Turning Cro into a
Transcriptional Activator
2
3
Figure by MIT OpenCourseWare.
Fred Bushman and Mark Ptashne
Cell (1988) 54:191-197
Presented by Natalie Kuldell
for 20.902
February 4th, 2009
Small patch of acidic residues is necessary
and sufficient for transcriptional activation
Figure 1
RNA
Polymerase
cl normally activates transcription
cro normally represses transcription
RNA
Polymerase
Figure by MIT OpenCourseWare.
cro/cl chimera activates transcription!
Site-directed mutagenesis of cro helix to make acidic patch
cartoon of cI binding DNA
Figure 2
Gln
Thr17
Lys
Glu
Thr
Asp22
Lys
Asp
Ala
Lys21
Leu
Gly
Val
Figure by MIT OpenCourseWare.
Gly
Tyr26
Cro67
fig from A Genetic Switch
Figure by MIT OpenCourseWare.
4 amino acid substitution --> cro67
Why might this work?
Repressor
Cro
Gln
Gln
Ser
Glu
Val
Ala
Lys
Met
Asp
Helix 3
Phe
Val
Gly Gly
Gly Gln
Ser
Figure by MIT OpenCourseWare.
Leu
ala
Helix 2
Thr
Ala
Asp
Met
Gly
Lys
Helix 2
Thr
Asn
Lys
Helix 3
Leu
Gly
Val
Ile
Ile
Ala Asn
Tyr Gln
Ser
Ala
Lys
His
Site-directed mutagenesis of cro helix to make acidic patch
Acidic and amide side chains
O
O
O
O
NH2 NH2
NH2
Aspartate
NH2
N
N
NH
H 2N
NH2
Arginine NH2
NH2
OH
NH2
Thr17
NH2
Lys
Alanine
O
_
O
NH2 Glycine
NH2
Leucine
Cyclic side chain
O
O
O
Isoleucine
Glu
Thr
NH2
NH2
Asp22
Lys
Asp
Ala
Lys21
Leu
Gly
Val
Gly
Tyr26
Cro67
N Proline
Figure by MIT OpenCourseWare.
Threonine
Methionine NH2
Serine
Gln
Hydroxyl or sulfur-containing
O side chains
HO
Tyrosine
Valine
Histidine
O Phenylalanine
NH2
O
O
_
O
Aliphatic side chains
Lysine
NH2
Glutamine NH
2
Tryptophan
Basic side chains
H 2N
H 2N
NH2
NH2
Asparingine
Glutamate
Aromatic side chains
HS
NH2
Cysteine
Figure by MIT OpenCourseWare.
4 amino acid substitution --> cro67
Site-directed mutagenesis of cro helix to make acidic patch
Acidic and amide side chains
O
O
O
O
O
O
NH2 NH2
NH2
Aspartate
N
N
NH
H2N
NH2
Histidine
Arginine NH2
NH2
OH
NH2
NH2
Alanine
O
O
Lys
Asp
Ala
Lys21
Leu
Gly
Val
Gly
NH2
Leucine
O
_
NH2 Glycine
Cyclic side chain
O
O
Isoleucine
_
Asp22
NH2
NH2
Tyr26
Cro67
Figure by MIT OpenCourseWare.
N Proline
Threonine
Methionine NH2
Serine
Glu
Thr
Hydroxyl or sulfur-containing
O side chains
HO
Lys
Tyrosine
Valine
Thr17
NH2
NH2
Gln
Aliphatic side chains
Lysine
O Phenylalanine
O
Glutamine NH
2
NH2
Tryptophan
Basic side chains
H 2N
_
O
H 2N
NH2
NH2
Asparingine
Glutamate
Aromatic side chains
HS
NH2
Cysteine
Figure by MIT OpenCourseWare.
4 amino acid substitution --> cro67
Site-directed mutagenesis of cro helix to make acidic patch
Acidic and amide side chains
O
O
O
O
O
O
NH2 NH2
NH2
Aspartate
Lysine
N
N
NH
H 2N
NH2
Arginine NH2
Histidine
O
NH2
OH
Threonine
NH2
O
_
Asp22
NH2
Alanine
NH2 Glycine
NH2
Glu
Lys
Asp
Ala
Lys21
Leu
Gly
Val
Gly
Leucine
Cyclic side chain
Tyr26
Cro67
O
O
Isoleucine
NH2
Figure by MIT OpenCourseWare.
N Proline
NH2
Methionine NH2
Serine
Lys
Thr
O
_
Hydroxyl or sulfur-containing
O side chains
HO
Thr17
Tyrosine
Valine
Gln
NH2
NH2
Aliphatic side chains
O
_
O Phenylalanine
O
Glutamine NH
2
NH2
Tryptophan
Basic side chains
H 2N
_
O
H 2N
NH2
NH2
Asparingine
Glutamate
Aromatic side chains
HS
NH2
Cysteine
Figure by MIT OpenCourseWare.
4 amino acid substitution --> cro67
Site-directed mutagenesis of cro helix to make acidic patch
Acidic and amide side chains
O
O
O
O
O
O
NH2 NH2
NH2
Aspartate
N
N
NH
H2N
NH2
Arginine NH2
Histidine
O
NH2
OH
Threonine
NH2
Lys
Alanine
NH2 Glycine
NH2
Lys
Asp
Ala
Lys21
Leu
Gly
Val
Gly
Leucine
Cyclic side chain
Tyr26
Cro67
O
O
O
_
Asp22
NH2
Glu
Thr
Isoleucine
NH2
Figure by MIT OpenCourseWare.
N Proline
NH2
Methionine NH2
Serine
Thr17
O
_
Hydroxyl or sulfur-containing
O side chains
HO
Gln
Tyrosine
Valine
O Phenylalanine
NH2
NH2
_
O
Aliphatic side chains
Lysine
NH2
Glutamine NH
2
Tryptophan
Basic side chains
H 2N
H 2N
NH2
NH2
Asparingine
Glutamate
Aromatic side chains
HS
NH2
Cysteine
Figure by MIT OpenCourseWare.
4 amino acid substitution --> cro67
Protein -helix recognizes sequence in DNA major groove
2
3
Courtesy of Timothy Paustian. Used with permission.
Figure by MIT OpenCourseWare.
model of lac repressor
binding lac operator
http://www.bact.wisc.edu/Microtextbook/index.php?module=Book&func=displaychapter&chap_id=35&theme=printer
Protein -helix recognizes sequence in DNA major groove
Wild type cro
binds OR3>>OR2 = OR1
binding to OR3 shuts off txn from PRM
Wild type cI
binds OR1>OR2>OR3
binding to OR2 activates txn from PRM
Protein -helix recognizes sequence in DNA major groove
Wild type cro
binds OR3>>OR2 = OR1
binding to OR3 shuts off txn from PRM
Wild type cI
binds OR1>OR2>OR3
binding to OR2 activates txn from PRM
cro67
binds? OR1>OR2>OR3
activates?
Figure 3
Courtesy Elsevier, Inc., http://www.sciencedirect.com. Used with permission.
Protein -helix recognizes sequence in DNA major groove
Wild type cro
binds OR3>>OR2 = OR1
binding to OR3 shuts off txn from PRM
Wild type cI
binds OR1>OR2>OR3
binding to OR2 activates txn from PRM
cro67
binds? OR1=OR2>OR3
activates?
Figure 3
Courtesy Elsevier, Inc., http://www.sciencedirect.com. Used with permission.
cro67 activates transcription in vitro
Figure 4
[cro67]
In vitro txn rxns
0
395 bases
+ buffer
+ DNA w/ PRM + PR
+ cro67 (purified)
250 bases
+ 32P-ATP, CTP, GTP or UTP
37 10
then + RNAP
37 10
then +formamide
to gel
Courtesy Elsevier, Inc., http://www.sciencedirect.com.
Used with permission.
cro67 activates transcription in vitro
Figure 4
cut out bands and count
~5x
395 bases
~5x
250 bases
Courtesy Elsevier, Inc., http://www.sciencedirect.com.
Used with permission.
Observe: txn of PR
as txn of PRM
when cro67 added
Qs: What are extra bands? Is cro67 bound in natural way?
cro67 binds operator sequences as expected
Figure 4
[cro67]
DNase footprint
+ buffer
+ 32P-DNA w/ PRM + PR
+ cro67 (purified)
37 10?
then + DNase
37 10?
then +formamide
to gel?
Courtesy Elsevier, Inc., http://www.sciencedirect.com.
Used with permission.
Observe:
OR1=OR2>OR3
Q: is assay sensitive to different conformations of bound prot?
cro67 activates transcription in vitro
Supporting data/controls
Figure 5
Wild type cro does not activate txn in vitro
using in vitro txn rxn, DNase ftpt
Figure 6
cro67 does not
activate txn from other promoters
Courtesy Elsevier, Inc., http://www.sciencedirect.com.
Used with permission.
cro67 in vivo expts hampered by low affinity for operators
(~100x < wt cro)
Summary of
434 cI data
Figures by MIT OpenCourseWare.
look at******
patch more acidic
cI
inc actn
patch more basic
operator occupancy
dec actn
satd
dec actn
satd
operator binding
normal
normal
** in vivo (-gal assays on lysogen)
vs
434 cI
inc actn
** in vivo DMS ftpt
** in vitro txn rxns, DNase ftpt
Turning cro into a transcriptional activator
key assumption
in vitro conclusions have meaning in vivo
biggest mistake
mixing the 434 work in
not pushing in vivo work
significance/meta-lessons
protein engineering by analogy (cro is like cI, thus)
small changes (e.g., individual AAs) are important
good data enables thoughtful experiments
be open to surprises (e.g., DNA binding)
ask the next question: does activation work the same way
in eukaryotic cells?
MIT OpenCourseWare
http://ocw.mit.edu
20.020 Introduction to Biological Engineering Design
Spring 2009
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