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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