Different cell types contain the same DNA but express different genes and proteins. Gene expression can be regulated at many steps from DNA to RNA to protein. Prokaryotes regulate gene expression through operons, where genes are organized together and transcribed as a single unit. The lac and trp operons are regulated by repressor proteins that bind to operator sites on the DNA and block transcription in the presence of effector molecules like lactose or tryptophan. This document discusses the mechanisms of repression and induction of the lac and trp operons through repressor proteins and RNA polymerase binding.

2. Different cell types
of multicellular
organisms contain
the same DNA…
…but
different
cell types
synthesize
different
sets of
proteins.

3.  A cell typically expresses only a fraction of its genes,
and the different types of cells in multicellular
organisms arise because different sets of genes are
expressed.
 Types of gene expression:
o Constitutive expression: some genes are essential
and necessary for life, and therefore are continuously
expressed; these genes are called housekeeping
genes.
o The expression levels of some genes fluctuate in
response to the external signals. Some genes
demonstrate higher expression level once being
activated (induced). Some genes are repressed and
their expression levels are lower (repressed).
 So how do cells know which kinds of proteins to
synthesize? …gene expression can be regulated at many
of the steps in the pathway from DNA to RNA to protein.

4. Prokaryotic
Gene Regulation
The best example of
genetic control is the
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well studied system
graw- of milk sugar
hill.com/olc/dl/1200 (lactose) inducible
77/bio25.swf catabolism in the
human
symbiote, Escherichi
a coli.

5. Regulation of the lac operon (dual control:
repression and promotion)
 Prokaryotic genes are polycistron systems, i.e. several relevant genes are organized
together to form a transcription unit called the operon.
 The lac operon includes 3 structural genes (lacZ, lacY and lacA) that are transcribed in
unison. Located near the lac operon, is the lacI gene regulates the operon by producing
the lac repressor protein.
 Both the regulatory gene and the lac operon itself contain: promoters (Pl and Plac) at
which RNA polymerase binds, and terminators at which transcription halts. Plac overlaps
with the operator site (O) to which the active form of the repressor protein binds.
 The operon is transcribed into a single long molecule of mRNA that codes for all three
polypeptides.

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 Transcription of the lac operon is down-regulated
through the binding of the lac repressor to the
operator.
 In the absence of lactose, the repressor remains
bound to the operator and preventing access of the
RNA polymerase to the promoter.
 Transcription is blocked and the operon is
repressed.

7.  In the presence of lactose, the repressor is
inactivated form and does not bind to the
operator.
 Thus the RNA polymerase may bind to the
promoter and transcribe the structural genes
into a single cistronic mRNA.

8.  The isomeric form of lactose that binds to the repressor is allolactose.
 The lac repressor is an allosteric protein capable of reversible
conversion between two alternative forms.
 In the absence of the effector allolactose, the repressor protein is in
the form that binds to the lac operator.
 In the effector’s presence, the repressor mostly exists in the
alternative and inactive state.

9.  Transcription of the lac operon is
up-regulated through the binding
of the cAMP Receptor Protein
(CRP) complex to the promoter.
 It is an allosteric protein that is
inactive in the free form but is
activated by binding to cAMP.
 The CRP-cAMP complex binds
the promoter of inducible
operons, increasing the affinity
of the promoter for RNA
polymerase to stimulate
transcription.
 The effects of active CRP on the
lac operon:
a. The CRP-cAMP complex
binds to the CRP recognition
site near the promoter
region
b. RNA polymerase binds to
the promoter and
transcribes the operon.

10. Together, the lac
repressor and CAP
provide a very
sensitive response
to the cell’s need to
utilize lactose-
metabolizing
enzymes.

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How the trp operon is controlled. The tryptophan repressor cannot bind the
operator (which is located within the promoter) unless tryptophan first binds to
the repressor. Therefore, in the absence of tryptophan, the promoter is free to
function and RNA polymerase transcribes the operon. In the presence of
tryptophan, the tryptophan-repressor complex binds tightly to the operator,
preventing RNA polymerase from initiating transcription.

12. The binding of tryptophan to the tryptophan repressor protein changes
its conformation. This structural change enables this gene regulatory
protein to bind tightly to a specific DNA sequence (t he
operator), thereby blocking transcription of the genes encoding the
enzymes required to produce tryptophan( the Trp operon). The 3-D
structure of this bacterial helix-turn-helix protein, as determined by x-
ray diffraction with and without tryptophan bound, is illustrated.
Tryptophan binding increases the distance between the two recognition
helices in the homodimer, allowing the repressor to fit snugly on the
operator.

13. Regulation of the trp
operon: a "riboswitch"
The trp operon includes 5 structural genes (trpE,
trpD, trpC, trpB, and trpA) as well as promoter (Ptrp),
operator (O), and leader (L) sequences.
The structural genes are transcribed and regulated
as a unit. The repressor protein, encoded by the trpR
gene is inactive (cannot recognize the operator site),
or in the free form when tryptophan is not abundant.

14. The polycistronic mRNA encodes for the enzymes
of the tryptophan biosynthetic pathway.
When complexed with tryptophan, the repressor
is active and binds tightly to the
operator, blocking access of RNA polymerase to
the promoter and keeping the operon repressed.

15. Absence of nuclear membrane separates transcription
and translation and the ribosomes will bind the nascent
message soon after it emerges from the RNA
polymerase. The close linkage of the processes can
lead to interdependent control mechanisms such as
the attenuation controlled by the trp leader sequence.
 The transcript of the trp operon includes 162 nucleotides upstream of the initiation
codon for trpE (the 1st structural gene). This leader mRNA includes a section encoding a
leader peptide (or sensor) of 14 amino acids.
 If tryptophan is present (in moderate amounts), the sensor peptide is easily made and
the long trp operon mRNA is NOT completed.
 If tryptophan is scarce, the leader peptide is not easily made and the full operon is
transcribed then translated into tryptophan synthetic enzymes.
 Two adjacent tryptophan (trp) codons within the leader mRNA sequence are essential
in the operon's regulation.
 The leader mRNA contains four regions capable of base pairing in various combinations
to form hairpin structures.

16.  Attenuation depends upon the ability of regions 1
and 2 and regions 3 and 4 of the trp leader
sequence to base pair and form hairpin secondary
structures.
 A part of the leader mRNA containing regions 3
and 4 and a string of eight U's is called the
attenuator.
 The region 3+4 hairpin structure acts as a
transcription termination signal; as soon as it
forms, the RNA and the RNA polymerase are
released from the DNA.

17. During periods of tryptophan scarcity, a ribosome translating the
coding sequence for the leader peptide may stall when it encounters
the two tryptophan (trp) codons because of the shortage of tryptophan-
carrying tRNA molecules.

18.  Because a stalled ribosome at this site blocks region 1, a
region 1+2 hairpin cannot form and an alternative, region
2+3 hairpin is formed instead.
 The region 2+3 base pairing prevents formation of the
region 3+4 transcription termination hairpin and therefore
RNA polymerase can move on to transcribe the entire
operon to produce enzymes that will synthesize tryptophan.
 When tryptophan is readily available, a ribosome can
complete translation of the leader peptide without stalling.
 As it pauses at the stop codon, it blocks region
2, preventing it from base pairing.
 As a result, the region 3+4 structure forms and terminates
transcription near the end of the leader sequence and the
structural genes of the operon are not transcribed (nor
translated).
 This is example of a "riboswitch", a mechanism which can
control transcription and translation through interactions of
substrate molecules with an mRNA.

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