The trp operon

1
The trp operon
2
Suggested readings on regulation/dna bp Voet
pp 1237-1253 Problems 2, 4
3
Figure 31-39 A genetic map of the E. coli trp
operon indicating the enzymes it specifies and
the reactions they catalyze.
4
MVA Fig.21.13
5
MVA Fig.21.14
6
(No Transcript)
7
Figure 31-31 X-Ray structure of an E. coli trp
repressor operator complex.
Page 1244
8
(No Transcript)
9
The Tryptophan Operon A Repressor

• When should the bacteria be transcribing genes
  for the synthesis of the amino acid tryptophan?
• When levels of tryptophan in the cell are low,
  the bacteria must make its own.
• However, if tryptophan is abundant in the cell or
  is being provided in the medium, it is a waste of
  energy for the bacteria to be synthesizing it.

10
(No Transcript)
11
Figure 31-40 The base sequence of the trp
operator. The nearly palindromic sequence is
boxed and its 10 region is overscored.
12
Alberts Fig. 7-34
13
Originally, regulation of the trp operon was
thought to occur solely through the
repressor-operator system until deletion mutants
located downstream of trpO were identified.
These mutants displayed increased expression of
the operon by six-fold which indicated the
presence of an additional transcriptional control
element.
Why is repression not the only mode of regulation?
14
Evidence 1. Trp-tRNA synthetase mutants had
regulatory anomalies. 2. Addition of trp to
trp-starved cells not only shut down initiation
of transcription but also inhibited transcription
already in progress on the initial segment of
the operon 3. Mutants lacking a functional
repressor could still respond to trp starvation
by increasing transcription of trp mRNA. 4 .
Deletion mutants in which both of the deletion
termini were within the transcribed region of the
operon had an unexpected six-fold increase in
expression of the remaining genes in the operon.
Obviously, repressor binding was unaffected.
15
5. Within the population of mRNAs produced in
vivo from the 5' end of the trp operon, RNAs
corresponding to the first 140 bp (the leader
sequence) of the operon were several times more
abundant than those from more distal regions,
therefore a transcription termination site was
located before the structural genes.
6. Starving bacteria of trp reduced termination
at this site (the trp attenuator).
7. Mutations altering trp-tRNA synthetase,
tRNAtrp or a tRNA trp modifying enzyme were
found to decrease transcription termination at
the trp attenuator. What does this suggest
about the mode of attenuation?
16
8. Ribosome binding experiments with the 140
base transcript demonstrated that ribosomes
protect a 20 base segment from nuclease attack.
A potential AUG start codon is located in the
center of this region. 9. A 14 residue peptide
(the leader peptide) could be synthesized from
this start codon and contained tandem trp
residues near its C-terminus. 10. The trp
leader ribosome binding site was shown to be an
efficient site for the initiation of translation
by fusing the leader to a structural gene and
demonstrating synthesis of the fused
polypeptide.
Examples?
17
(No Transcript)
18
(No Transcript)
19
(No Transcript)
20
11. Two classes of termination defective leader
mutants have been isolated. One type terminates
at less than normal frequency and has bp changes
in the 34 bp region. In vivo, these mutants
have a 2-4 fold increase in operon expression.
12. The second class of mutants have increased
termination of the attenuator. These prevent the
relief from termination that is associated with
trp starvation. One of these mutants has
an altered start codon for the leader peptide.
Another has a G to A conversion at position 75,
which would prevent 23 pairing and cause
formation of a 34 termination structure.
21
Genetic analysis indicated that the new control
element was located in trpL, a 162 nt region
30-60 nt upstream from trpE. When trp is
scarce, the entire 6720 nt polycistronic trp,
including trpL, is synthesized. As the trp
concentration increases, the rate of trp
transcription decreases as a result of the trp
r epressor-corepressor's greater abundance.
With increasing trp, the mRNA synthesized
consists more and more of a 140 nt segment
corresponding to trpL sequences only. The
availability of trp results in the premature
termination of transcription of the operon.
22
MVA Fig. 26.35
23
Figure 31-41 The alternative secondary structures
of trpL mRNA.
Page 1252
24
(No Transcript)
25
MVA Fig. 26.36
26
(No Transcript)
27
Figure 31-42a Attenuation in the trp operon. (a)
When tryptophanyltRNATrp is abundant, the
ribosome translates trpL mRNA.
28
Figure 31-42b Attenuation in the trp operon. (b)
When tryptophanyltRNATrp is scarce, the ribosome
stalls on the tandem Trp codons of segment 1.
29
Trp operon
And again all the cool animation files
30
DNA Binding Proteins
How does a repressor find its operator in a sea
of other sequences?
It is not enough just for the regulatory protein
to recognize the correct DNA site. The protein
must also find it rapidly and bind to it
sufficiently tightly to discriminate it from the
millions of competing and overlapping
nonspecific sites that are explored in the
course of specific target localization.
31
One point to keep in mind while considering
protein-DNA interactions is that such an
interaction represents a dynamic
equilibrium Whether an operator has its (or a)
particular repressor protein bound to it depends
on
1. the concentration of the regulatory protein
in the cell,
2. the affinity between the repressor and the
operator sequence and
3. the affinity between the repressor and other
non-specific DNA binding sites.
32
Association constants lac repressor DNA to
R-DNA complex Repressor lac operator 1-2 X
1013 M-1 other DNA 2-3 X 106 M-1 (specificity
KA(s)/KA(non-specific) 107) Repressor bound to
inducer lac operator 2 X 1010 M-1--or some
references suggest this is even lower other DNA
2 X 106 M-1 When repressor is bound to
allosteric regulator (allolactose in this case)
non-specific binding competes more effectively
with specific binding.
33
How a repressor recognizes and binds to an
operator The interaction between repressor and
operator is often taken as a paradigm for
sequence-specific DNA-protein interactions.
Each regulatory protein in E. coli must select
its operator site (or sites) from among the five
million or so base pairs of DNA in the cell.
Examples?
For this organism, an operator (or any other cis
acting site) must be at least 11-12 bases long
in order to form a site that reoccurs at random
less than once per genome.
Accordingly, regulatory proteins in E. coli bind
tightly to specific DNA sequences that are about
15-20 base pairs long.
34
Operator Sequence and Structure
A large number of operator sites have been
identified and their DNA sequence has been
determined.
One feature that is common to all operators is an
imperfect two-fold axis of symmetry.

• A perfectly symmetrical sequence is shown below.
• gt---- G C C A T G C G C A T G G C ----gt
• lt---- C G G T A C G C G T A C C G ----lt

35
Cap binding site
Link to view structure
36
Lac repressor
lac operator binding site for the lac repressor
protein (lac I gene product)
37
Structure of Regulatory Proteins Many
DNA-binding regulatory proteins share features in
common that reflect a common mode of DNA
binding. Some of these features are
(1) The active binding unit is a dimer of two
identical globular polypeptide chains oriented
oppositely in space to give a molecule with a
two-fold axis of symmetry
phage lambda cI repressor protein alpha helical
region in contact with the major groove is in
red.
38
(2) The critical contacts between the protein
and the DNA are made by adjacent a helices
located at the binding face of each monomer. The
helices are connected by a turn in the protein
secondary structure. This helix-turn-helix
motif is common to many regulatory proteins.
HTH
39
(No Transcript)
40
Cro
Why do the recognition helices contact the major
groove? What determines the specificity of
interaction?
41
Binding motifs http//www.umass.edu/molvis/freich
sman/Site642/page_dnab/menu.html again http//ww
w.web-books.com/MoBio/Free/Ch4F2.htm4F1
42
AH bond acceptor DH bond donor
43
(No Transcript)
44
One of the most common DNA-protein interactions.
Because of its specific geometry of H-bond
acceptors, guanine can be unambiguously
recognized by the side chain of arginine
45
Stereo view for phage lambda repressor
46
(No Transcript)
47

• Specificity of protein-DNA interaction of due to
• ability of amino acid side chains in the
  recognition helix to form hydrogen bonds with
  specific bases in its cis-acting site
• multiple complementary interactions between the
  protein and the DNA that are dependent on the
  deformation of the helix and which increase the
  number of contact points

48
Main features of interactions between DNA and
the helix-turn-helix motif of DNA binding
proteins
49
(No Transcript)
50
(No Transcript)
51
Phage Lambda
52
Binding tutuorial
http//www.biochem.arizona.edu/classes/bioc462/462
a/NOTES/ Nucleic_Acids/prodna.html
53
Important Points a handshake leads to a bear-hug
Specific recognition of DNA targets by the
helix-turn-helix motif involves interactions
between sides of the recognition helix and bases
in the major groove of the DNA
But, specific recognition of DNA sequences is to
a large extent governed by other interactions
within complementary surfaces between the protein
and the \
These interactions frequently involve H-bonds
from protein main-chain atoms to the DNA backbone
in both the major and the minor groove and are
dependent on the sequence-specific deformability
of the target DNA
54
DNA deformation induced by protein-binding.
The ease with which a stretch of DNA can be
deformed can affect the affinity of protein
binding to a specific sequence
55
CAP binding to its cis-acting site cAMP binding
domain in blue red -- DNA phosphates whose
ethylation interferes with cap binding
blue hypersenstivie to DNase I-- these
phosphates bridge the cap-induced where the
minor groove has been widened
56
Here the lac repressor tetramer is shown binding
to two operators. Each dimer contacts one
operator (either dark or light blue). The
operators are 21 bases long.
57
MVA Fig.21.15
58
No direct H-bonding with bases!
All specific H-bonds occur via bridging water
molecules!
Only direct contacts are H-bonds to the
phosphate backbone!!!!
Yet mutations of these non-contact bases alter
binding specificity
This suggests that the operator assumes a
sequence-specific conformation that makes
favorable contacts with the repressor known as
Indirect Readout
59
When tryptophan is added to crystals of
aporepressor, the crystals shatter. When the
tryptophan wedges itself into the protein, it
changes the shape of the protein enough to break
the lattices of the crystal The orientation of
the recognition helix shifts when tryptophan is
bound.
60
trp repressor (HTH allosteric)
61
trp repressor (HTH allosteric)
62
(No Transcript)
63
(No Transcript)
64
(No Transcript)
65
His
Cys
66
?
?
67
mutations that affect DNA binding are oncogenic
minor groove
major groove
p53 DNA binding domain
68
arc repressor from phage P22
(beta sheet recognition element)
69
arc repressor from P22
70
bZip
71
bZip homo- and heterodimers
72
max (bHLHzip)
73
max (bHLHzip)
74
Gal4
75
Gal4