Turning λ 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
λcl normally activates transcription
λcro normally represses transcription
RNA
Polymerase
Figure by MIT OpenCourseWare.
λcro/cl chimera activates transcription!
Site-directed mutagenesis of λcro helix to make acidic patch
cartoon of λcI binding DNA
Figure 2
2
Gln
Thr17
3
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 --> “λcro67”
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 λcro helix to make acidic patch
Acidic and amide side chains
O
O
O
O
_
O
_
O
NH2 NH2
NH2
Aspartate
O
O
O
O
_
O
_
NH2
N
N
NH
H 2N
O
NH2
_
Arginine NH2
O
O
_
NH2
OH
NH2
Thr17
_
O
NH2
Lys
Alanine
_
O
_
O
_
O
NH2 Glycine
NH2
Leucine
Cyclic side chain
O
O
O
O
Isoleucine
_
O
Glu
Thr
_
NH2
NH2
S
Asp22
Lys
Asp
Ala
Lys21
Leu
Gly
Val
Gly
Tyr26
Cro67
_
N Proline
Figure by MIT OpenCourseWare.
O
O
Threonine
O
O
_
Methionine NH2
Serine
Gln
O
O
Hydroxyl or sulfur-containing
O side chains
HO
_
Tyrosine
Valine
Histidine
O
N
O Phenylalanine
NH2
O
O
O
O
_
O
Aliphatic side chains
O
Lysine
NH2
O
Glutamine NH
2
_
O
Tryptophan
Basic side chains
H 2N
O
_
O
H 2N
NH2
NH2
N
Asparingine
O
Glutamate
Aromatic side chains
O
O
O
_
HS
NH2
O
_
Cysteine
Figure by MIT OpenCourseWare.
4 amino acid substitution --> “λcro67”
Site-directed mutagenesis of λcro helix to make acidic patch
Acidic and amide side chains
O
O
O
O
O
O
_
O
_
O
NH2 NH2
NH2
Aspartate
O
O
O
O
_
O
_
O
N
N
NH
H2N
O
NH2
_
Histidine
Arginine NH2
O
O
_
NH2
OH
NH2
O
NH2
Alanine
O
O
O
_
Lys
Asp
Ala
O
_
Lys21
Leu
Gly
Val
Gly
NH2
Leucine
O
_
_
NH2 Glycine
Cyclic side chain
O
O
O
Isoleucine
_
Asp22
_
NH2
NH2
S
Tyr26
Cro67
Figure by MIT OpenCourseWare.
N Proline
O
O
Threonine
_
O
_
Methionine NH2
Serine
O
Glu
Thr
O
O
Hydroxyl or sulfur-containing
O side chains
HO
Lys
Tyrosine
Valine
O
N
Thr17
NH2
O
O
O
NH2
Gln
_
Aliphatic side chains
O
Lysine
_
O
O Phenylalanine
O
Glutamine NH
2
_
NH2
Tryptophan
Basic side chains
H 2N
O
_
O
O
H 2N
NH2
NH2
N
Asparingine
O
Glutamate
Aromatic side chains
O
_
HS
NH2
O
_
Cysteine
Figure by MIT OpenCourseWare.
4 amino acid substitution --> “λcro67”
Site-directed mutagenesis of λcro helix to make acidic patch
Acidic and amide side chains
O
O
O
O
O
O
_
O
_
O
NH2 NH2
NH2
Aspartate
O
O
O
O
_
O
_
Lysine
N
N
NH
H 2N
O
NH2
_
Arginine NH2
Histidine
O
O
_
NH2
OH
_
Threonine
O
NH2
O
O
_
Asp22
O
NH2
Alanine
O
_
_
NH2 Glycine
NH2
Glu
Lys
Asp
Ala
Lys21
Leu
Gly
Val
Gly
Leucine
Cyclic side chain
Tyr26
Cro67
O
O
O
_
Isoleucine
NH2
S
_
O
_
Figure by MIT OpenCourseWare.
N Proline
O
O
NH2
O
O
Methionine NH2
Serine
Lys
Thr
O
_
O
Hydroxyl or sulfur-containing
O side chains
HO
Thr17
Tyrosine
Valine
O
N
Gln
NH2
O
O
O
NH2
_
Aliphatic side chains
O
_
_
O
O Phenylalanine
O
Glutamine NH
2
O
NH2
Tryptophan
Basic side chains
H 2N
O
_
O
O
H 2N
NH2
NH2
N
Asparingine
O
Glutamate
Aromatic side chains
O
_
HS
NH2
O
_
Cysteine
Figure by MIT OpenCourseWare.
4 amino acid substitution --> “λcro67”
Site-directed mutagenesis of λcro helix to make acidic patch
Acidic and amide side chains
O
O
O
O
O
O
_
O
_
O
NH2 NH2
NH2
Aspartate
O
O
O
O
_
O
_
O
N
N
NH
H2N
O
NH2
_
Arginine NH2
Histidine
O
O
_
NH2
OH
_
Threonine
O
NH2
Lys
Alanine
O
_
_
NH2 Glycine
NH2
Lys
Asp
Ala
Lys21
Leu
Gly
Val
Gly
Leucine
Cyclic side chain
Tyr26
Cro67
O
O
O
O
O
_
Asp22
O
NH2
Glu
Thr
_
Isoleucine
NH2
S
_
O
_
Figure by MIT OpenCourseWare.
N Proline
O
O
NH2
O
O
Methionine NH2
Serine
Thr17
O
_
O
Hydroxyl or sulfur-containing
O side chains
HO
Gln
_
Tyrosine
Valine
O
N
O Phenylalanine
NH2
O
O
O
NH2
_
O
Aliphatic side chains
O
Lysine
NH2
O
Glutamine NH
2
_
O
Tryptophan
Basic side chains
H 2N
O
_
O
H 2N
NH2
NH2
N
Asparingine
O
Glutamate
Aromatic side chains
O
_
HS
NH2
O
_
Cysteine
Figure by MIT OpenCourseWare.
4 amino acid substitution --> “λcro67”
Protein α-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 α-helix recognizes sequence in DNA major groove
Wild type λcro
• binds OR3>>OR2 = OR1
• binding to OR3 shuts off tx’n from PRM
Wild type λcI
• binds OR1>OR2>OR3
• binding to OR2 activates tx’n from PRM
Protein α-helix recognizes sequence in DNA major groove
Wild type λcro
• binds OR3>>OR2 = OR1
• binding to OR3 shuts off tx’n from PRM
Wild type λcI
• binds OR1>OR2>OR3
• binding to OR2 activates tx’n from PRM
λcro67
• binds? OR1>OR2>OR3
• activates?
Figure 3
Courtesy Elsevier, Inc., http://www.sciencedirect.com. Used with permission.
Protein α-helix recognizes sequence in DNA major groove
Wild type λcro
• binds OR3>>OR2 = OR1
• binding to OR3 shuts off tx’n from PRM
Wild type λcI
• binds OR1>OR2>OR3
• binding to OR2 activates tx’n from PRM
λcro67
• binds? OR1=OR2>OR3
• activates?
Figure 3
Courtesy Elsevier, Inc., http://www.sciencedirect.com. Used with permission.
λcro67 activates transcription in vitro
Figure 4
[λcro67]
In vitro tx’n rxn’s
0
395 bases
+ buffer
+ DNA w/ PRM + PR
+ λcro67 (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.
λcro67 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 λcro67 added
Q’s: What are extra bands? Is λcro67 bound in natural way?
λcro67 binds operator sequences as expected
Figure 4
[λcro67]
DNase footprint
0
+ buffer
+ 32P-DNA w/ PRM + PR
+ λcro67 (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?
λcro67 activates transcription in vitro
Supporting data/controls
Figure 5
Wild type λcro does not activate txn in vitro
using in vitro txn rxn, DNase ftpt
Figure 6
λcro67 does not
activate txn from other promoters
Courtesy Elsevier, Inc., http://www.sciencedirect.com.
Used with permission.
λcro67 in vivo exp’ts hampered by low affinity for operators
(~100x < wt λcro)
Summary of
434 cI data
2
2
3
3
Figures by MIT OpenCourseWare.
look at******
patch more acidic
λ cI
inc act’n
patch more basic
operator occupancy
dec act’n
sat’d
dec act’n
sat’d
operator binding
normal
normal
** in vivo (β-gal assays on lysogen)
vs
434 cI
inc act’n
** in vivo DMS ftpt
** in vitro txn rxns, DNase ftpt
Turning λcro 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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