Overview

•  Regulon
•  mal regulon
•  trp operon
•  ara operon
–  Repression
–  Activation
–  Attenuation
–  Autoregulation
 REGULON

Definition - Collection of genes not in the same

operon that are co-regulated

Examples:
1.  SOS response regulon: DNA repair genes
induced by DNA damaging agents.
2.  Maltose (mal) regulon- genes needed to
metabolize maltose (glucose-glucose
disaccharide).
 The mal Regulon
•  Encodes genes needed for maltose utilization
•  Involves multiple promoters, some regulated
by:
–  MalT (a regulatory protein that also
requires ATP and the mal regulon inducer,
maltotriose)
–  MalT and CAP
–  CAP alone
 Regulatory regions of the divergently transcribed malEp
and malKp operons: CAP + MalT activate both operons

Two sets of binding sites for MalT on the

Blue boxes: CAP binding sites malKp promoter:
Red arrows: MalT binding sites 3, 4 and 5--has higher affinity
Yellow box: -10 region 3’, 4’ and 5’ (better suited to promote
RNAP binding to the -10 region).
Hypothesis: CAP binding to the blue sites pushes
MalT to bind to the prime sites, so it is closer to the
RNA polymerase to activate transcription.
Two sets of binding sites for MalT on the
Blue boxes: CAP binding sites malKp promoter:
Red arrows: MalT binding sites 3, 4 and 5--has higher affinity
Yellow box: -10 region 3’, 4’ and 5’ (better suited to promote
RNAP binding to the -10 region).
 Footprinting DNA-Protein
Interactions
•  Powerful and fairly rapid methods for
mapping where and how proteins bind
tightly to DNA
•  2 examples:
1.  DNAse I footprinting
2.  DMS footprinting
DNAse I Footprinting

1.  Prepare end-labeled

DNA.
2.  Bind protein.
3.  Do a mild digestion with
DNAse I (DNase I
randomly cleaves DS
DNA on each strand)
4.  Separate DNA
fragments on
denaturing acrylamide
gels (sequencing gels)
5.  Expose gel to X-Ray
film.
Fig. 5.37a
Sample of a DNase I Fig. 5.37b
footprinting gel (for a
DNA-binding protein).

Footprint

Samples in lanes 2-4

had increasing
amounts of the DNA-
binding protein
(lambda protein cII);
lane 1 had none.
Dimethylsulfate (DMS) Footprinting

1.  End-label DNA fragment.

2.  Bind protein.
3.  Treat with dimethylsulfate,
which methylates purines.
4.  Partially cleave DNA at the
methylated bases using
piperidine.
5.  Separate DNA fragments on
DNA sequencing gels.

Fig. 5.38a
Sample of DMS footprinting.

Lanes 1 and 4 had no protein

Lanes 2 and 3 had 2 different
amounts of protein.

Protein binding protects most purines

from modification by DMS, but it can
stimulate modification of those in
regions where the helix is distorted or
partially melted (indicated by *) .

Fig. 5.38b
DMS footprinting of MalT
on the regulatory region
of malKp in the presence
(+) and absence (-) of
CAP.

CAP forces MalT to

bind to the 3’, 4’,
Filled arrows-enhancement
and 5’ boxes
upstream of malKp
promoter, thereby
activating
transcription.
 Tryptophan operon: Regulation
by repression and attenuation
•  Genes for tryptophan synthesis
•  Repressed by end-product of pathway,
Tryptophan.
•  Repression requires Operator sequence,
Aporepressor (trpR gene product) & Co-
repressor (Tryptophan).

•  Also controlled by attenuation in the

“Leader” peptide region of the transcript.
Figure 7.27

Fig. 7.25
The trp operon is also controlled by attenuation.
P P/O L E D C B A

Moderate trp 140 bp

AUG 7000 bp
Low trp
AUG

Transcription stops in the leader-attenuator region

(leader encodes a small peptide) when [tryptophan] is
elevated.
 The trp Leader peptide has two key tryptophan codons.

The ribosome stalls at the trp codons when [Tryptophan]

is too low.
The stalled ribosome prevents a downstream
transcription terminator (IR + U-rich sequence) from
forming.
Fig. 7.29
http://www.youtube.com/watch?
v=8aAYtMa3GFU
Why do we need attenuation over repression?
70-fold repression X 10-fold attenuation
Total 700-fold regulation from active to inactive.

Trp synthesis is very expensive

 How is translation of the trp Operon genes
achieved with the leader peptide there to stop
ribosomes?
Each gene in the polycistronic mRNA has
its own ribosome binding site!

The trp operon is also controlled by attenuation.

P P/O L E D C B A

Moderate trp 140 bp

AUG 7000 bp
Low trp
AUG
ara operon
DNA Looping
Autoregulation
Figure 7.21
 Arabinose breaks the loops
between araO2 and araI

Figure 7.23
 Effects of mutations in araO2 and araI on the
stability of looped complexes with AraC

Added AraC to form the complex and then added excess amount of wild type araI as
a competitor over time

Figure 7.22
 Autoregulation of araC

Figure 7.24
 A Regulatory Theme for
Metabolic Control:
•  Small molecules binding to and
changing activity of a transcription
regulator:
1. lacI and allolactose
2.  CAP and cAMP
3.  MalT and maltotriose
4.  trpR and tryptophan
5.  araC and arabinose