Abstract
Viruses constitute the most abundant genetic pool on earth and are divided into several orders. One orders is the bacteriophages. A common characteristic of this order is the ability to infect bacteria. The order is further divided into families, subfamilies, genuses and species. Some species are virulent such as the T4 bacteriophage and some are temperate as the well studied λ bacteriophage. Temperate bacteriophages inherently have the ability to be bistable and thus can be in a lytic state or in a lysogenic state.
A genetic switch determines the decision for lytic or lysogenic cycle.
Temperate bacteriophage 901-1 (TP901-1) hosts a genetic switch, which is until present unique in many aspects compared to other investigated genetic switches such as those found in λ - and 186 bacteriophage. In TP901-1 the genetic switch consists of Clear I repressor (CI), the antirepressor Modulator Of Repression (MOR), three Operator sites, Right, Left and Distance (OR, OL and OD) and two Promoters, Right and Left (PR and PL). The CI repressor is generally the major regulator of temperate bacteriophages genetic switches. In general CI repressors consist of two domains, the N-Terminal Domain (NTD) and C-Terminal Domain (CTD), separated by a linker of various lengths. The NTD is responsible for DNA binding and the CTD is responsible
for oligomerization of the CI protein. A Helix-turn-Helix motif is usually responsible for DNA binding.
This thesis presents a structural characterization of the CI repressor from
TP901-1 obtained by employing complementary techniques: X-ray crystallography, Nuclear Magnetic Resonance (NMR) spectroscopy and Small Angle X-ray Scattering (SAXS). Employing crystallography, NMR and SAXS we show that NTD in solution and when crystallizes exists as a monomer. Furthermore, we here show the interaction with DNA by crystallography and NMR. The binding are found to be similar to previously found pattern of HTH motifs DNA binding supporting the assumption that NTD is a HTH motif. The HTH motif itself revealed an extended scaffold helix, which we hypothesize might be an interaction site for the antirepressor MOR.
To investigate the flexibility of CI we truncated it C-terminally by 58 amino
acids. Thus truncated CI, CIΔ58, was of a size that could be investigated by NMR and was also investigated by SAXS. We here show by NMR experiments that the dimeric construct of CI, CIΔ58, is highly flexible with a three domains arrangement, two NTDs and one dimerization region. SAXS analysis by the EOM approach showed that CIΔ58 exists in equilibrium between preferred compact and more rare extended conformations resulting variable distance between NTDs in CIΔ58 We further present preliminary crystallization condition of the CTD, responsible for the arrangement of the hexameric CI repressor. The arrangement of the CI repressor for TP901-1 has previously been described to have 32 point group symmetry based on SAXS studies. The CTD crystallizes with P3121 or P3221 symmetry with one molecule in the asymmetric unit. The symmetry is thus reminiscent of the previously suggested symmetry. Indicating that the arrangement of the hexamer is as suggested previously.
The results obtained have structurally characterized the NTD interactions with
DNA and described the dimeric arrangement, but how each dimer is arranged in the full-length CI can only be answered unambiguously by solving the CTD crystal structure or by use of alternative techniques on the full-length CI.
A genetic switch determines the decision for lytic or lysogenic cycle.
Temperate bacteriophage 901-1 (TP901-1) hosts a genetic switch, which is until present unique in many aspects compared to other investigated genetic switches such as those found in λ - and 186 bacteriophage. In TP901-1 the genetic switch consists of Clear I repressor (CI), the antirepressor Modulator Of Repression (MOR), three Operator sites, Right, Left and Distance (OR, OL and OD) and two Promoters, Right and Left (PR and PL). The CI repressor is generally the major regulator of temperate bacteriophages genetic switches. In general CI repressors consist of two domains, the N-Terminal Domain (NTD) and C-Terminal Domain (CTD), separated by a linker of various lengths. The NTD is responsible for DNA binding and the CTD is responsible
for oligomerization of the CI protein. A Helix-turn-Helix motif is usually responsible for DNA binding.
This thesis presents a structural characterization of the CI repressor from
TP901-1 obtained by employing complementary techniques: X-ray crystallography, Nuclear Magnetic Resonance (NMR) spectroscopy and Small Angle X-ray Scattering (SAXS). Employing crystallography, NMR and SAXS we show that NTD in solution and when crystallizes exists as a monomer. Furthermore, we here show the interaction with DNA by crystallography and NMR. The binding are found to be similar to previously found pattern of HTH motifs DNA binding supporting the assumption that NTD is a HTH motif. The HTH motif itself revealed an extended scaffold helix, which we hypothesize might be an interaction site for the antirepressor MOR.
To investigate the flexibility of CI we truncated it C-terminally by 58 amino
acids. Thus truncated CI, CIΔ58, was of a size that could be investigated by NMR and was also investigated by SAXS. We here show by NMR experiments that the dimeric construct of CI, CIΔ58, is highly flexible with a three domains arrangement, two NTDs and one dimerization region. SAXS analysis by the EOM approach showed that CIΔ58 exists in equilibrium between preferred compact and more rare extended conformations resulting variable distance between NTDs in CIΔ58 We further present preliminary crystallization condition of the CTD, responsible for the arrangement of the hexameric CI repressor. The arrangement of the CI repressor for TP901-1 has previously been described to have 32 point group symmetry based on SAXS studies. The CTD crystallizes with P3121 or P3221 symmetry with one molecule in the asymmetric unit. The symmetry is thus reminiscent of the previously suggested symmetry. Indicating that the arrangement of the hexamer is as suggested previously.
The results obtained have structurally characterized the NTD interactions with
DNA and described the dimeric arrangement, but how each dimer is arranged in the full-length CI can only be answered unambiguously by solving the CTD crystal structure or by use of alternative techniques on the full-length CI.
| Originalsprog | Engelsk |
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| Forlag | Department of Chemistry, Faculty of Science, University of Copenhagen |
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| Antal sider | 172 |
| Status | Udgivet - 2013 |