No description available.

No description available.

No description available.

No description available.

Secondary metabolism occurs after the main growth phase in Streptomyces. A 'transition phase' occurs to remodel global patterns of gene expression at the onset of physiological and developmental differentiation. Many different signals influence this transition phase, integrating, for example, information on nutritional status, growth rate, and stress responses. Several pleiotropic transcription factors that regulate the transition phase have been identified, but aspects of epigenetic control of gene expression are not well understood. This study focused on the characterisation of a novel gene sco2075 in S. coelicolor encoding a protein that combines a histone-like domain with a conserved DksA-like domain, the latter considered a ppGpp cofactor. The protein is important for integrating responses to both oxidative and osmotic stresses. The sco2075- mutant strain is sensitive to oxidative stress at least in part due to reduced induction of the alternative sigma factor sigmaR. SCO2075, similarly to E. coli DksA, may play a possible role in the liberation of core RNA polymerase to bind alternative sigma factors such as sigmaR. In addition DSCO2075 has an altered topological profile of a reporter plasmid under osmotic stress, showing little alteration in negative supercoiling when compared to the significant increase in wildtype. DSCO2075 also has a reduction in aerial hyphae and a possible reduction in actinorhodin production when grown with osmolyte. The histone-like domain of SCO2075 binds DNA non-specifically. SCO2075 expression appears to coincide with diffused FtsZ expression prior to Z-ring formation when SCO2075 appears to become nucleoid associated. Analysis of pre-spore compartment lengths showed SCO2075 is one of several nucleoid associated proteins involved in nucleoid compaction during aerial hyphal erection and sporulation. Absence of sco2075, however, does not affect the production of unigenomic spore chains. Finally, over-expression of SCO2075 suppresses defects in secondary metabolism of a relA mutant affected in ppGpp synthesis. SCO2075 could potentially be a new type of regulator, likely acting as a node to integrate stress and physiological cues by modulating DNA topology/compaction and RNA polymerase activity.

No description available.

No description available.

Zinc finger (ZnF) domains are prevalent in eukaryotes and play crucial roles in mediating protein-DNA and protein-protein interactions. This Thesis focuses on the molecular details underlying interactions mediated by two ZnF domains. The GATA-1 protein is vital for the development of erythrocytes and megakaryocytes. Pertinent to the protein function is the N-terminal ZnF. In particular, this domain mediates interaction with DNA containing GATC motifs and the coactivator protein FOG. The importance of these interactions was illustrated by the findings in Chapter 3 that naturally occurring mutations identified in patients suffering from blood disorders affect the interaction of the N-terminal ZnF with either DNA (R216Q mutation) or FOG (V205M and G208S mutations). In addition to the interaction FOG makes with GATA-1, it also interacts with the centrosomal protein TACC3. In Chapter 4, this interaction is characterised in detail. The solution structure of the region of FOG responsible for the interaction is determined using NMR spectroscopy, revealing that it is a true classical zinc finger, and characterisation of the interaction domain of TACC3 showed that the region is a dimeric coiled-coil. The FOG:TACC3 interaction appears to be mediated by a-helices from the two proteins. The data presented here represent some of the first described molecular details of how a classical ZnF can contact a protein partner. Interestingly, the a-helix used by the FOG finger to bind TACC3 is the same region utilised by DNA-binding classical zinc fingers to contact DNA. In addition to the multiple roles played by ZnFs, this domain is also known for its robustness and versatility. In Chapter 5, incomplete ZnF sequences were assessed for its ability to form functional zinc-binding domains. Remarkably, CCHX sequences (in the context of BKLF finger 3) were able to form discrete zinc-binding domains and also, mediate both protein-DNA and protein-protein interactions. This result not only illustrates the robust nature of ZnFs, it highlights the need for expanding ZnF sequence criteria when searching for functional zinc-binding modules. Together, the data presented here help further our understanding of zinc finger domains. Similar to the use of DNA-binding ZnFs in designer proteins, these data may start us on the path of designing novel protein-binding ZnFs.

Zinc finger (ZnF) domains are prevalent in eukaryotes and play crucial roles in mediating protein-DNA and protein-protein interactions. This Thesis focuses on the molecular details underlying interactions mediated by two ZnF domains. The GATA-1 protein is vital for the development of erythrocytes and megakaryocytes. Pertinent to the protein function is the N-terminal ZnF. In particular, this domain mediates interaction with DNA containing GATC motifs and the coactivator protein FOG. The importance of these interactions was illustrated by the findings in Chapter 3 that naturally occurring mutations identified in patients suffering from blood disorders affect the interaction of the N-terminal ZnF with either DNA (R216Q mutation) or FOG (V205M and G208S mutations). In addition to the interaction FOG makes with GATA-1, it also interacts with the centrosomal protein TACC3. In Chapter 4, this interaction is characterised in detail. The solution structure of the region of FOG responsible for the interaction is determined using NMR spectroscopy, revealing that it is a true classical zinc finger, and characterisation of the interaction domain of TACC3 showed that the region is a dimeric coiled-coil. The FOG:TACC3 interaction appears to be mediated by a-helices from the two proteins. The data presented here represent some of the first described molecular details of how a classical ZnF can contact a protein partner. Interestingly, the a-helix used by the FOG finger to bind TACC3 is the same region utilised by DNA-binding classical zinc fingers to contact DNA. In addition to the multiple roles played by ZnFs, this domain is also known for its robustness and versatility. In Chapter 5, incomplete ZnF sequences were assessed for its ability to form functional zinc-binding domains. Remarkably, CCHX sequences (in the context of BKLF finger 3) were able to form discrete zinc-binding domains and also, mediate both protein-DNA and protein-protein interactions. This result not only illustrates the robust nature of ZnFs, it highlights the need for expanding ZnF sequence criteria when searching for functional zinc-binding modules. Together, the data presented here help further our understanding of zinc finger domains. Similar to the use of DNA-binding ZnFs in designer proteins, these data may start us on the path of designing novel protein-binding ZnFs.

SgrAI is a restriction endonuclease (ENase) that cuts a long recognition sequence and exhibits self-modulation of cleavage activity and sequence specificity. Previous research has shown that SgrAI forms large oligomers when bound to particular DNA sequences and under the same conditions where SgrAI exhibits accelerated DNA cleavage kinetics. However, the detailed structure and stoichiometry of SgrAI:DNA as well as the basic building block of the oligomers, has not been fully characterized. Ion mobility mass spectrometry (IM-MS) was employed to analyze SgrAI/DNA complexes and show that the basic building block of the oligomers is the DNA-bound SgrAI dimer (DBD). The oligomers are heterogeneous containing a mixture of species with variable numbers of DBD. The collision cross sections (CCS) of the oligomers were found to have a linear relationship with the number of DBD. Models of the SgrAI/DNA oligomers were constructed and a head-to-tail arrangement was most consistent with the experimental CCS.