The Wonderful World of Repressors Supriya Pokhrel, Firras Garada, Sonia Sharma, Kristen Wade, Trevor...
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Transcript of The Wonderful World of Repressors Supriya Pokhrel, Firras Garada, Sonia Sharma, Kristen Wade, Trevor...
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
CONIDENTIAL21
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
CONFIDENTIAL25
CONFIDENTIAL26
CONFIDENTIAL27
CONFIDENTIAL28
Graphical map of e-lambda/che12
Click icon to add SmartArt graphic
CONFIDENTIAL29
CONFIDENTIAL30
CONFIDENTIAL31
CONFIDENTIAL32
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