Transcriptional and post transcriptional regulation of gene expression

Transcriptional and post transcriptional
regulation of gene expression
Presented by :
Kirti
Ph.D. (MBB)

What is gene expression ?
• It is the process by which information from a gene is used in the
synthesis of a functional gene product.
• These products are often proteins, but in non coding genes such
as rRNA genes or tRNA genes, the product is functional RNA.
• Prokaryotic organisms regulate gene expression in response to
their environment.
• Eukaryotic cells regulate gene expression to maintain
homeostasis in the organism.

Classification of genes with respect to their expression
Constitutively expressed genes ( housekeeping genes ) :
• Are expressed at fixed rate, irrespective of cell condition.
• The genes that are involved in vital biochemical processes
such as respiration.
• Their structure is simpler.
Regulated gene expression (Controllable genes) :
• Are expressed only as needed. Their amount may increase or
decrease with respect to their basal level in different
condition.
• Their structure is relatively complicated with some response
elements.

An overview of Transcription
.

How is gene expression controlled?
• Gene activity is controlled first and foremost at the level of
transcription.
• Much of this control is achieved through the interplay between
proteins that bind to specific DNA sequences and their DNA
binding sites.
• Gene-regulation mechanisms in prokaryotes particularly in E. coli
have been extensively investigated.

Transcription is more selective
During Replication the entire chromosome is usually copied but
transcription is more selective
Only particular genes or group of genes are transcribed at any
one time and some portions of DNA genome are never
transcribed
Thus cell restricts the expression of genetic information to the
formation of gene products needed at any particular moment

Modern microarray analysis of transcription pattern has
revealed that much of genome of humans and other mammals
is transcribed into RNA
involved in regulation of gene expression
3 Phases of Transcription : Initiation
Elongation
Termination
Initiation phases of DNA binding
initiation of RNA synthesis

Structure and
nomenclature of a typical
gene
Most genes are made up
of 3 regions
Structural gene
• Centre sequence that is copied in the form of RNA
• Concerned with the synthesis of polypeptide chain or a number of
polypeptide chains
• In Eukaryotes, these occur in spilt-form, segmented into introns and
exons
• In Prokaryotes, these are continuous
Structural gene
Promoter region
Terminator region

Promoter sequences in DNA
Regulatory region of DNA usually located to the 5 ’ end or upstream from
structural gene
 well characterised in prokaryotes such as E.coli
 binding of transcription factors
 comprise two highly conserved sequences
1. Pribnow box or the TATA box (six nucleotides consensus sequence
TATAAT) 10 bp upstream from the transcription initiation site (-10 )
 essential to start transcription in prokaryotes
 involved in the formation of transcription initiation complex

2. the - 35 region -25 bp upstream from the TATA box has
the consensus sequence TTGACA important in initial recognition
and binding of RNA polymerase to the DNA the first nucleotide
transcribed is usually a purine.
Eukaryotic promoter
Eukaryotic promoters are extremely diverse and are difficult to
characterize.
They typically lie upstream of the gene and can have regulatory
elements several kilobases away from the transcriptional start
site.

.
Many eukaryotic promoters also have a CAAT box with a
GGNCAATCT consensus sequence centered at about -75.
Eukaryotic genes encoding proteins have promoter sites with a
TATAAA consensus sequence, called a TATA box or Hogness box,
centered at about - 25.

Promoters differ markedly in their efficacy
Genes with strong promoters are transcribed frequently as often as
every 2 seconds in E. coli.
In contrast, genes with very weak promoters are transcribed about
once in 10 minutes.
The -10 and -35 regions of most strong promoters have sequences
that correspond closely to the consensus sequences
Weak promoters tend to have multiple substitutions at these sites.

Promoter element serve as binding site for trans-acting
element (transcription factor or DNA-binding proteins that
control gene expression)
have DNA-binding domain for interaction with promoters
and an activation domain to allow interaction with other
transcription factors
The distance between these conserved sequences is also important;
a separation of 17 nucleotides is optimal. Thus, the efficiency or
strength of a promoter sequence serves to regulate transcription.
Mutation of a single base in either the -10 sequence or the -35
sequence can diminish promoter activity.

Enhancer element
Transcription of eukaryotic genes is stimulated by enhancer
sequence, which can be quite distant (several kb) from the start
site, on either its 5’ or 3’ side.
DNA between the enhancer and the promoter loops out to allow
the proteins bound to the enhancer to interact directly either with
one of the general transcription factors or with RNA polymerase
itself.

Transcription begins when DNA- dependent RNA polymerase
binds to promoter region and moves along the DNA to the
transcription unit.
• In prokaryotes only single enzyme, RNA polymerase governs the
synthesis of all cellular RNAs.
• There are 3 different eukaryotic RNA polymerases that are
transcribed by 3 different sets of genes and are distinguished by
their sensitivity to a fungal toxin α–amanitin :
• located in nucleolus
• Synthesises rRNAs
• Sensitive to α -amanitin
RNA
polymerase I
• located in nucleoplasm
• Synthesises snRNA
• Very sensitive to α -amanitin
RNA
polymerase I I
• located in nucleoplasm
• Synthesises tRNA, 5SrRNA
• Moderately sensitive to α -amanitin
RNA polymerase
I I I

Structure of Prokaryotic RNA Polymerase
Upstream
DNA
Downstream
DNA

RNA polymerase elongates an RNA strand by adding ribonucleotide
units to the 3’ hydroxyl end, building RNA in the 5’ 3’ direction
RNA polymerase from E. coli is a very large (~400 kd) and complex
enzyme consisting of four kinds of subunits: α2, β, β’, σ constituting an
holoenzyme.
The σ subunit helps find a promoter site where transcription begins,
participates in the initiation of RNA synthesis and then dissociates
from the rest of the enzyme
RNA polymerase without this subunit (α2, β, β’) is called the
core enzyme
contains catalytic site 


Template
The template strand is the strand from which the RNA is actually
transcribed. It is also termed as antisense strand.
The coding strand is the strand whose base sequence specifies the
amino acid sequence of the encoded protein. It is also called as
sense strand.
G C A G T A C A T G T C5' 3'
3' C G T C A T G T A C A G 5' template
strand
coding
strand
RNAG C A G U A C A U G U C5' 3'
transcription

Figure 7-9 Essential Cell Biology (© Garland Science 2010)
Promoter and terminator sequences of a gene tell the RNA
polymerase where to start and stop transcription

‘the promoter site is encountered by a random walk in
one dimension rather than in three dimensions’
The holoenzyme binds to duplex DNA and moves along the
double helix in search of a promoter, forming transient
hydrogen bonds with exposed hydrogen-donor and -acceptor
groups on the base pairs.
The search is rapid because RNA polymerase slides along DNA
instead of repeatedly binding and dissociating from it.

The σ subunit contributes to specific initiation in two ways:
1. It decreases the affinity of RNA polymerase for general
regions of DNA by a factor of 104.
In its absence, the core enzyme binds DNA indiscriminately
and tightly.
2. The σ subunit enables RNA polymerase to recognize
promoter sites.
A large fragment of a σ subunit was found to have an a helix
on its surface; this helix has been implicated in recognizing the
5’-TATAAT sequence of the -10 region.

Different types sigma factor specific for sets of genes :
Sigma factor 70 (MW = 70 kDa) is most common form -initiates
transcription at most promoters.
Sigma factor 32 (MW = 32 kDa) is produced after heat shock –
initiates transcription at promoters of genes needed for responding
to heat.
Sigma factor 54 turns on genes for nitrogen utilization.
Bacteriophage produces a powerful sigma factor that
preferentially transcribes the phage DNA instead of the bacterial
DNA.

Promoters of heat shock proteins
Products of this set of genes are made at higher levels
when the cell receives a sudden increase in
temperature
RNA polymerase bind to the promoters of these genes
only when σ70 is replaced with σ32 subunit, specific
for heat shock promoters

RNA synthesis can start de novo, without the requirement
for a primer.
RNA Polymerase unwind the template double helix for over
a short distance, forming transcription ‘bubble’.
Elongation takes place at transcription bubbles that move
along the DNA template.
RNA polymerase stays bound to its template until a
termination signal is reached
The region containing RNA polymerase, DNA and
nascent RNA is called a transcription bubble.

E. coli generally keeps about 17 bp unwind.
Elongation of transcript by E.coli
RNA polymerase proceeds at a rate
of 50-90 nucleotides/sec.

The RNA-DNA helix formed is about 8 bp long and corresponds to
nearly one turn of a double helix.
The σ factor dissociates at random as polymerase enters the
elongation phase.
The NusA protein binds to the elongating RNA polymerase,
competitively with the σ subunit.
Once the transcription is complete, NusA dissociates from the
enzyme, the RNA polymerase dissociates from the DNA and
σ factor can now bind to the enzyme to initiate
transcription.

Termination of RNA synthesis
1. Intrinsic Termination –
RNA transcript with self-complementary sequences
Permitting the formation the formation of hairpin loop structure
Centered 15-20 nucleotides before the projected end of the RNA
Highly conserved string of the A residues in the template strand
are transcribed into U residues near the 3’ end of the hairpin.
Formation of hairpin structure in the RNA disrupts several A=U
bp in the RNA-DNA hybrid segment
Disrupt interaction between RNA and the RNA polymerase

2. rho – dependent termination (-dependent termination).
Lack the sequence of repeated A residues in the template
strand.
Contain CA-rich sequence – rut (rho utilization element).
ρ protein associates with the RNA at the binding sites and
migrates in 5’-3’ direction (reaches transcription complex
that is paused).
ρ factor has an ATP-dependent RNA-DNA helicase activity
that promotes translocation of the protein along RNA and
ATP is hydrolysed.

Control of Eukaryotic
Gene Expression
Eukaryotes have more
complex means to
regulate gene
expression because
they have
compartments (e.g.,
nucleus) within cells
and multicellular
structures that require
cell differentiation.

Successful binding of active RNA polymerase II
holoenzyme at one of its promoter usually requires the
action of other proteins:
1. Transcription activators
2. Coactivators
3. Basal transcription factors
4. Chromatin modification and remodeling

Transcription activators
Mediate positive gene regulation.
Binds to specific regulatory DNA sequences (e.g. enhancers) &
enhance the RNA polymerase -promoter interaction.
It actively stimulates transcription.
Common in eukaryotes.
On binding to repressor, it brings confirmational changes which
leads to dissociation of repressor from the operator & increase in
transcription.

How do the activators function at a distance?
The intervening DNA is looped so that the various protein
complexes can interact directly.
Looping is promoted by certain nonhistone proteins that are
abundant in chromatin and bind nonspecifically to DNA
High mobility group (HMG)
play an important structural role in chromatin remodeling and
transcriptional activation

Coactivator Protein Complexes
Eukaryotic coactivator consists of 20 to 30 or more polypeptides in a
protein complex called mediator.
Mediator binds tightly to the carboxyl-terminal domain (CTD) of
largest subunit of Pol II.
Transcription activators interact with one or more components of the
mediator complex.
Coactivator complexes function at or near the promoter’s TATA box.

Activators
DNA
Enhancer Distal control
element
Promoter
Gene
TATA box
General
transcription
factors
DNA-
bending
protein
Group of mediator proteins
RNA
polymerase II
RNA
polymerase II
RNA synthesis
Transcription
initiation complex
Figure 18.10-3

What are Basal and Regulatory transcription factors?
The basal transcription factors bind to the nearby promoter
region to the start site.
For example TATA element or initiator sequence.
They are the minimal complement of proteins necessary to
reconstitute accurate transcription from a minimal
promoter.

So the whole TFIIA,B,C,D,E,H,K family of factors are basal
transcription factors. They are the ones that bind in and
out of the RNA polymerase region.
Regulatory transcription factors are TF factors that bind to
sequences farther away from the initiation site.
Serve to modulate levels of transcription.
They are also called activators and bind to specific
enhancer sequences way upstream from the start site.

Protein-DNA interaction
Regulatory proteins generally bind to specific DNA
sequences.
Affinity is 104 to 106 times higher than their affinity for any
other DNA sequences.
Typically, a protein-DNA interface consists of 10 to 20
contacts that involves different amino acids
each contributing to the binding energy of the protein-DNA
interaction.

How the different
base pairs in DNA
can be recognized
from their edges
without the need
to open the double
helix?

The binding of a gene regulatory protein to the major groove of
DNA
Most of chemical groups that differ among the
four bases, permit discrimination between
base pairs are hydrogen bond donor and
acceptor groups exposed in major
groove and most of the protein-DNA
contacts that impart specificity are hydrogen
bonds.
Within regulatory proteins, the amino acid
side chains most often hydrogen-bonding to
bases in the DNA are those of Asn, Gln, Glu,
lys, arg residues.

Regulatory proteins possess DNA-binding motifs
- Helix-Turn-Helix motif
homeodomain motif
- Zinc finger motif
Regulatory proteins having protein-protein interaction domains
- Leucine Zipper motif
- Helix-Loop-Helix

Helix-Turn-Helix Motif
All of the proteins bind DNA as dimers in which the two copies of the recognition
helix are separated by exactly one turn of the DNA helix (3.4 nm).

First DNA-binding protein motif identified
It comprises about 20 amino acids in two short α helices, each 7-9 amino
acids residues long, separated by β-turn and is found in many proteins that
regulate gene expression.
First identified in 3 prokaryotic proteins: two repressor proteins (Cro and
cI) and the E. coli catabolite activator protein (CAP).
This structure generally is not stable by itself, it is simply the reactive
portion of somewhat larger DNA-binding domain.
One of the 2 helices is recognition helix because it usually contains many of
the amino acids that interact with the DNA in a sequence specific way.
When bound to DNA, the recognition helix is positioned in or nearly in the
major groove.
The lambda repressor and Cro proteins control bacteriophage lambda gene
expression, and the tryptophan repressor and the catabolite activator
protein (CAP) control the expression of sets of E. coli genes.

Homeodomain motif
The homeodomain is folded into 3 alfa helices, packed tightly together by
hydrophobic interactions (A) The part containing helix 2 and 3 closely resembles the
helix-turn-helix motif, with the recognition helix (red) making important contacts
with the major groove (B). The Asn of helix 3, for example, contacts an adenine.
Nucleotide pairs are also contacted in the minor groove by a flexible arm attached
to helix 1. The homeodomain shown here is from a yeast gene regulatory protein.

The homeodomain is a DNA-binding domain of 60 amino acids that has 3 α-
helices.
The C-terminal α-helix-3 is 17 amino acids, binds in the major groove of DNA.
The N-terminal arm of the homeodomain projects into the minor groove of DNA.
Proteins containing homeodomains may be either activators or repressors of
transcription i.e. they function as transciptional regulators, especially during
eukaryotic development.
it was discovered in homeotic genes (genes that regulate the development of
body patterns)
highly conserved, identified in proteins from wide variety of organisms
including humans

Zinc finger motif
(A) The gene regulatory protein bound to a specific DNA site. This protein recognizes
DNA using three zinc fingers of the Cys-Cys-His-His type arranged as direct repeats.
(B) The three fingers have similar amino acid sequences and contact the DNA in
similar ways. In both (A) and (B) the zinc atom in each finger is represented by a
small sphere.

• About 30 amino acids residues form an elongated loop held together at
the base by a single Zn2+ ion, coordinated to four of the 2 residues
(four Cys, or two Cys and two His).
• Zinc does not itself interact with DNA.
• The coordination of Zinc with the amino acid residues stabilizes this
small structural motif.
• The interaction of a single Zinc finger with DNA is typically weak.
• DNA-binding proteins like Zif268 have multiple Zinc fingers that
enhance binding by interacting simultaneously with DNA.

 Zinc finger domain exists in two
forms:
1. C2H2 zinc finger: a loop of 12 amino
acids anchored by two cysteine and two
histidine residues that tetrahedrally co-
ordinate a zinc ion.
This motif folds into a compact
structure comprising two β-strands and
one α-helix.
The α-helix containing conserved basic
amino acids binds in the major groove
of DNA.

Examples:
(1) TFIIIA, the RNA Pol III transcription factor, with C2H2 zinc
finger repeated 9 times.
(2) SP1, with 3 copies of C2H2 zinc finger.
Usually, three or more C2H2 zinc fingers are required for DNA
binding.
2. C4 zinc finger: zinc ion is coordinated by 4 cysteine residues.
Example:
Steriod hormone receptor transcription factors consisting of
homo- or hetero-dimers, in which each monomer contains two C4
zinc finger.

• This motif is an amphipathic α-helix with a series of
hydrophobic amino acid residues concentrated on one side
the hydrobhobic surface forming an area of contact between the two
polypeptides of a dimer.
• Stricking feature :
Leu residue at every seventh position form a straight chain along
the hydrophobic surface.
• Regulatory proteins with leucine zippers
DNA binding domain with a high concentration of Lsy or Arg residues
interact with negatively charged phosphates of the DNA backbone.
• Leucine zippers have been found in many eukaryotic and a few
bacterial proteins.

• Structural motif in eukaryotic regulatory proteins implicated
in control of gene expression during the development of
multicellular organisms.
• They share a conserved region of about 50 amino acid
residues important in DNA binding and protein
dimerization.
• Two short amphipathic α-helices linked by a loop of variable
length.
• The Helix-Loop-Helix motifs of two polypeptides interact to
form dimers.
• DNA binding is mediated by an adjacent short amino acid
sequence rich in basic residues.

Eukaryotic gene expression is regulated at many stages
Transcriptional ground state is restrictive – strong promoters
are inactive in vivo in absence of regulatory proteins.
In bacteria, it is nonrestrictive, RNA polymerase has access to
every promoter , bind and initiate transcription at some level of
efficiency in the absence of activators or repressor.

Distinguish the regulation of gene expression in
eukaryotes from that in bacteria
1. Access to eukaryotic promoter is restricted by structure of
chromatin.
2. Positive mechanisms predominate – transcriptional ground state
is restrictive – every gene requires activation in order to
transcribe.
3. Eukaryotic cells have larger, more complex multimeric
regulatory proteins than do bacteria.
4. Transcription in the eukaryotic nucleus is separated from
translation in cytoplasm in both space and time.

Transcriptionally active chromatin is structurally
distinct from inactive chromatin
It is darkly stained region of the
chromatin.
It is compactly coiled regions
and with more DNA.
It is genetically inert as can not
transcribe mRNA due to tight
coiling.
It is late replicative.
About 10% of chromatin in
typical eukaryotic cell is in
more condensed form.
Associated with centromeres.
It is lightly stained region.
It is loosely coiled region and
with less DNA.
It is genetically active.
It is early replicative.
Heterochromatin Euchromatin

Chromatin Remodeling
is the modification of chromatin
architecture to allow access of condensed
genomic DNA to the regulatory
transcription machinery proteins, and
thereby control gene expression.
 Covalent histone modifications
 ATP-dependent chromatin remodeling complexes
Each of the core histones has two distinct structural domains:
1. Central domain is involved in histone-histone interaction
and wrapping of DNA around the nucleosome.
2. Lysine rich amino-terminal domain – positioned
exterior of nucleosome assembly.

Methylation of specific lysine residues in H3 and H4 causes
condensation of DNA around histones preventing binding of
transcription factors to the DNA leading to gene repression.
Methylation of lysines H3K4 and H3K36 is correlated with
transcriptional activation while demethylation of H3K4 is
correlated with silencing of the genomic region.
These methylations facilitates the binding of histone
acetyltransferase (HATs) - acetyl groups are attached to
positively charged lysines in histone tails (N-terminal).
This loosens chromatin structure, thereby promoting the
initiation of transcription.

Histone
tails
DNA
double
helix
Nucleosome
(end view)
Amino acids
available
for chemical
modification
(a) Histone tails protrude outward from a nucleosome
Unacetylated histones
Acetylated histones
(b) Acetylation of histone tails promotes loose chromatin structure
that permits transcription

Acetylation induces a conformational change in
the core histones
Note: acetylation neutralizes
the positive charge of lysine
HAT: Histone Acetyltransferase

DNA Methylation
• Methylation near gene promoters varies considerably depending
on cell type, with more methylation of promoters correlating with
low or no transcription (Suzuki & Bird, 2008).
• Proteins that bind to methylated DNA also form complexes with
the proteins involved in deacetylation of histones.
• Therefore, when DNA is methylated, nearby histones are
deacetylated, resulting in compounded inhibitory effects on
transcription. Likewise, demethylated DNA does not attract
deacetylating enzymes to the histones, allowing them to remain
acetylated and more mobile, thus promoting transcription.

• In Neurospora crassa (Tamaru & Selker, 2001) and Arabidopsis
thaliana (Jackson et al., 2002), H3-K9 methylation (methylation of a
specific lysine residue in the histone H3) is required in order for
DNA methylation to take place.
• When transcription of a gene is no longer required, the extent of
acetylation of nucleosomes in that vicinity is reduced by the
activity of histone deacetylation (HDACs).
• Methylation of lysines H3K9 and H3K27 is correlated with
transcriptional repression (Rosenfeld et al., 2009 "Determination of
enriched histone modifications in non-genic portions of the human genome."
BMC Genomics 10: 143).
• H3K9 is highly correlated with constitutive heterochromatin
(Hublitz et al., 2009) "Mechanisms of Transcriptional Repression by Histone
Lysine Methylation" The International Journal of Developmental Biology
(Basel) 10 (1387): 335–354).

In vertebrates and plants, many genes contain CpG islands near their
promoters
1,000 to 2,000 nucleotides long
In housekeeping genes
The CpG islands are unmethylated
Genes tend to be expressed in most cell types
In tissue-specific genes
The expression of these genes may be silenced by the
methylation of CpG islands

ATP-dependent chromatin remodeling complexes
The three best-characterized classes of ATP-dependent chromatin-
remodelling enzyme are the SWI/SNF, CHD (chromodomain and
helicase-like domain) and ISWI (imitation SWI) families.
Each has a unique domain (bromo, chromo), known to interact with
specific chromatin substrates.
NURF, member of ISW1 family, remodels chromatin in a way that
complement and overlap the activity of SWI/SNF.
SWI/SNF complex contain bromodomain near the carboxy terminus of
active ATPase subunit, which interacts with acetylated histone tails.

SWI/SNF opens up DNA region where RNA
Pol II, transcription factors and co-activators
bind to turn on gene transcription.
In the absence of SWI/SNF, nucleosomes can
not move farther and remain tightly aligned to
one another.
Additional methylation by methylase and
deacetylation by HDAC proteins condenses
DNA around histones, make DNA unavailable
for binding by RNA Pol II and other activators,
leading to gene silencing.

ATP-dependent chromatin-remodeling complexes regulate gene
expression by either moving, ejecting or restructuring nucleosomes.
These protein complexes have a common ATPase domain and energy
from the hydrolysis of ATP allows these remodeling complexes to
reposition (slide, twist or loop) nucleosomes along the DNA, expel
histones away from DNA or facilitate exchange of histone variants,
and thus creating nucleosome-free regions of DNA for gene
activation (Wang et al., 2007 "Chromatin remodeling and cancer, Part II: ATP-
dependent chromatin remodeling." Trends Mol Med. 13 (9): 373–80).

Examples of transcriptional regulation
1. Constitutive transcription factors: SP1
Binds to a GC-rich sequence with the consensus sequence
GGGCGG.
Binding site is in the promoter of many housekeeping genes.
It is a constitutive transcription factor present in all cell types.
Contains three zinc finger motifs and two glutamine-rich
activation domains.

2. Hormonal regulation: steroid hormone receptors
Many transcription factors are activated by hormones.
Steroid hormones: lipid soluble and can diffuse through cell
membranes to interact with transcription factors called
steroid hormone receptors.
In the absence of steroid hormone, the receptor is bound to
an inhibitor, located in the cytoplasm.

In the presence of steroid hormone,
the hormone binds to the receptor and releases the
receptor from the inhibitor,
receptor dimerize and translocate to the nucleus.
receptor interacts with its specific DNA-binding sequence
(response element) via its DNA-binding domain,
activating the target gene.

Case Study
Positive and Negative Regulation of Transcription of the Yeast
Galactose Utilization Genes
Three genes encode enzymes for metabolizing galactose
GAL1 encodes galactokinase
GAL7 encodes galactose transferase
GAL10 encodes galactose epimerase
Yeast cells have no operon - each of the GAL gene is transcribed
separately.

In the absence of galactose, a Gal4p dimer binds the UASG
(upstream activator sequence) along with the repressor protein
Gal80p. No transcription occurs (quenching).
In the presence of galactose, Gal80p is bound to the inducer. A shift
occurs, exposing the Gal4p activation domain; transcription
proceeds
Gal4p – transactivator
Gal80p – repressor
Galactose – effector
Glucose is the preferred carbon source for yeast. When glucose is
present, most of the GAL genes are repressed – whether galactose
is present or not.

An operon is a cluster of bacterial genes along with an adjacent
promoter that controls the transcription of those genes.
They usually control an important biochemical process.
They are only found in prokaryotes.
Francois Jacob and Jacques Monod (1962) -
first proposed the operon model of gene regulation
For their significant contribution in field of
biochemistry, they were awarded Nobel Prize in
Medicine in 1965.
The first system of gene regulation that
was understood was the lac operon in
E. coli.
Jacob, Monod & Lwoff

What are inducible genes ?
• They can be turned on or off
– depending on the environment they are in.
• An Inducer acts as a ‘switch’ to turn the gene on or off.
– a chemical substance in the nutrient medium
• The Inducer influences the transcription of the inducible
gene(s) via controlling sites called Operators
– on the DNA adjacent to the coding sequence of the gene(s).
• The Operator is usually where a regulatory protein binds.

General Organization of an Inducible Gene

Regulatory Proteins can activate or block
transcription of inducible genes

The Lac operon - showing its genes and its binding sites.
The promoter is a specific DNA sequence to which the RNA Polymerase binds.

You can classify genes in a simple way in two classes
structural genes
• are those that produce the enzyme required for lactose
metabolism
regulatory elements
• determine whether transcription of the structural genes
will occur. They monitor and respond to environmental
conditions (presence of lactose)
The structural genes and the regulatory elements form a
functional genetic unit called the Lac Operon.

The lac operon
The lac operon consists of three genes each involved in
processing the sugar lactose:
1. lacZ encodes β-galactosidase (LacZ), an intracellular enzyme
that cleaves the disaccharide lactose into glucose and
galactose.
2. lacY encodes β-galactoside permease (LacY), a membrane-
bound transport protein that pumps lactose into the cell.
3. lacA encodes β-galactoside transacetylase (LacA), an enzyme
that transfers an acetyl group from acetyl-CoA to β-
galactosides.

The primary enzymatic function of β-galactosidase relevant to its
role as a biotechnological tool is to cleave the chemical bond
between the anomeric carbon and glycosyl oxygen of appropriate
substrates (Serebriiskii & Golemis, 2000).
The lac operon regulatory elements are distributed along the DNA
chain as (Reznikoff, 1992; Muller-Hill, 1998): the lac promoter is
located between bp - 36 (relative to the starting point of gene lacZ,
bp +1) and bp -7.

Prokaryotic genes are polycistron systems
All 3 genes of the lac operon are transcribed on the same
messenger RNA.
Ribosomes translate the 3 proteins independently.
Unique feature of prokaryotes that is only very rarely seen in
eukaryotes, where 1 gene per mRNA is the rule.

Adapting to the environment
• E. coli can use either glucose (monosaccharide) or
lactose (disaccharide).
• Glucose needs to be hydrolysed (digested) first
So the bacterium prefers to use glucose when it can.
• It would be energetically wasteful for E. coli if the lac
genes were expressed when lactose was not present.
• It achieves with the lac repressor which halts the
production in the absence of lactose and EIIAGlc, which
shuts down lactose permease when glucose is being
transported into the cell.

Lac operon uses a two-part control mechanism
Diauxic dual control mechanism causes the sequential
utilization of glucose and lactose in two distinct growth
phases.
This phenomenon was originally studied by Monod (1941).
The first control mechanism is the regulatory response to
lactose, which uses an intracellular regulatory protein
called the lactose repressor to hinder production of β-
galactosidase in the absence of lactose.

Diauxic growth curve
The existence of two
different exponential
growth phases, separated
by a short interval in
which the culture does not
grow. The first phase
corresponds to the
bacterial culture feeding
on glucose (lactose), while
the interval with no
growth corresponds to the
time the bacteria need to
turn on the genes needed
to metabolize lactose after
glucose exhaustion.

At low glucose concentrations, phosphorylated EIIA accumulates
and this activates membrane-bound adenylate cyclase.
Intracellular cyclic AMP levels rise and this then activates CAP
(catabolite activator protein), which is involved in the catabolite
repression system, also known as glucose effect.
When the glucose concentration is high, EIIA is mostly
dephosphorylated and this allows it to inhibit lactose permease.

When the genes in an operon are transcribed, a single
mRNA is produced for all the genes in that operon. This
mRNA is said to be polycistronic because it carries the
information for more than one type of protein.

The regulatory gene lacI (constitutive) produces an mRNA
that produces a Lac repressor protein, which can bind to
the operator of the lac operon.
The Lac regulatory protein is called a repressor because it
keeps RNA polymerase from transcribing the structural
genes
Thus the Lac repressor inhibits transcription of the lac
operon

Structure of Lac repressor
The binding sites for regulatory proteins are often inverted
repeats of short DNA sequences (a palindrome) at which
multiple (usually two) subunits of a regulatory protein bind.
The lac repressor is a homotetramer (dimer of dimers consisting
of two functional homodimers of 37-kd subunits ) of lacI
polypeptides (Lewis, 2005; Wilson et al., 2007).
An E. coli cell usually contains about 20 tetramers of the Lac
repressor.
Each of the tethered dimers separately binds to a palindromic
operator sequence, in contact with 17 bp of a 22 bp region in the
lac operon.

Each dimer binds to a
distinct DNA sequence at –
82 and +11 respective to
transcription start site.
This results in DNA
looping, preventing the DNA
polymerase from binding to
–35 and –10 sequences.

In the absence of lactose, the Lac repressor binds to the operator
and keeps RNA polymerase from transcribing the lac genes.
Effect of Lac repressor on the lac genes is referred to as negative
regulation.

When lactose is present, the lac genes are expressed because
allolactose binds to the Lac repressor protein and keeps it physically
from binding to the lac operator.
In the "induced" state, the lac repressor is NOT bound to the
operator.

Allolactose is an isomer of lactose. Small amounts of allolactose are
formed when lactose enters E. coli.
Allolactose binds to an allosteric site on the repressor protein
causing a conformational change
repressor can no longer bind to the operator region and falls off
RNA polymerase can then bind to the promoter and transcribe the
lac genes

Allolactose is called an inducer because it turns on or induces the
expression of the lac genes.
The presence of lactose (and thus allolactose) determines whether
or not the Lac repressor is bound to the operator.

When the enzymes encoded by the lac operon are produced, they
break down lactose and allolactose, eventually releasing the
repressor to stop additional synthesis of lac mRNA.
Whenever glucose is present, E. coli metabolizes it before using
alternative energy sources such as lactose, arabinose, galactose,
and maltose.
Glucose is the preferred and most frequently available
energy source for E. coli.
The enzymes to metabolize glucose are made constantly by
E. coli.

When both glucose and lactose are available, the genes for lactose
metabolism are transcribed at low levels.
Sometimes the transport of glucose blocks the transport of the
inducer of the lac operon
Inducer exclusion
External glucose decreases the efficiency of lac permease to
transport lactose (Reznikoff 1992), and by doing so negatively
affects the induction of the operon genes.
Only when the supply of glucose has been exhausted does RNA
polymerase start to transcribe the lac genes efficiently, which
allows E. coli to metabolize lactose.

Maximal transcription of the lac operon occurs only when glucose
is absent and lactose is present.
The action of cyclic AMP and a catabolite activator protein produce
this effect.
Catabolite Activator Protein (CAP; also known as cAMP
receptor protein, CRP) :
 transcriptional activator, exists as a homodimer in solution, with
each subunit comprising a ligand-binding domain at the N-
terminus and a DNA-binding domain at the C-terminus
 helix-turn-helix structure - binding is mediated by a helix-turn-
helix motif in the protein’s DNA binding domain.

CAP binds a specific DNA site upstream from the lac promoter, and
by doing so it increases the affinity of the RNA polymerase for this
promoter (Reznikoff 1992). This regulatory mechanism is known as
catabolite repression.
It binds to successive major grooves on DNA
this opens the DNA molecule up
allowing RNA polymerase to bind
transcribe the genes involved in lactose catabolism
Thus, CAP enhances the expression of the lac operon when lactose
is present, but not glucose.

The presence or absence of glucose affects the lac operon by
affecting the concentration of cyclic AMP.
The concentration of cyclic AMP in E. coli is inversely proportional
to the concentration of glucose: as the concentration of glucose
decreases, the concentration of cyclic AMP increases.

When the concentration of glucose is low, cAMP accumulates in
the cell. The binding of cAMP and the catabolite activator protein
to the lac promoter increases transcription by enhancing the
binding of RNA polymerase to the lac promoter.

In the presence of lactose and absence of glucose, cyclic AMP
(cAMP) joins with a catabolite activator protein that binds to the
lac promoter and facilitates the transcription of the lac operon
and stimulates RNA transcription 50 fold.

CAP-cAMP is a positive regulatory element responsive to glucose
levels whereas the lac repressor is a negative regulatory element
responsive to lactose.
The open complex of RNA polymerase and the promoter does not
form readily unless CAP-cAMP is present.
CAP interacts directly with RNA polymerase through the
polymerase’s α-subunit.

There are two ways of making a mutant strain where
the lac operon is always on, regardless of whether
lactose is present or not.
1. i- mutation: the lacI gene does not produce a functional
repressor protein no repressor to bind to the operator RNA
polymerase is never inhibited lac operon is always
transcribed.
i- mutants are recessive: an i+ / i- heterozygote has normal
gene regulation, because the wild type allele produces a
normal repressor.
2. Many operator mutants are constitutive, oc
The operator is mutated so that the repressor can no longer
bind to it. Transcription occurs and the lac operon is on when
no repressor is bound to the operator.

trp operon
Structure of trp operon
• .

trp operon
The E. coli operon includes 5 genes, 7-kb mRNA transcript for the
enzymes required to convert chorismate to tryptophan.
Two of the enzymes catalyse more than one step in the pathway.
The mRNA from the try operon has a half-life of only about 3 min,
allowing the cell to respond rapidly to changing needs for this
amino acids.
The trp operon is a repressible operon.

A repressible operon is one that is usually on; binding of a
repressor to the operator shuts off transcription.
Repression is associated with anabolic pathways -focus is on the
"end products" of anabolic pathways.
Regulatory gene codes for a protein (the repressor) that is
inactive in absence of trp, repressor is inactive and RNA
polymerase transcribes the structural genes.
The trp repressor is a homodimer, each subunit containing 107
amino acid residues.

A corepressor is a molecule that cooperates with a repressor
protein to switch an operon off.
For example, E. coli can synthesize the amino acid tryptophan.
By default the trp operon is on and the genes for tryptophan
synthesis are transcribed.
When tryptophan is present, it binds to the trp repressor protein,
which turns the operon off.
The repressor is active only in the presence of its corepressor
tryptophan; thus the trp operon is turned off (repressed) if
tryptophan levels are high.

Promoter
DNA
Regulatory
gene
mRNA
trpR
5
3
Protein Inactive
repressor
RNA
polymerase
Promoter
trp operon
Genes of operon
Operator
mRNA 5
Start codon Stop codon
trpE trpD trpC trpB trpA
E D C B A
Polypeptide subunits that make up
enzymes for tryptophan synthesis
(a) Tryptophan absent, repressor inactive, operon on
(b) Tryptophan present, repressor active, operon off
DNA
mRNA
Protein
Tryptophan
(corepressor)
Active
repressor
No RNA
made
Figure 18.3

The trp operator site overlaps the promoter, so binding of the
repressor blocks binding of RNA polymerase.
Repressor lowers transcription 70-fold (as compared to
derepressed state)  attentuation permits another 10-fold
control  total dynamic range of control = 700-fold.

The regulation of the trp operon is achieved by means of two
mechanisms controlling successive stages of expression:
1. Repression regulates initiation of transcription by blocking
attachment of RNAP.
2. Attenuation is responsible for premature termination of
transcription due to conformational changes in nascent mRNA.
Attenuation in the control of expression of bacterial operons
(Yanofsky C, 1981 Nature 289(5800):751-8).

…
…
Attenuator Region of Trp Operon

Low tryptophan: transcription of trp operon genes RNA
polymerase reads through attenuator.

High tryptophan: attenuation, premature termination 
attenuator causes premature termination of transcription. The
terminator consists of an inverted repeat followed by string of eight
A-T pairs. The inverted repeat forms a hairpin loop.

When RNA polymerase reaches string of U’s, the polymerase
pauses, the hairpin forms Transcript is released

 Termination occurs before transcription reaches the trp
(structural) genes.
preventing hairpin formation would destroy termination signal
 transcription would proceed

Mechanism of Attenuation
The trp operon attenuation mechanism uses signals encoded in
four sequences within 162 nucleotide leader region at the 5’ end of
the mRNA, preceding the initiation codon of the first codon.
Within the leader lies a region Attenuator, made up of
sequences 3 and 4
base pair to form a G C rich stem-and-loop structure closely followed
by a series of U residues
attenuation sequence acts as a transcription terminator
sequence 2 is a alternative complement for sequence 3

mRNA produced from attenuator region can fold into two different
secondary structures
Stem loops: 1-2, 3-4 Stem loop: 2-3

Formation of stem loop structures; 1-2 and 3-4 is more stable and
results in the formation of a termination (hairpin loop)
structure/signal.
Formation of stem loop structure 2-3 would result in the disruption
of stem loops 1-2/3-4.
The stem loop structure formed between 2-3 does not result in
termination signal  attenuator structure cannot form,
transcription would proceed.

Posttranscriptional Regulation
Control of gene expression usually involves the control of
transcription initiation.
But gene expression can be controlled after transcription, with
mechanisms such as:
– RNA editing
– RNA interference
– alternative splicing
(different mRNA molecules are produced from the same primary
transcript, depending on which RNA segments are treated as exons
and which as introns)
– mRNA degradation

RNA editing
Some mRNAs are edited before translation.
RNA editing has been observed in mRNA, tRNA and rRNA.
It has been detected in mitochondria and chloroplasts and in
nuclear encoded RNAs but as yet not in procaryotes.
RNA editing can be divided into two categories.
1. Insertion/Deletion RNA editing- nucleotides are inserted or
deleted as occurs in mitochondrial mRNAs of trypanosomes, a
kind of protozoan.

The initial transcripts of the genes that encode cytochrome oxidase
subunit II in some protist mitochondria provide an example of
editing by insertion.
These do not correspond precisely to the sequence needed at the
carboxyl terminus of the protein product.
A number of U nucleotides are inserted or deleted to create the
translatable mRNAs.
Specific small RNAs, guide RNAs interact with the mRNA to
define the position of editing.
Base pairing between the initial transcript and the guide RNA
involves a number of G=U base pairs, common in RNA molecules.

2. Nucleotides are modified to change one nucleotide into another.
One example is the de-amination of cytidine which occurs in
mammalian apolipoprotein B mRNA in the intestine.
Here, a specific C is changed into a U, introducing a stop codon for
translation and thereby a shorter version of the protein.
The cytidine deaminations are carried out by the apoB mRNA
editing catalytic peptide family of enzymes (APOBEC).

A second example is de-amination of adenosine to inosine.
Inosine is read as guanosine by the translation machinery.
The adenosine is present in a double stranded region of the mRNA
and the enzyme adenosine de-aminase that acts on RNA (ADAR)
catalyzes the reaction.
It is common in transcripts derived from the genes of primates (90%
or more of the editing occurs in the short interspersed elements
(SINES) called Alu elements).

Concentrated near protein-encoding genes, often appearing in
introns and untranslated regions at the 3’ and 5’ ends of transcripts.
The ADAR enzymes bind to and promote A-to-I editing only in
duplex regions of RNA.
Defects in ADAR function have been associated with a variety of
human neurological conditions, including amyotrophic lateral
sclerosis (ALS), epilepsy, and major depression.

dsRNA as a regulator of gene expression
 dsRNA has role in several chromatin and/ or genomic DNA
modifications, which lead in the regulation of specific genes.
 dsRNA dependent mechanism can act at both transcriptional
as well as post transcriptional levels.
This type of gene expression is given different names in different
organisms.
▫ RNA interference (RNAi) , in case of animals.
▫ Post transcriptional gene silencing (PTGS) , in case of plants.
▫ Quelling, in case of filamentous fungi.

Inhibitors of gene expression
Rifamycin
Streptovarcins
Streptolydigin
they tightly bind with
β-subunit of RNA
polymerase (rpoB) and
inhibit initiation and
transcription

Transcriptional regulation network of cold-responsive
genes in higher plants
(Tongwen Yang, Lijing Zhang , Tengguo Zhang , Hua Zhang , Shijian Xu ,
Lizhe An, 2005; Plant science 169 (2005) 987–995).
• ABA, abscisic acid;
• CBF, C-repeat-binding factor;
• COR, cold-regulated; CRT, C-repeat; DRE,
• dehydration-responsive element;
• DREB, DRE-binding factor; DREB,
• DRE-binding protein;
• ICE, inducer of CBF expression;

CBF (DREB1) genes act as nodes of regulatory network in Arabidopsis
response to cold stress.
With the use of genetic and molecular approaches, a series of regulatory
genes involved in CBF cold response pathway have been isolated and
analyzed.
Many plants increase freezing tolerance in response to low, nonfreezing
temperature, a phenomenon known as cold acclimation.
With Arabidopsis as model plant, many cold response genes were
isolated are of CBF family key components in transcriptional
regulation of cold-responsive genes.

The expression of CBFs in Arabidopsis is also regulated by ABA, light and
the circadian clock.
Arabidopsis encodes a small family of cold-responsive transcriptional
activators known either as CBF1, CBF2, and CBF3 or DREB1b, DREB1c,
and DREB1a.
The CBF transcription factors are members of the AP2/EREBP family of
DNA-binding proteins, recognize the cold- and dehydration-responsive
DNA regulatory element designated the CRT/DRE.
Have a conserved 5 bp core sequence of CCGAC, are present in the
promoter regions of many cold- and dehydration-responsive genes of
Arabidopsis, including COR.

Multiple biochemical changes that are associated with cold acclimation and
thought to contribute to increased freezing tolerance, occur in non-
acclimated transgenic Arabidopsis plants that constitutively express
CBF3.
The homologous components of the Arabidopsis CBF cold response
pathway have been found in many plants, including soybean, broccoli,
alfalfa, tobacco, wheat, corn, rice and barley.
These CBF-like proteins from different species not only have high
conserved 60 amino acid AP2/EREBP DNA-binding domain but also
present conserved amino acid sequences.
Constitutive over-expression of the Arabidopsis CBF genes in other plants
resulted in increase freezing tolerance.

Many cold-regulated genes of Arabidopsis are inducible by ABA as well as
by cold.
This occurs via two separate signaling pathways, the ABA-dependent
pathway and ABA-independent pathway.
DREB2 plays a role in drought adaptation in an ABA-independent manner.
Later three other CBF-related genes in Arabidopsis have been identified
DREB1D, E, and F.
The DREB1D (CBF4) have been demonstrated to be inducible by ABA
and drought but not by cold.
CBF1-3 transcript levels also increase in response to elevated ABA levels
and suggest that both the cold-inducible CBF transcriptional factors and the
non-cold-inducible CBF4 could be involved in activation of the CRT/DRE
by ABA.

Cold-induced gene expression through CRT/DRE is greatly enhanced by
light signaling, in which phytochrome B is required.
Light enhanced the induction kinetics of CBF1-3 encoding the transcription
factors in a consecutive manner compared to the dark condition in the cold
suggest that the connection between cold and light signaling mediated by
phytochrome is at higher step than the expression of CBF gene.
The circadian clock also gates expression of the CBF1-3 genes in response
to low temperature in Arabidopsis.
CBF3 transcripts accumulate to maximum levels in the early morning and
reach minimum levels in the early evening in Arabidopsis grown on a 12 h
photoperiod.

Transcriptome studies suggest a diversity and complexity of the cold
response pathways.
By microarrays technology and transcriptional profiling analysis
approach, 8000 genes were determined at multiple times after plants were
transferred from warm to cold temperature and in warm grown plants that
constitutively expressed CBF1, CBF2, or CBF3.
These results indicate that extensive down regulation of gene expression
occurs during cold acclimation.
CBF expression at warm temperatures repressed the expression of eight
genes that also were down-regulated by low temperature, indicating that in
addition to gene induction, gene repression is likely to play an integral
role in cold acclimation.

Potential application of regulatory factors in crop anti-freezing
engineering
Understanding the molecular mechanisms that plants have evolved to
tolerate environmental stresses has the potential to provide new tools and
strategies to improve the environmental stress tolerance of crops.
Since freezing tolerance is a multigenic trait, transformation of a single
functional gene appears to have a limited effect on crop freezing tolerance.
Because many aspects of cold adaptation process are under transcriptional
control, many transcription regulatory factors were chosen as one of the
best targets for engineering crops to achieve enhanced cold tolerance.

Constitutive over expression of the CBF genes using the cauliflower
mosaic virus 35S promoter can result in undesirable agronomic traits.
In Arabidopsis, CBF over expression can cause a ‘‘stunted’’ growth
phenotype, a decrease in seed yield and a delay in flowering.
Using stress-inducible or artificial cold-inducible promoters may be a
ideal approach to improve cold tolerance without causing negative
agronomic effects.
Though many transcription regulatory factors were cloned and identified,
only CBF genes have been successfully used to engineering cold stress
tolerance in several species.

RegulonDB (version 6.0): gene regulation model of Escherichia coli
K-12 beyond transcription, active (experimental) annotated
promoters and Textpresso navigation
(Gama-Castro et.al., 2008; Nucleic Acids Research)
RegulonDB (http://regulondb.ccg.unam.mx/) is the primary reference
database offering curated knowledge of the transcriptional regulatory
network of Escherichia coli K12, currently the best-known
electronically encoded database of the genetic regulatory network of
any free-living organism.
RegulonDB contains detailed information of the different elements
that conform the known regulatory network of the cell, such as
transcription factors (TFs), small RNAs (sRNAs) and operon
structures with their associated regulatory elements: promoters, TF
binding sites and terminators.

RegulonDB is complemented with computational analyses and genome
wide predictions of operons, promoters, TF binding sites, ribosome-
binding sites and, for the first time, RNA regulatory target sites.
Visualizing tools in RegulonDB allow the user to navigate in the genome
(Genome browser), to identify co-regulators for a particular TF, to locate
the genes’ immediate neighbors in the regulatory network, and to identify
sets of genes predicted to be functionally related (Nebulon tool).
Moreover, it also incorporates tools for the analysis of the transcriptional
regulation of global gene expression experiments made in E. coli K12
(GET tools), as well as for exhaustive analyses focused on the detection
of regulatory signals in upstream regulatory regions (RSA tools).

RegulonDB is mainly a manual database of regulatory information in E.
coli incorporated by a team of curators from the primary literature.
PubMed abstracts are selected using a set of pertinent key words related to
gene regulation.
When there is direct or suspected new relevant information, the full text of
the articles is analyzed and the data are added to RegulonDB.
Starting on January 2008, every release of RegulonDB and EcoCyc will
contain up-to-date curation with a delay of no more than 3 months.
The evidences associated to all RegulonDB objects are now classified as
‘strong’ or ‘weak,’ based on the confidence level of the experiment.
The evidences associated to all RegulonDB objects are now classified as
‘strong’ or ‘weak,’ based on the confidence level of the experiment.

Examples of strong evidences are DNA binding of purified TF for
regulatory interactions, mapping of TSSs for promoters, and length of
mRNA for transcription units.
On the other hand, gene expression analyses and computational
predictions are considered weak evidences.
Generation of computational predictions for four different promoters of
the σ70 family: those of σ 24, 28, 32 and 38.
The putative +1 of transcription initiation along with the -35 and -10
boxes can be downloaded from RegulonDB.

The active and inactive conformation of TFs is regulated by specific cell
signals (‘effectors’) that can be metabolites, ions or other chemical-
signaling molecules, through covalent or allosteric interactions.
The origin of these effectors can be endogenous (synthesized inside the
cell), exogenous (incorporated or transported from outside the cell) or
both (hybrid).
This feature has been added to the TFs in the database and a link to a
specific web page that shows details of the cell sensing properties of the
transcriptional regulators has been created.
RegulonDB v.6.0 has an expanded conceptual and relational model that
includes other levels and mechanisms of regulation of gene expression,
such as transcriptional elongation, posttranscriptional modification and
translational initiation.

The first elements that are now modeled and populated are RNA
regulatory elements, specifically riboswitches and attenuators, and small
RNAs.
The user interface has a graphic representation and textual information
about their sequences, location, evidences and references.
Riboswitches and attenuators are cis-regulatory elements that can
modulate transcription elongation or translation initiation.
A riboswitch is part of the 50 non-translated region in specific bacterial
mRNAs that can modulate gene expression in direct response to small
molecules without the need for a protein intermediate.
These regulatory elements are highly conserved, both in structure and
sequence, due to the constraints of forming a highly structured binding
pocket for the effector.

Riboswitches are usually found associated with transcription or
translation attenuators.
Several of these riboswitches have been experimentally described and
their sequences are obtained from Rfam, a database of RNA families.
Attenuators are segments of RNA in the untranslated regions of some
mRNAs that can form several mutually exclusive secondary structures,
contrary to riboswitches, are rarely conserved at the sequence level.
The sRNAs genes code for RNA sequences of <350 nucleotides long can
have intrinsic catalytic activity, modify a protein activity.
RegulonDB includes 49 interactions between sRNAs and their target
genes.

RegulonDB literature can now be searched with the Textpresso text-
mining engine, customized for E. coli.
Textpresso allows direct exploration of the curated literature, both at the
level of highly specific key words and with entire categories.
The user can, for example, search for a type of regulation in which a gene
or operon and a specific TF are mentioned within sentences of different
papers.
The tool can search through 2472 full-text papers, 3125 paper abstracts,
and more than 4200 curator notes.
The addition of this text-mining tool to RegulonDB will expand the
possibilities, for the end user, of traversing the knowledge space of E. coli
metabolism and gene regulation and will allow our curators to refine and
confirm their annotations.

Transcriptional and post transcriptional regulation of gene expression

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