Part:BBa_K613017
TetR Y42F mutant
This is a TetR mutant that carries the Y42F mutation. Part of the EPFL2011 muTetR collection.
In vitro characterization
Using the MITOMI technique we determined the DNA binding landscape of the TetR Y42F mutant. To do so, first we designed and generated the library of double stranded DNA sequences that cover all possible single base substitution within the tetO operator sequence. Based on that library we measured the dissociation constants of the mutant to variable tetO-like sequences and determined the specificity of the mutant to the tet operator sequence (expressed as a PWM). For the Y42F mutant we observed the decrease of the specificity compared to the wild-type tetR sequence.
WebLogo we obtained for the Y42F mutant:
The measured binding affinities show no symmetry conservation, these experiment will have to be repeated in order to get a clearer idea of the binding affinities.
Workman CT, Yin Y, Corcoran DL, Ideker T, Stormo GD, Benos PV. enoLOGOS: a versatile web tool for energy normalized sequence logos. Nucleic Acids Res. 2005 Jul 1;33:W389-92.
Position Weight Matrix
| PO | A | T | C | G |
| 1 | 0.385241 | 0.85619 | 0 | 0.693793 |
| 2 | 1.30965 | 0.385241 | 0.629213 | 1.2542 |
| 3 | 1.18851 | 1.22909 | 0.385241 | 1.09825 |
| 4 | 1.1084 | 0.385241 | 1.57045 | 1.20206 |
| 5 | 0.385241 | 1.3955 | 1.10171 | 1.2069 |
| 6 | 1.80251 | 0.385241 | 1.37974 | 1.23728 |
| 7 | 1.25285 | 1.54821 | 0.385241 | 1.43937 |
| 8 | 0.385241 | 0.475161 | 0.93954 | 0.488479 |
| 9 | 0.341937 | 0.385241 | 0 | 0.053757 |
| 10 | 0.973293 | 0.385241 | 0.426141 | 1.06563 |
| 11 | 1.1587 | 1.50073 | 1.5542 | 0.385241 |
| 12 | 0.385241 | 1.30762 | 1.11351 | 1.39762 |
| 13 | 1.23638 | 0.385241 | 1.11282 | 1.24361 |
| 14 | 0.385241 | 1.09074 | 0.75983 | 0.933921 |
| 15 | 0.679315 | 1.33124 | 1.09937 | 0.385241 |
| 16 | 0 | 0.467396 | 1.08277 | 0.385241 |
| 17 | 0.664957 | 0.130021 | 0.385241 | 0 |
Each row represents the changes in binding energy, ΔΔG, compared to the reference sequence upon the substitution to the indicated nucleotide at certain position within the target DNA element. Values are indicated in kcal/mol.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Designed sequence (1 mutation in yellow):
MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLFWHVKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPLLRQAIELFDHQGAEPA
FLFGLELIICGLEKQLKCESGS*
| None |