Ch. 11: Molecular Mechanisms of Gene Regulation
Gene expression is regulated at many levels:
 Transcription
 RNA processing
 Translation
 mRNA stability
 post-translation
 DNA rearrangements
11.1 Prokaryotic gene regulation
Gene activity in bacteria is usually controlled at the level of transcription: the production
of the mRNA transcript, which is subsequently translated into protein – an up-regulation
in the transcription of a given gene is also called turning the gene “on”
However, many genes are still transcribed at a low level even when they “turned off,” or
are not transcriptionally activated
Several enzymes that work in a common pathway may be regulated together – all are
produced at the same time or all are not produced – “coordinate regulation”
Types of transcriptional regulation in proks:
Negative regulation: the gene is “on” in the default state and must be
turned off by a repressor protein
Inducible transcription: a DNA-binding repressor protein is
removed from the DNA when it binds to a small molecule called
an inducer
e.g. catabolic (degradation) pathways often have the initial
substrate as an inducer
Repressible transcription: an aporepressor has no DNA-binding
activity until it binds to a a small molecule corepressor, upon
binding the gene is repressed by the activated repressor – presence
of the substrate activates its own degradation
e.g. anabolic (synthesis) pathways often have the final product as a
co-repressor – the product down-regulates its own production in a
feedback loop
Positive regulation: in the default state, the gene is turned “off” – so a
regulatory protein is necessary to turn the gene “on” – this is called a
transcriptional activator
Negative regulation is more common in prokaryotes
Positive regulation is more common in eukaryotes
Autoregulation: the protein product of a gene regulates its own transcription –
can be positive or negative feedback loop
Many genes have both positive and negative regulatory elements
11.2 Operons
Operon: a group of several genes located in tandem and a single regulatory region with a
promoter, an operator, and a repressor gene. The repressor gene product binds to the
operator region to turn off the expression of genes in the operon. An inducer protein
binds to the repressor protein, releasing it from the operator and allowing RNA
polymerase to bind to the promoter and begin transcription and subsequent expression of
the genes in the operon.
11.3 Transcription Termination
Transcription is down-regulated via the involvement of the stalling of a ribosome
translating the mRNA and variable base-pairing within the mRNA causing an RNA
stemloop structure called an attenuator that represses downstream transcription
11.4 Regulation in Bacteriophage Lambda
Competition between action of cro and cI proteins results in lytic or lysogenic pathway
11.5 Transcriptional Regulation in Eukaryotes
The galactose metabolic pathway converts galactose to glucose-1-phosphate in yeast.
Regulation of the galactose metabolism pathway involves regulation of the transcription
of GAL7, GAL10 and GAL1 transcripts by the transcriptional activator protein GAL4
and a repressor of GAL4 named GAL80. The GAL81c mutation within the GAL4 coding
region results in a constitutively active GAL4 that cannot bind to GAL80. When the
inducer galactose is present, it binds to the repressor GAL80, releasing GAL4 and
activating the pathway.
The GAL4 protein has DNA binding motifs also seen in other transcriptional
activator proteins:
The helix-turn-helix motif binds in the grooves of double-helical DNA
The zinc finger motif is a sequence specific DNA-binding motif which
involves a two zinc divalent cations complexed to six cysteine amino
acids.
The region of DNA to which the transcriptional activators bind are often
enhancers – these short sequences can be upstream of the transcription
start site or very distant and at either the 5’ or 3’ end or even within
introns (non-coding segments within the coding sequence). Enhancers
may activate by causing DNA looping, bringing together different parts of
the DNA, and recruiting protein complexes involved in transcription.
There are also DNA sequences that act as transcriptional silencers, and are
bound by proteins which recruit complexes that shut down transcription of
a given gene
Deletion scanning is an experimental approach - it determines which sequences in a
regulatory region have enhancer or silencer activity, also used to determine promoters.
This approach often places the regulatory region upstream of a known reporter gene
such as lacZ, which encodes -galactosidase which can be observed by a blue color
produced in the presence of the substrate X-Gal. Deletional analysis of the GAL1
regulatory region revealed a binding site for the GAL4 activator consisting of two tandem
repeats of a 17 base pair sequence, to which the symmetrical GAL4 dimer binds – this is
called the upstream activating sequence (UAS) of GAL1.
The Eukaryotic Transcription Complex
Basal transcription factors – are the minimal proteins necessary for transcription:
TFIIB, TFIID, TFIIE, TFIIF, PolII (an RNA polymerase), and TFIIA (where TF
stands for transcription factor) – many of these come together in a complex called the
PolII holoenzyme
TBP: TATA-box-binding protein: bind to the TATA box, a region of the promoter with
the sequence TATA
TAF’s: TBP-associated factors – about 10 other proteins that associate with TBP and
respond to activator proteins
A complex containing TFIID (“TFIID complex”) is thought to assemble together and be
recruited by a transcriptional activator protein, and then in turn recruit the PolII
holoenzyme
An example in Drosophila involves the transcriptional activator proteins BCD (bicoid)
and HB (hunchback). These proteins bind to enhancer sequences on DNA. They then
recruit the TFIID complex by protein-protein interactions with different specific proteins
in the complex, BCD with a 110kD TAF and HB with a 60kD TAF. The large complex
recruits the PolII holoenzyme, containing all the transcriptional machinery, to the
promoter, so that the gene is transcribed.
Chromatin-remodeling complexes
For transcriptional activation of genes within packaged chromatin, additional factors are
necessary to allow transcription factors access to the DNA.
CRCs (chromatin-remodeling complexes) use ATP to remodel chromatin and allow
transcription – the molecular mechanism of chromatin remodeling is unknown – some
models propose repositioning of nucleosomes to make DNA sites more accessible
11.6 Epigenetic mechanisms
Epigenetic regulation is caused by something outside of the nucleotide sequences of
genes
One type of epigenetic control is the methylation of DNA
Methylation is catalyzed by DNA methylase, which adds a CH3 group at the C5 position
of cytosine bases – this preferentially occurs at 5’-CG-3’ sites (also called “CpG
islands”), usually upstream of the coding region of a gene
methylation of DNA can be detected by digesting with MspI which recognizes all
CCGG sites whether methylated or not, and HpaII which only recognizes non-methylated
CCGG sites
There is a strong correlation between heavy methylation and transcriptional silencing, but
there may be an initial signal for silencing before methylation occurs
Genomic imprinting is a gamete-of-origin dependent change to the transcriptional
activity of a given gene. It occurs in a small fraction of genes – probably only 100-200
genes in a much larger genome (e.g. human genome has ~ 40,000 genes), imprinted
genes are heavily methylated – still not known exact mechanism or role of methylation in
imprinting and silencing
o e.g. if gene A is imprinted, it will be either active or silenced depending on
whether it is in an egg or sperm, i.e. the maternal or paternal copy that is
inherited by an organism
New imprinting occurs during gametogenesis – – the maternal and paternal copies of the
genome must first by demethylated and then imprinted/remethylated according to the
formation of ova or sperm
o if a gene is said to be maternally imprinted, then the gene is
transcriptionally silenced when inherited from the mother
o if a gene is paternally imprinted, then the copy of the gene that was
inherited from the father will be transcriptionally silenced
 In terms of advantage to a species, this process seems to be risky because only
one copy of the gene will be active – if that copy is defective, this organism will be at
risk.
 Based on the imprinting of the Igf-1 gene in mice (Insulin-like growth factor), it is
has been suggested that paternal imprinting conveys some advantage to the growth of
a developing fetus, whereas maternal imprinting counteracts the paternal expression
pattern
 Prader-Willi and Angelman syndrome are two disease resulting from a deletion in
a region of the chromosome with imprinted genes. Because there is not a second copy
of the gene present when some of the genes are maternally or paternally imprinted, the
patient is completely lacking the gene, leading to the symptoms of Prader-Willi or
Angelman syndromes.
11.7 RNA processing and stability
Alternative splicing
mRNA Stability
deadenylation dependent pathway: polyA tail length – if too short (25-60nt), the mRNA
is unstable, i.e. it is susceptible to degradation by first a decapping mechanism and then
digestion by exonucleases
deadenylation independent pathway – may also involve decapping and exonucleases, or
endonucleases – truncated or unspliced mRNA’s are degraded this way
NMD: nonsense mediated decay – if a mRNA contains a stop codon upstream of a splice
site, the mRNA will be degraded
RNAi
dsRNA (double-stranded RNA) of a specific gene sequence will direct the degradation of
that gene’s mRNA
In C. elegans this is propagated throughout the organism from the site of entry of dsRNA,
C.elegans has enzyme activity Dicer which cuts the RNA into 21-22 nt dsRNA chunks
the 21-22 nt duplexes. The duplexes enter a protein complex called RISC (RNA-induced
silencing complexes). The RISC complexes unwind the duplex, allow the RNA to bind to
its complementary sequence in cellular mRNA, and then cleave the mRNA to form a new
duplex
RNAi is a very useful tool in genetic studies – it is an easy way to knock out any gene.
RNAi works in many other mammalian tissues – even humans! For it to work in humans
and other mammals, the dsRNA must be small – 21-22 nt. Longer dsRNA activates an
antiviral pathway that will destroy the dsRNA and not allow the RNAi pathway to be
activated. Because dsRNA in human cells will selectively turn off a particular gene, this
is an area of intense research and may prove to be very useful and important in the
treatment of various diseases.
Many small RNA’s (around 20-25nt) are naturally expressed in humans and other
organisms – they have a regulatory role in development and tissue-specific expression of
genes. Their mechanism is still an area of active research and some disagreement – the
mechanism may be at the level of RNA stability and there is also evidence for a role in
translational control.
11.8 Translational control
Proteins, small regulatory RNAs, and elements in the 5’and 3’UTR (untranslated region)
of mRNA are involved in the control of the translation of mRNAs. They may control
expression of a gene by:
o Preventing initiation of translation of an mRNA, or activating the initiation of
translation of an RNA
o Changing the rate of protein synthesis (or elongation phase of translation)
o Halting translation in the middle of the coding region – this could also lead to
subsequent degradation of the mRNA
Many different mechanisms – many involve proteins that bind to the mRNA directly, also
involve RNA’s that base pair with regions of the mRNA – these RNA structures may
recruit proteins that in turn affect translation
11.9 DNA Rearrangements
o In Xenopus (toad) genes for the ribosomal RNA (“rDNA”) are amplified 4000x
during oogenesis, in order to increase the number of ribosomes, which are needed
for the huge amount of protein production taking place. The genes are replicated
in the form of extrachromosomal rolling circles of replicating DNA.
o In the antibody-producing cells of the immune system, the B cells, DNA
rearrangement takes place in the genes for the heavy and light chain of
immunoglobulin protein (antibody). Differential joining of V segments and J
segment leads to a large number of possible combinations, creating a huge array
of different immunoglobulin molecules, each one with a different antigen-binding
site.
o Mating-type interconversion in yeast occurs by DNA rearrangement, initiated
when an endonuclease makes a double stranded DNA cut to excise the MAT gene
and insert a copy of a cassette containing the  or a mating type genes. The genes
within each region then direct the mating type of the yeast cell through
transcriptional activation or repression.