The Wonderful World of RepressorsSupriya Pokhrel, Firras Garada, Sonia Sharma, Kristen Wade, Trevor Faske, Mandi Feinberg

Background

- Repressor protein binds to upstream promoter regions- blocks transcription of downstream genes- allows for regulation of lytic vs. lysogenic cycles

Conserved Domains of Repressor Proteins in Mycobacteriophages

By Supriya Pokhrel

Repressor and its domains

Is the repressor conserved

mostly in the C-terminal domain

or N-terminal domain of

various phages?

LIST OF PHAGES THAT HAVE SIMILAR REPRESSOR PROTEIN

Motifs compared to lambda phage

Protein Sequence of λ Repressor

CTD Sequence

Conserved regions were found mostly in the C-Terminal domain

ReferencesTimothy L. Bailey and Charles Elkan, "Fitting a mixture

model by expectation maximization to discover motifs in biopolymers", Proceedings of the Second International Conference on Intelligent Systems for Molecular Biology, pp. 28-36, AAAI Press, Menlo Park, California, 1994.

Bell, Charles E. “Crystal Structure of the λ Repressor C-Terminal Domain Provides a Model for Cooperative Operator Binding.” Cell Press, June 23 2000. Volume 101, 801-811.

Ganguly, Tribid and Bandhu, Amitava. “Repressor of temperate mycobacteriophage L1 harbors a stable C-terminal domain and binds to different operator DNAs with variable affinity.” Virology Journal 2007, 4:64.

CI Repressor Protein in Lactococcus Phage TP901 and like Proteins

By Firras Garada

Background Info

• Mor First Protein Transcribed = Lytic State• CI First Protein Transcribed = Lysogenic Stage

Claim and So What

Claim – When the CI repressor protein was mutated it can block transcription of MOR protein

So what – Do other phages have a protein similar to the CI repressor protein that may function the same?

Top Hits for Proteins Similar to Repressor Protein of TP901 (tp901-1.p-tp901-1p04)

Motif Results

Data

• From the Motif Comparison there are 5 other proteins that are closely related to the Repressor Protein of tp901

• However 4 of those are other Lactococcus Phages

• BUT one was an Enterococcus Phage

Alignment of Comparison

Conclusion

• It is highly probably that other Lactococcus phages have a protein similar to the original CI repressor protein

• It is probable that Enterococcus Phage phiEf11 may also have a protein very similar to the original Ci repressor and may also be mutated to control what pathway the phage takes

References

• Characterization of the CI Repressor Protein Encoded by the Temperate Lactococcal Phage TP901-1 Margit Pedersen, Małgorzata Ligowska, Karin Hammer J Bacteriol. 2010 April; 192(8): 2102–2110. Published online 2010 January 29. doi: 10.1128/JB.01387-09

• http://biobike.csbc.vcu.edu/

Locating the Repressor Binding Sites in the

Bacteriophages

• Presented by Sonia Sharma

• BNFO 301

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Repressors What are They

• Repressor are proteins when present can lead to Lysogenic pathway• Block the PR (lytic) promoter, facilitating the binding of RNA

polymerase to the PRM (lysogenic) promoter• Leading to synthesis of CI (orange) Repressor• They bind to specific, upstream sequences

22CONFIDENTIAL

Model- E.Coli phage lambdaRepressor-CI

• Ideal binding sites in e-lambda OR1 OR2• Repressor binds as a Dimer at specific palindromic sequences

Phage RepressorAnti-Repressor

CONFIDENTIAL23

Intergenic sequence

CATACGTTAAATCTATCACCGCAAGGGATAAATATCTAACACCGTGCGTGTTGACTATTTTACCTCTGGCGGTGATAATGGTTGCATGTACTAAGGAGGTTGTATG

CONFIDENTIAL24

Method

Similar sequences were found in known phages- e-lambda, L5, Che12 BXb1Two unknown phages- Packman Shaka

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Graphical map of e-lambda/che12

Click icon to add SmartArt graphic

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Observations

• CI repressor in e-lambda has fewer binding sites• L5 and Bxb1 - Genome contains large number of binding site (29

and 34 each) in intergenic regions in only one orientation relative to transcription direction.

• Che12 has 16 such sites also located in non coding region close to start and stop codon, orientation of sites correlates to the direction of transcription

• Shaka and Packman each show 16 and 18 putative sites each 21 nucleotides wide

• Protein alignment- L5, Bxb1 and Che12 more similar then e-lambda

CONFIDENTIAL33

Concluding thoughtDo majority of Phages follow e-lambda style

or L5 yet to be analyzed

CONFIDENTIAL34

ReferencesSource- Jeff Elhai, Gene Regulation and Phage, Center for the Study of Biological Complexity, Virginia Commonwealth University 

  Gomathi, N S, Sameer, H, Kumar, V, et al. (2007). In silico analysis of mycobacteriophage che12 genome: Characterization of genes required to lysogenise mycobacterium tuberculosis. Computational biology and chemistry, 31(2), 82-91.

Oppenheim, A B, Kobiler, O, Stavans, J, et al. (2005). Switches in bacteriophage lambda development. Annual review of genetics, 39, 409-429.

Indirect Readout Sites of the Repressor-Operator Complex

By: Kristen Wade

Repressor-DNA Interaction

Repressor Protein (two subunits)

Image adapted from: http://www.acsu.buffalo.edu/~koudelka/

Repressor-DNA Interaction

Repressor Protein (two subunits)

Contacted bases

Contacted bases

Image adapted from: http://www.acsu.buffalo.edu/~koudelka/

Repressor-DNA Interaction

Repressor Protein (two subunits)

Contacted bases

Contacted bases

Bases NOT contacted by protein

Image adapted from: http://www.acsu.buffalo.edu/~koudelka/

Repressor-DNA Interaction

Contacted bases

Contacted bases

Bases NOT contacted by protein

Image adapted from: http://www.acsu.buffalo.edu/~koudelka/

Do these non-contacted nucleotides have a function in protein-DNA binding?

Repressor binding sites of phage P22From: Wu et al (1992) J Biol Chem 267:9234-9239

Importance

Essential for structural adjustment of DNA that allows protein interaction with contacted bases

▪ Wu et al, 1992

Can these sites be identified and given

functional significance in phages whose

repressors are unrelated to P22?

Identification

1. Centrally located within operator motifs

Identification

1. Centrally located within operator motifs

2. Surrounded by a palindromic sequence A T AAG--------CTT

A T

Identification

1. Centrally located within operator

motifs2. Surrounded by a

palindromic sequence

3. Display greater sequence

diversity than surrounding

positions

Nu

cle

oti

de

Fre

qu

en

cy

Nucleotide Position

A = solid line C = _._. G = _ _ _ _ T = ----

Application

Operators were collected using Motifs-In Upstream-Sequences-Of Repressor Protein of specific phage

E. Coli phage Lambda

Lambda

Lambda

Lambda

Lambda

Chi Squared:

Sum of (O-E)^2 E

Lambda

A: 5.4120

C: 17.3505

G: 0.1916

T: 0.0256

Predicted Chi square values for each

nucleotide:

Lambda

A: 5.4120

C: 17.350

5G:

0.1916T:

0.0256

Predicted Chi square values for each

nucleotide:

Degrees of freedom: 3Critical value: 0.05

Significance score must be > 7.815

Lambda

A: 5.4120

C: 17.350

5G:

0.1916T:

0.0256

Predicted Chi square values for each

nucleotide:

Degrees of freedom: 3Critical value: 0.05

Significance score must be > 7.815

Mycobacteriophage U2

Observed:A C G

T1 4 2

2

Expected: A C G

T.5840 .9160 .9160 .5

840

Mycobacteriophage U2

Observed:A C G

T1 4 2

2

Expected: A C G

T.5840 .9160 .9160 .5

840A: 0.2963C: 10.3828G: 1.2828T: 3.4333

Next direction:

Apply method used on Lambda and U2 to predicted Indirect Readout Sites of other, unrelated phages

Greater number of sequences = greater likelihood of significance

References

Indirect readout of DNA sequence by p22 repressor: roles of DNA and protein functional groups in modulating DNA conformation.

Lydia-Ann Harris, Derrick Watkins, Loren Dean Williams, Gerald B Koudelka (2013) Journal of molecular biology 425 (1) p. 133-43

Non-contacted bases affect the affinity of synthetic P22 operators for P22 repressor.

L Wu, A Vertino, G B Koudelka (1992)The Journal of biological chemistry 267 (13) p. 9134-9

Image: http://www.acsu.buffalo.edu/~koudelka

Incomplete Repressors and Characteristics in PhagesMandi FeinbergBNFO 301Spring 2013

Phage

Bacteria

attP

attB

C Terminal end Repressor

N Terminal end Repressor

Complete Repressor

Phages

Bacteria

Charlie

“TGGTGCCCCCAGCTGGGCTCGAACCAGCGACCTGCGGATTACCAG"Acintobacteriophage Acj9

TCGAACCAGCGACCTGCGGATTbetween tRNA – Cys & Gly

Mycobacteriophage LeBronGGTGCCCCCAGCAGGACTCGAACCTGCGACC

tRNA-cys (Lys)Mycobacteriophage UPIE

GGTGCCCCCAGCAGGACTCGAACCTGCGACCTGtRNA- cys/ lys

Brujita

“TGGGAGCCGCCTGGGGGAATCGAACCCCCGACCTATTCATTATCA”Mycobacteriophage Island3 TGATAATGAATAGGTCGGGGGTTCGATTCCCCCAGGCGGCTCCCA

phage antirepressor proteintyrosine integrase

Mycobacterium Phage Babsiella TGATAATGAATAGGTCGGGGGTTCGATTCCCCCAGGCGGCTCCCA

integrase (Y-int)

BPS

"AAGTGCGCCCGGAGGGATTCGAACCCCCAACCTTCTGTTT"Mycobacteriophage Angel

AAACAGAAGGTTGGGGGTTCGAATCCCTCCGGGCGCACTTphage repressor phage integrase

Mycobacteriophage HopeAAACAGAAGGTTGGGGGTTCGAATCCCTCCGGGCGCACTTintegraserepressor

Mycobacteriophage HaloAAACAGAAGGTTGGGGGTTCGAATCCCTCCGGGCGCACTT

The End?• Tried same techniques with Acintobacteriophage Acj9 • Characteristics• Use characteristics to find more in other types of phages

Works Cited• http://

www.sciencedirect.com/science/article/pii/S1097276512009434• Broussard, Gregory W., Lauren M. Oldfield, Valerie M. Villanueva,

Bryce L. Lunt, Emilee E. Shine, and Graham F. Hatfull. "Integration-Dependent Bacteriophage Immunity Provides Insights into the Evolution of Genetic Switches." Molecular Cell 49 (2013): 237-48.

• http://biobike.phantome.org/ajax/vpl.html?PKG=FEINBERGMA39619• Intro to BNFO 301 Exam 2• http://www.ncbi.nlm.nih.gov/pmc/articles/PMC94198/• The Genetic Switch Regulating Activity of Early Promoters of the Temp

erate Lactococcal Bacteriophage TP901-1

• Peter Lynge Madsen, Annette H. Johansen, Karin Hammer, Lone Brøndsted

• J Bacteriol. 1999 December; 181(24): 7430–7438.

TREVOR FASKE

Phage cI Repressors: the effects on transcriptional/translational

direction

Enterobacteria Phage P22

Similar cI Repressors

Q-START Q-END TARGET T-START T-END E-VALUE %ID T-ORGANISM 1. Ent-P22.p-P22p26 1 216 Ent-P22.p-P22p26 1 216 4.0d-116 100.0 Enterobacteria-phage-P22 2. Ent-P22.p-P22p26 1 216 DE3.p-ECD_10021 1 216 7.0d-109 93.52 Enterobacteria-phage-DE3 3. Ent-P22.p-P22p26 28 215 ST64T.p-ST64Tp25 41 234 1.0d-54 56.7 Salmonella-phage-ST64T 4. Ent-P22.p-P22p26 25 211 P27.p-P27p11 23 214 1.0d-43 45.31 Escherichia-phage-P27 5. Ent-P22.p-P22p26 28 211 Ent-1717.p-Stx2-1717_gp20 25 210 6.0e-28 36.36 Stx2-phage-1717 6. Ent-P22.p-P22p26 86 211 VT2-Sa.p-VT2-Sap24 36 162 3.0e-27 46.09 Escherichia-phage-VT2-Sa 7. Ent-P22.p-P22p26 35 214 VP882.p-VPVV882_gp59 61 241 1.0e-22 35.29 Vibrio-phage-VP882 8. Ent-P22.p-P22p26 35 208 VHML.p-VHMLp06 43 215 5.0e-11 26.7 Vibrio-phage-VHML 9. Ent-P22.p-P22p26 25 208 Phi-47.p-Phi-47-0035 20 197 1.0e-6 27.08 Staphylococcus-phage-47 10. Ent-P22.p-P22p26 34 210 2638A.p-2638A-0026 31 202 8.0e-4 25.68 Staphylococcus-phage-2638A

Surrounding Proteins

Does cI repressors role in directional also play a

part in alignment and function

of proteins up- and down stream?

Motif and Alignment of cI

(<- Ent-P22.P22p21 Phage protein) 0 (<- Ent-P22.P22p22 Phage protein) 36 (<- Ent-P22.P22p23 Restriction alleviation ral # )214 (-> Ent-P22.P22p24 Phage superinfection exclusion) 20 (<- Ent-P22.P22p25 Phage antitermination protein ) 353 (<- Ent-P22.P22p26 Phage cI repressor # ACLAME 5) 80 (-> Ent-P22.P22p27 Phage repressor # ACLAME 146)106(-> Ent-P22.P22p28 Phage repressor protein CII) 34 (-> Ent-P22.P22p29 Phage protein) 0 (-> Ent-P22.P22p30 Origin specific replication in) 0 (-> Ent-P22.P22p31 Phage replicative DNA helicase)

(<- DE3.ECD_10016 Phage repressor protein CIII) 72 (<- DE3.ECD_10017 Single stranded DNA-binding pr) 182 (<- DE3.ECD_10018 Restriction alleviation ral # ) 0 (<- DE3.ECD_10019 Phage protein) 8 (<- DE3.ECD_10020 Phage antitermination protein ) 314 (<- DE3.ECD_10021 Phage cI repressor (ACLAME 5)) 80 (-> DE3.ECD_10022 Putative phage repressor (ACLA) 115 (-> DE3.ECD_10023 Phage repressor protein CII) 32 (-> DE3.ECD_10024 Origin specific replication in) 0 (-> DE3.ECD_10025 Origin specific replication bi) 0 (-> DE3.ECD_10026 Serine/threonine protein phosp)

(<- ST64T.ST64Tp20 Phage repressor protein CIII) 63 (<- ST64T.ST64Tp21 Phage superinfection exclusion) 171 (<- ST64T.ST64Tp22 Phage pentapeptide repeat fami) 0 (<- ST64T.ST64Tp23 Restriction alleviation ral # ) 78 (<- ST64T.ST64Tp24 Phage antitermination protein ) 557 (<- ST64T.ST64Tp25 Phage cI repressor # ACLAME 5) 76 (-> ST64T.ST64Tp26 Phage repressor) 110 (-> ST64T.ST64Tp27 Phage repressor protein CII) 173 (-> ST64T.ST64Tp28 Origin specific replication in) 0 (-> ST64T.ST64Tp29 DNA helicase (EC 3.6.1.-), pha) 74 (-> ST64T.ST64Tp30 Phage protein # ACLAME 1442)

(<- Ent-1717.Stx2-1717_gp15 Phage repressor protein CIII) 72 (<- Ent-1717.Stx2-1717_gp16 Single stranded DNA-binding pr) 182 (<- Ent-1717.Stx2-1717_gp17 Phage protein) 58 (<- Ent-1717.Stx2-1717_gp18 Phage anti-termination protein) 656 (<- Ent-1717.Stx2-1717_gp19 Phage protein) 501 (<- Ent-1717.Stx2-1717_gp20 Phage cI repressor # ACLAME 5) 116 (-> Ent-1717.Stx2-1717_gp21 Phage repressor) 141 (-> Ent-1717.Stx2-1717_gp22 Phage repressor protein CII) 32 (-> Ent-1717.Stx2-1717_gp23 Phage protein) 0 (-> Ent-1717.Stx2-1717_gp24 Origin specific replication in) 0 (-> Ent-1717.Stx2-1717_gp25 Phage replicative DNA helicase)

(<- VT2-Sa.VT2-Sap19 Phage protein) 0 (<- VT2-Sa.VT2-Sap20 Phage protein) 58 (<- VT2-Sa.VT2-Sap21 Putative anti-termination prot) 384 (-> VT2-Sa.VT2-Sap22 Phage protein) 110 (<- VT2-Sa.VT2-Sap23 Phage protein) 501 (<- VT2-Sa.VT2-Sap24 Phage cI repressor # ACLAME 5) 57 (<- VT2-Sa.VT2-Sap25 Phage cI repressor # ACLAME 5) 75 (-> VT2-Sa.VT2-Sap26 Phage repressor) 141 (-> VT2-Sa.VT2-Sap27 Phage repressor protein CII) 171 (-> VT2-Sa.VT2-Sap28 Phage replication initiation p) 0 (-> VT2-Sa.VT2-Sap29 DNA helicase (EC 3.6.1.-), pha)

P22

DE3 ST64T

STX2

VT2

Protein Alignment

Potential further discoveries

Upstream/Downstream sequences using cI repressor motif

Motifs in predicted “like” proteins

Use this information for annotations

References

http://biobike.phantome.org/ajax/vpl.html?PKG=FEINBERGMA39619

Jeff Elhai, Introduction to Bioinformatics 301, Center for the Study of Biological Complexity, VCU