon
(bacterial typrically use negative regulation – this is an example of positive regulation)
Also known as cAMP receptor protein (CRP)
• CAP Only binds to DNA in the presence of cAMP.
• Facilitated when high levels of cAMP (low levels of glucose)
• Bends the structure of the DNA by 90o

CAP-cAMP activates gene expression

• CAP binds to CAP operator – can facilitate binding of the RNA polymerase by
interacting with c-terminal domains of alpha subunits of RNA polymerase. Seen in
regulation of lac operon and other areas.
• CAP-cAMP binding upstream of the promoter facilitates RNA polymerase
holoenzyme binding
• It activates more than 100 different E. coli promoters when glucose levels are low….

Summary: lac operon regulated by a combination of negative inducible regulation (Lac repressor) and positive
regulation (via cAMP-CAP)
Most expression of lac operon in circumstances where have low glucose
and the lactose is available.
Derepression of lac repressor and binding of the cap
Where glucose and no lactose – cell needs to respond – but no point
transcribing lac operon as no lactose to metabolise. Lac repressor is
binding so genes not transcribed even though you have CAP bound – lac
repressor overrides that CAP signal.
High glucose and lactose available- derepression of lac operator – as no
CAP binding only get weak levels of transcription of lac operion.
When high glucose and no lactose – binding of lac repressor so no
transcription.

Negative regulation by repression

• Operons for anabolic (biosynthetic) pathways (synthesizing biomolecules) turned off when end product
is readily available – repressible regulation.
• e.g. trp operon (two mechanisms for controlling expression) that combine together to ensure that is
can produce right amount of mRNA for situation it is in.
• 5 genes encoding enzymes for tryptophan biosynthesis are expressed in an operon as a polycistronic
mRNA – turned off by excess tryptophan
• Controlled by trp operator – downstream from promotor sequence.
• In contast to lac operon. Gene for regulatory protein – trp repressor protein
– maps away from operon -located at different site on E.coli chromosome to
the trp operon.
* Regulatory gene for trp operon is trpR – maps away from operon

The trp Operon of E. coli

If amino acids are available in the medium, E. coli will import them rather than make them, and
the genes for amino acid biosynthesis are repressed. When amino acids are absent, the genes
are expressed and biosynthesis occurs.

Tryptophan mediated repression

BIOL253: Genetics L14: Regulation of Transcription
trpR encodes an aporepressor:
normal circumstances -doesn’t/cant bind to DNA.
A. In absence of tryptophan no binding of TrpR aporepressor to operator. RNA pol can bind. Transcription
occurs.
B. In presence of excess tryptophan, aporepressor activated by binding tryptophan (acts as co-repressor).
Binds to operator, prevents transcription.
Regulation acts as simple on/off switch
Repression reduces transcription of the trp operon about 70-fold.
Excess tryptophan binds the TrpR protein causing an allosteric shift allowing binding to the operator
sequence which then inhibits transcription.

Expression of the trp operon in presence of low concentrations of tryptophan

Sometimes it needs to be able to produce a little bit of the mRNA to ptroduce a little typtophan.
Second regulatory mechanism:
Tryptophan starvation – maximal expression of trp genes
Tryptophan limitation – less than maximal expression of trp genes
Controls ratio of full-length transcripts to short 140 bp trunkated transcripts (truncate before any of those
genes for the biosynthesis of expression of tryptophan)terminated within trpL leader region – attenuation.
Attenuation can reduce trp operon transcrioption 10 fold.
Mechanism dependent on translation. Thiese two together can reduce tracroption 100 fold.
Transcription and translation in bacteria occur in the same space in the cell and can occur at the same time.
So as soon as mRNA – activing immediatel a substrate for translation.

Expression of the trp Operon in the Presence of Low Concentrations of

Tryptophan
3. When tryptophan is limited, transcription is also controlled by attenuation.
a. Attenuation produces only short (140 bp) transcripts that do not encode structural
proteins.
b. Termination occurs at the attenuator site within the trpL region.
c. The proportion of attenuated transcripts to full-length ones is related to
tryptophan levels, with more attenuated transcripts as the tryptophan concentration
increases.
d. Attenuation can reduce trp operon transcription 8- to 10-fold. Together,
repression and attenuation regulate trp gene expression over a 560- to 700-fold
range.

Four regions of the trp leader mRNA can form alternative secondary structures
Secondary structures formed depend on ribosome progression as mRNA translated
Sequence that encodes very short polypeptide – in this polypeptide have codons that encode tryptophan =
leader peptide.

When low levels of tryptophan – ribosome stalls – no tRNAs with trp coming to ribosome so stops at trp
codons.
Rest of mRNA has some regions of self complimentarity. Formation of hairpin strucutres though
complementarity further down mRNA. RNA pol carries on transcribing.
High levels of trp – ribosome does not stall, proceeds, to create leader peptide. As carries on , now sitting on
sequence 2, sequence 2 is no longer can bp with reigon 3 – canot form hair pin and instread region 3 can bp
with region 4 instead – another hairpin, this produced under conditions of high trp = attenuator structure.
Creates structure to terminate transcription – produced only 140 nucloetide mRNA as transcription stpped.
Low levels of trp – continues transciribnf becaseof the pausing so get full length transcrop of polycistronic
RNA.

The molecular model for attenuation:

a. Translation of the trpL gene produces a short polypeptide. Near the stop codon are two tryptophan codons.
BIOL253: Genetics L14: Regulation of Transcription
b. Within the leader mRNA are four regions that can form secondary structures by complementary base-pairing
(Figure 17.16).
i.Pairing of sequences 1 and 2 creates a transcription pause signal.
ii.Pairing of sequences 3 and 4 is a transcription termination signal (a rho-independent terminator).
iii.Pairing of 2 and 3 is an antitermination signal, and so transcription will continue.
c.Tight coupling of transcription and translation in prokaryotes makes control by attenuation possible.
i.RNA polymerase pauses when regions 1 and 2 base-pair just after they are synthesized (Figure 17.16).
ii.During the pause, a ribosome loads onto the mRNA and begins translation of the leader peptide. Ribosome
position is key to attenuation:

Summary: trp operon regulation

At the level of initiation:
• ‘On/Off’ switch by negative repressible regulation:
• Absence of tryptophan: No repression – transcription
• Presence of excess tryptophan: TrpR aporepressor + tryptophan co-repressor bind operator – no
transcription
At the level of production of full length transcript:
• Presence of tryptophan - attenuation at second regulatory site in response to Trp-tRNA levels
terminates transcription before structural genes transcribed
• Absence of tryptophan – stalling of ribosome at Trp codons results in antitermination – transcription
proceeds to produce full length mRNA encoding biosynthesis enzymes.
Both repressible regulation and attenuation are common methods of regulating expression of amino acid
biosynthetic operons

Riboswitches
The RNA can interact with ribosome to control how much of the RNA Is produced.
Small molecule (adenine) interacting directly with RNA itself
• Riboswitches are portions of a transcript that can directly bind a small molecule that
controls the RNA secondary structure, regulating transcription or translation
• Riboswitches have two regions – the aptamer (aptimeric region) that binds to the
metabolite, and an expression platform which controls transcription or translation
Controlled by regions of self-complimentarity allowing hairpin strucutres to form.
The Adenine Riboswitch
• The B. subtilis (bacteria) adenine riboswitch regulates adenine synthesis and
transport. Gene expression depends on whether a terminator or anti-terminator
forms
When adeinine is available bacteria doesn’t need to produce it, when adenine is not available it needs to
be able to transcribe the genes involved in the biosynthesis of adenine.
• In low adenine, the RNA structure is as in (b) form hairpin strucutres – regions 2 and 3 form an anti-
terminator and transcription proceeds. Transcriptin carries on.
Full length transcript.
• High adenine allows (c) to form – regions 3 and 4 bp form a
terminator – note sequcen of uridine.
In presence of adenine form 3d structure – froming antiterminatin oin
low adenine, and type.1 termiantor in high adeinne