Turning λ Cro into a

Transcription

1 Turning λ Cro into a 3 Transcriptional Activator Figure by MIT pencourseware. Fred Bushman and Mark Ptashne Cell (1988) 54: Presented by atalie Kuldell for 0.90 February 4th, 009

2 Small patch of acidic residues is necessary and sufficient for transcriptional activation Figure 1 RA Polymerase λcl normally activates transcription λcro normally represses transcription RA Polymerase λcro/cl chimera activates transcription! Figure by MIT pencourseware.

3 Site-directed mutagenesis of λcro helix to make acidic patch cartoon of λci binding DA Figure 3 Thr17 Glu Gln Thr Asp Asp Ala 1 Leu Val Figure by MIT pencourseware. fig from A Genetic Switch Tyr6 Figure by MIT pencourseware. Cro67 4 amino acid substitution --> λcro67

4 Why might this work? Repressor Cro Met Met Asp Glu Ser Val Ala Gln Gln Ser Helix Helix 3 Val Phe Leu Asn ala Asp Leu Val Thr Thr Ala Tyr Gln Ala Ser Gln Helix Ile Helix 3 Asn Ala Ile His Figure by MIT pencourseware.

5 Site-directed mutagenesis of λcro helix to make acidic patch Acidic and amide side chains H H Aspartate Asparingine Tryptophan Phenylalanine _ H _ H _ Glutamate Glutamine H H Tyrosine H H ine H Serine H Threonine Basic side chains H _ Arginine S Valine Histidine _ Hydroxyl or sulfur-containing side chains Methionine HS Cysteine Aromatic side chains Aliphatic side chains Alanine _ Isoleucine Leucine _ Cyclic side chain _ Proline _ cine _ Asp Thr17 Leu Val Asp Glu Thr Ala Tyr6 Figure by MIT pencourseware. Gln 1 Cro67 Figure by MIT pencourseware. 4 amino acid substitution --> λcro67

6 Site-directed mutagenesis of λcro helix to make acidic patch Acidic and amide side chains H H Aspartate Asparingine Tryptophan Phenylalanine _ H _ H _ Glutamate Glutamine H H Tyrosine H H ine H Serine H Threonine Basic side chains H _ Arginine S Valine Histidine _ Hydroxyl or sulfur-containing side chains Methionine HS Cysteine Aromatic side chains Aliphatic side chains Alanine _ Isoleucine Leucine _ Cyclic side chain _ Proline _ cine _ Asp Thr17 Leu Val Asp Glu Thr Ala Tyr6 Figure by MIT pencourseware. Gln 1 Cro67 Figure by MIT pencourseware. 4 amino acid substitution --> λcro67

7 Site-directed mutagenesis of λcro helix to make acidic patch Acidic and amide side chains H H Aspartate Asparingine Tryptophan Phenylalanine _ H _ H _ Glutamate Glutamine H H Tyrosine H H ine H Serine H Threonine Basic side chains H _ Arginine S Valine Histidine _ Hydroxyl or sulfur-containing side chains Methionine HS Cysteine Aromatic side chains Aliphatic side chains Alanine _ Isoleucine Leucine _ Cyclic side chain _ Proline _ cine _ Asp Thr17 Leu Val Asp Glu Thr Ala Tyr6 Figure by MIT pencourseware. Gln 1 Cro67 Figure by MIT pencourseware. 4 amino acid substitution --> λcro67

8 Site-directed mutagenesis of λcro helix to make acidic patch Acidic and amide side chains H H Aspartate Asparingine Tryptophan Phenylalanine _ H _ H _ Glutamate Glutamine H H Tyrosine H H ine H Serine H Threonine Basic side chains H _ Arginine S Valine Histidine _ Hydroxyl or sulfur-containing side chains Methionine HS Cysteine Aromatic side chains Aliphatic side chains Alanine _ Isoleucine Leucine _ Cyclic side chain _ Proline _ cine _ Asp Thr17 Leu Val Asp Glu Thr Ala Tyr6 Figure by MIT pencourseware. Gln 1 Cro67 Figure by MIT pencourseware. 4 amino acid substitution --> λcro67

9 Protein α-helix recognizes sequence in DA major groove 3 Figure by MIT pencourseware. Courtesy of Timothy Paustian. Used with permission. model of lac repressor binding lac operator

10 Protein α-helix recognizes sequence in DA major groove Wild type λcro binds R 3>> R = R 1 binding to R 3 shuts off tx n from P RM Wild type λci binds R 1> R > R 3 binding to R activates tx n from P RM

11 Protein α-helix recognizes sequence in DA major groove Wild type λcro binds R 3>> R = R 1 binding to R 3 shuts off tx n from P RM Wild type λci binds R 1> R > R 3 binding to R activates tx n from P RM λcro67 binds? R 1> R > R 3 activates? Figure 3 Courtesy Elsevier, Inc., Used with permission.

12 Protein α-helix recognizes sequence in DA major groove Wild type λcro binds R 3>> R = R 1 binding to R 3 shuts off tx n from P RM Wild type λci binds R 1> R > R 3 binding to R activates tx n from P RM λcro67 binds? R 1= R > R 3 activates? Figure 3 Courtesy Elsevier, Inc., Used with permission.

13 λcro67 activates transcription in vitro Figure 4 + buffer In vitro tx n rxn s + DA w/ P RM + P R [λcro67] bases + λcro67 (purified) + 3 P-ATP, CTP, GTP or UTP then + RAP then +formamide to gel 50 bases Courtesy Elsevier, Inc., Used with permission.

14 λcro67 activates transcription in vitro Figure 4 cut out bands and count ~5x 395 bases ~5x 50 bases Courtesy Elsevier, Inc., Used with permission. bserve: txn of P R as txn of P RM when λcro67 added Q s: What are extra bands? Is λcro67 bound in natural way?

15 λcro67 binds operator sequences as expected Dase footprint + buffer Figure P-DA w/ P RM + P R + λcro67 (purified) 37 10? then + Dase 37 10? then +formamide [λcro67] 0 to gel? Courtesy Elsevier, Inc., Used with permission. bserve: R 1= R > R 3 Q: is assay sensitive to different conformations of bound prot?

16 Figure 5 λcro67 activates transcription in vitro Supporting data/controls Wild type λcro does not activate txn in vitro using in vitro txn rxn, Dase ftpt Figure 6 λcro67 does not activate txn from other promoters Courtesy Elsevier, Inc., Used with permission. λcro67 in vivo exp ts hampered by low affinity for operators (~100x < wt λcro)

17 Summary of 434 ci data 3 3 Figures by MIT pencourseware. look at****** λ ci vs 434 ci patch more acidic inc act n inc act n patch more basic operator occupancy operator binding dec act n sat d normal dec act n sat d normal ** in vivo (β-gal assays on lysogen) ** in vivo DMS ftpt ** in vitro txn rxns, Dase ftpt

18 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., DA binding) ask the next question: does activation work the same way in eukaryotic cells?

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