Transcript The Wonderful World of Repressors

The Wonderful
World of Repressors
Supriya 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 Cterminal 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
References
Timothy 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 CTerminal 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
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
21
CONIDENTIAL
Model- E.Coli phage lambda
Repressor-CI
AntiRepressor
Phage Repressor
• Ideal binding sites in e-lambda OR1 OR2
• Repressor binds as a Dimer at specific palindromic sequences
22
CONFIDENTIAL
Intergenic sequence
CATACGTTAAATCTATCACCGCAAGGGATAAATATCTAACACC
GTGCGTGTTGACTATTTTACCTCTGGCGGTGATAATGGTTGCA
TGTACTAAGGAGGTTGTATG
23
CONFIDENTIAL
Method
Similar sequences were found in known phages- e-lambda,
L5, Che12 BXb1
Two unknown phagesPackman
Shaka
24
CONFIDENTIAL
25
CONFIDENTIAL
26
CONFIDENTIAL
27
CONFIDENTIAL
Graphical map of elambda/che12
28
CONFIDENTIAL
29
CONFIDENTIAL
30
CONFIDENTIAL
31
CONFIDENTIAL
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 elambda
32
CONFIDENTIAL
Concluding thought
Do majority of Phages
follow e-lambda style or L5
yet to be analyzed
33
CONFIDENTIAL
References
Source- 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.
34
CONFIDENTIAL
By: Kristen Wade
Repressor Protein
(two subunits)
Image adapted from: http://www.acsu.buffalo.edu/~koudelka/
Contacted bases
Repressor Protein
(two subunits)
Contacted bases
Image adapted from: http://www.acsu.buffalo.edu/~koudelka/
Contacted bases
Repressor Protein
(two subunits)
Bases NOT contacted
by protein
Contacted bases
Image adapted from: http://www.acsu.buffalo.edu/~koudelka/
Do these non-contacted nucleotides
have a function in protein-DNA binding?
Contacted bases
Bases NOT contacted
by protein
Repressor binding sites of phage P22
Contacted bases
From: Wu et al (1992) J Biol Chem 267:9234-9239
Image adapted from: http://www.acsu.buffalo.edu/~koudelka/

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?
1. Centrally located within operator motifs
1. Centrally located within operator motifs
2. Surrounded by a palindromic sequence
A T AAG--------CTT A T
Nucleotide Frequency
1. Centrally located
within operator motifs
2. Surrounded by a
palindromic sequence
3. Display greater
sequence diversity than
surrounding positions
A = solid line
C = _._.
G=____
T = ----
Nucleotide Position

Operators were collected using Motifs-In
Upstream-Sequences-Of Repressor
Protein of specific phage
Chi Squared:
Sum of (O-E)^2
E
Predicted Chi square values
for each nucleotide:
A: 5.4120
C: 17.3505
G: 0.1916
T: 0.0256
Predicted Chi square values
for each nucleotide:
A: 5.4120
C: 17.3505
G: 0.1916
T: 0.0256
Degrees of freedom: 3
Critical value: 0.05
Significance score must be > 7.815
Predicted Chi square values
for each nucleotide:
A: 5.4120
C: 17.3505
G: 0.1916
T: 0.0256
Degrees of freedom: 3
Critical value: 0.05
Significance score must be > 7.815
Observed:
A C G T
1 4 2 2
Expected:
A
C
G
T
.5840 .9160 .9160 .5840
Expected:
A
C
G
T
.5840 .9160 .9160 .5840
Observed:
A C G T
1 4 2 2
A: 0.2963
C: 10.3828
G: 1.2828
T: 3.4333

Apply method used on Lambda and U2 to
predicted Indirect Readout Sites of other,
unrelated phages

Greater number of sequences = greater
likelihood of significance

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
Phages
Mandi Feinberg
BNFO 301
Spring 2013
Phage
attP
Bacteria
attB
N Terminal end
Repressor
Complete Repressor
C Terminal end
Repressor
Phages
Bacteria
Charlie
“TGGTGCCCCCAGCTGGGCTCGAACCAGCGACCTGCGGATTACCAG"
Acintobacteriophage Acj9
TCGAACCAGCGACCTGCGGATT
between tRNA – Cys & Gly
Mycobacteriophage LeBron
GGTGCCCCCAGCAGGACTCGAACCTGCGACC
tRNA-cys (Lys)
Mycobacteriophage UPIE
GGTGCCCCCAGCAGGACTCGAACCTGCGACCTG
tRNA- cys/ lys
Brujita
“TGGGAGCCGCCTGGGGGAATCGAACCCCCGACCTATTCATTATCA”
Mycobacteriophage Island3
TGATAATGAATAGGTCGGGGGTTCGATTCCCCCAGGCGGCTCCCA
phage antirepressor protein
tyrosine integrase
Mycobacterium Phage Babsiella
TGATAATGAATAGGTCGGGGGTTCGATTCCCCCAGGCGGCTCCCA
integrase (Y-int)
BPS
"AAGTGCGCCCGGAGGGATTCGAACCCCCAACCTTCTGTTT"
Mycobacteriophage Angel
AAACAGAAGGTTGGGGGTTCGAATCCCTCCGGGCGCACTT
phage repressor
phage integrase
Mycobacteriophage Hope
AAACAGAAGGTTGGGGGTTCGAATCCCTCCGGGCGCACTT
integrase
repressor
Mycobacteriophage Halo
AAACAGAAGGTTGGGGGTTCGAATCCCTCCGGGCGCACTT
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/S1097276512009
434
• 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=FEINBERGMA3961
9
• 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
Temperate Lactococcal Bacteriophage TP901-1
• Peter Lynge Madsen, Annette H. Johansen, Karin Hammer, Lone
Brøndsted
• J Bacteriol. 1999 December; 181(24): 7430–7438.
Phage cI Repressors: the effects on
transcriptional/translational
direction
TREVOR FASKE
Enterobacteria Phage P22
Similar cI Repressors
Q-START Q-END TARGET
ORGANISM
1. Ent-P22.p-P22p26
P22
2. Ent-P22.p-P22p26
DE3
3. Ent-P22.p-P22p26
ST64T
4. Ent-P22.p-P22p26
5. Ent-P22.p-P22p26
6. Ent-P22.p-P22p26
VT2-Sa
7. Ent-P22.p-P22p26
8. Ent-P22.p-P22p26
9. Ent-P22.p-P22p26
phage-47
10. Ent-P22.p-P22p26
phage-2638A
1 216 Ent-P22.p-P22p26
T-START T-END E-VALUE
1 216
1 216 DE3.p-ECD_10021
1 216
28 215 ST64T.p-ST64Tp25
41 234
4.0d-116
100.0
7.0d-109
93.52
1.0d-54
56.7
25 211 P27.p-P27p11
23 214
1.0d-43
28 211 Ent-1717.p-Stx2-1717_gp20 25 210 6.0e-28
86 211 VT2-Sa.p-VT2-Sap24
36 162 3.0e-27
%ID
T-
Enterobacteria-phageEnterobacteria-phageSalmonella-phage-
45.31
Escherichia-phage-P27
36.36
Stx2-phage-1717
46.09
Escherichia-phage-
35 214 VP882.p-VPVV882_gp59 61 241
35 208 VHML.p-VHMLp06
43 215
25 208 Phi-47.p-Phi-47-0035
20 197
1.0e-22
5.0e-11
1.0e-6
35.29
26.7
27.08
Vibrio-phage-VP882
Vibrio-phage-VHML
Staphylococcus-
34 210 2638A.p-2638A-0026
8.0e-4
25.68
Staphylococcus-
31 202
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
P22
(<- Ent-P22.P22p21
(<- Ent-P22.P22p22
(<- Ent-P22.P22p23
(-> Ent-P22.P22p24
(<- Ent-P22.P22p25
(<- Ent-P22.P22p26
(-> Ent-P22.P22p27
(-> Ent-P22.P22p28
(-> Ent-P22.P22p29
(-> Ent-P22.P22p30
(-> Ent-P22.P22p31
STX2
DE3
Phage protein) 0
Phage protein) 36
Restriction alleviation ral # )214
Phage superinfection exclusion) 20
Phage antitermination protein ) 353
Phage cI repressor # ACLAME 5) 80
Phage repressor # ACLAME 146)106
Phage repressor protein CII) 34
Phage protein) 0
Origin specific replication in) 0
Phage replicative DNA helicase)
(<- Ent-1717.Stx2-1717_gp15
(<- Ent-1717.Stx2-1717_gp16
(<- Ent-1717.Stx2-1717_gp17
(<- Ent-1717.Stx2-1717_gp18
(<- Ent-1717.Stx2-1717_gp19
(<- Ent-1717.Stx2-1717_gp20
(-> Ent-1717.Stx2-1717_gp21
(-> Ent-1717.Stx2-1717_gp22
(-> Ent-1717.Stx2-1717_gp23
(-> Ent-1717.Stx2-1717_gp24
(-> Ent-1717.Stx2-1717_gp25
ST64T
(<- 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)
Phage repressor protein CIII) 72
Single stranded DNA-binding pr) 182
Phage protein) 58
Phage anti-termination protein) 656
Phage protein) 501
Phage cI repressor # ACLAME 5) 116
Phage repressor) 141
Phage repressor protein CII) 32
Phage protein) 0
Origin specific replication in) 0
Phage replicative DNA helicase)
(<- 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)
(<- 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)
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