Transcript Regulation-of-Gene

Information flow and
regulatory factors
1)
The number of copies of the gene
2)
The efficiency with which the gene is transcribed,
which is mainly determined by the level of initiation
of transcription by RNA polymerase (promoter
activity).
3)
4)
The stability of the mRNA
5)
6)
The efficiency with which the mRNA is translated
into protein
The stability of the protein product
Post-translational effects
Transcriptional control
Promoters
Most E. coli promoters, for example, have two key parts
(motifs) that are involved in the recognition by the RNA
polymerase and resemble TTGACA and TATAAT at
positions that are centred at 35 bases and 10 bases before
(upstream from) the transcriptional start site and are
hence referred to as the -35 and -10 positions respectively.
The latter is also known as the Pribnow box
The structure of typical E. coli promoters
Importance of the distance between the -35 and -10 regions of a promoter
Operons and regulons
Structure of the lac operon
Operons and regulons
divergent genes
In some cases, co- ordinated control of several genes is
achieved by a single operator site that regulates two
promoters facing in opposite directions
In one example, the genes ilvC (coding for an enzyme
needed for isoleucine and valine biosynthesis) and ilvY
(which codes for a regulatory protein) are transcribed in
opposite directions , but transcription of both genes is
controlled by a single operator site
This provides an exception to the general rule that genes on
an operon are transcribed into a single mRNA
Structure of operons
and regulons
Alternative promoters and σ-factors
The promoter consensus described above is recognized by the
primary s-factor, commonly referred to as σ70 (because it is
about 70 kDa in size in E. coli).
This subunit is responsible for recognition of the promoters
used for transcription of most of the genes required in
exponentially growing cells. These are sometimes called
‘housekeeping’ genes since they encode essential functions
needed for the cell cycle and for normal metabolism such as
glycolysis, the TCA cycle and DNA replication
Alternative sigma factors and promoter recognition sequences.
In B.subtilis. All others are in E. coli
Sporulation in Bacillus
At the onset of starvation, the primary
σ-factor (σA) and a low abundance factor
called σH direct the transcription of a set
of genes whose products cause an
asymmetric
invagination
of
the
membrane, thus separating the forespore
from the mother cell
Another σ-factor, σF, is present before the
septum forms, but is inactive. σF becomes
active but only in the forespore
Following this, a third sporulation specific
σ-factor, σE, also becomes active, but only
in the mother cell
Anti-s-factors
The regulation of flagella
filament production (FliC) by the
anti sigmafactor FlgM
Terminators, attenuators and anti-terminators
* Attenuation
within an operon. The presence of a weak transcriptional
termination site (t1) within an operon leads to reduced expression of the
distal genes (c and d). The strong terminator t2 causes termination at the
end of th full-length mRNA
Structure of the operator/promoter region of the lac operon
*
The lac repressor is a multimeric protein, consisting of four identical subunits,
showing a secondary structure feature consisting of two a-helices separated by a
few amino acids that place the two a-helices at a defined angle to each other.
This conformation, known as a helix-turn-helix motif, is characteristic of DNAbinding proteins and enables this part of the protein to fit into the major groove of
the DNA and make specific contacts with the operator DNA
Regulation of the
lac operon
The lac repressor protein also has affinity for allolactose (a derivative of lactose)
gratuitous inducer is the synthetic analogue iso-propyl-thiogalactoside (IPTG).
The converse is also true: some compounds are substrates for breakdown by bgalactosidase but are not able to act as inducers since they are not recognized by the
repressor. as X-gal (which gives a blue colour after hydrolysis by b- galactosidase)
Regulatory mutants of the lac operon
Constitutive mutants
(a) lacI mutants which are defective in the production of the repressor (or the
repressor cannot bind to the operator) (capable of acting in trans)
(b) operator-constitutive (Oᶜ) mutants in which the change is in the operator itself,
preventing recognition by the repressor protein (These mutations are described as
cis-dominant)
Non-inducible mutants, which again are unaffected by the presence or absence
of the inducer, but in this case the level of the enzymes is always low.
The most significant one from our point of view is a different type of lacI
mutation that abolishes the. ability of the repressor protein to recognize and
respond to the inducer
Super-repressor (lacIq) mutants. These cells are characterized by an
overproduction of the repressor, commonly due to a mutation in the promoter
of the lacI gene (remember that the lacI gene is not part of the lac operon and is
transcribed from a different promoter).
lacI mutation is recessive
lacOc mutation is cis-dominant
Catabolite repression
In the presence of glucose the level of ATP within the cell rises as the
glucose is broken down to release energy; at the same time, the level
of cyclic AMP (cAMP), a cellular alarm molecule, decreases due to
activation of cAMP phosphodiesterase
In the absence of glucose, adenylate cyclase is activated and levels of
cAMP rise.
Binding of cAMP to CRP causes a conformational change in the
protein which allows it to recognize and bind to, specific sites on the
DNA. The cAMP–CRP complex binds to a DNA site upstream from
the promoter (-72 to -52)
Despite the term catabolite repression, it should be clear that the role
of the CRP is a positive one; it is an activator (when bound to cAMP)
not a repressor. Hence, some people prefer to call it catabolite
activator protein (CAP)
The cAMP–CRP complex causes DNA bending
Arabinose operon
Repression and
activation of the
arabinose operon
NH2-terminal domain binds arabinose and mediates dimerization, while the COOHterminal domain contains the regions that bind to the DNA.
When arabinose binds to the NH2-terminal domain, it alters the way that the dimer
forms and hence its ability to contact different sites on the DNA.
Thus in the absence of arabinose AraC is a negative regulator while in the presence
of the substrate it acts as a positive regulator
Structure of the trp operon
Attenuation: trp operon
Structure of the trp operon
The operon contains a sequence (of 162 bases), known as the leader sequence,
between the transcription start point and the start of the first structural gene
Attenuation control of the trp operon.
(a) In the absence of protein synthesis, the
terminator stem–loop 3:4 is able to form, and
the operon is not transcribed.
(b) If protein synthesis occurs in the presence of
limiting amounts of tryptophan, ribosomes will
stall at the tryptophan codons in the leader
region, blocking formation of the 1:2 stem–
loop. When the RNA polymerase transcribes
region 3, it will pair with region 2. The 2:3
structure is not a terminator, but it sequesters
region 3, thus preventing formation of the 3:4
terminator and allowing transcription of the
operon.
(c) In the presence of sufficient tryptophan, the
ribosomes will proceed as far as the stop codon,
thus blocking both regions 1 and 2. This allows
the 3:4 termination structure to form,
preventing transcription of the operon
Attenuation control of the trp operon in
Bacillus subtilis.
(a) The leader region contains two pairs of
complementary sequences, enabling two
alternative stem–loop structures A:B and
C:D. The partial overlap of B and C
prevents both structures forming. The
leader also contains 11 repeats of GAG or
UAG, which can bind TRAP (trp RNAbinding Attenuation Protein) in the
presence of tryptophan.
(b) In the absence of tryptophan, TRAP
does not bind, the A:B stem–loop forms
and the terminator C:D cannot form. Thus
transcription of the operon occurs.
(c) In the presence of tryptophan, TRAP
binds to the GAG/UAG repeats, which
blocks region A and prevents the
formation of the A:B stem–loop. Region
C is free to pair with region D to form the
terminator
structure,
preventing
transcription of the operon. Shaded circles
indicate regions that are blocked from
forming stem–loop structures