No abstract

The bacteriophage lambda replication initiation protein P exhibits a toxic effect on its Escherichia coli (E. coli) host, likely due to the formation of a dead-end P-DnaB complex, sequestering the replicative DnaB helicase from further activity. Intracellular expression of P triggers SOS-independent cellular filamentation and rapidly cures resident ColE1 plasmids. The toxicity of P is suppressed by alleles of P or dnaB. We asked whether P buildup within a cell can influence E. coli replication fidelity. The influence of P expression from a defective prophage, or when cloned and expressed from a plasmid was examined by screening for auxotrophic mutants, or by selection for rifampicin resistant (RifR) cells acquiring mutations within the rpoB gene encoding the β-subunit of RNA polymerase (RNAP), nine of which proved unique. Using fluctuation assays, we show that the intracellular expression of P evokes a mutator effect. Most of the RifR mutants remained PS and localized to the Rif binding pocket in RNAP, but a subset acquired a PR phenotype, lost sensitivity to ColE1 plasmid curing, and localized outside of the pocket. One PR mutation was identical to rpo*Q148P, which alleviates the UV-sensitivity of ruv strains defective in the migration and resolution of Holliday junctions and destabilizes stalled RNAP elongation complexes. The results suggest that P-DnaB sequestration is mutagenic and supports an earlier observation that P can interact with RNAP.

Homeotic selector (HOX) transcription factors (TFs) regulate gene expression that determines the identity of Drosophila segments along the anterior-posterior (A-P) axis. The current challenge with HOX proteins is understanding how they achieve their functional specificity while sharing a highly conserved homeodomain (HD) that recognize the same DNA binding sites. One mechanism proposed to regulate HOX activity is differential posttranslational modification (PTM). As a first step in investigating this hypothesis, the sites of PTM on a Sex combs reduced protein fused to a triple tag (SCRTT) extracted from developing embryos were identified by Tandem Mass Spectrometry (MS/MS).

Cholesterol-dependent cytolysins (CDCs), a family of β-barrel pore-forming toxins produced by various pathogens create pores on membranes of mammalian cells. Inhibition of pore formation by these toxins is considered as an effective prevention method of bacterial infection. However, the structural and mechanistic determinants of pore formation are not completely understood. Pneumolysin (Ply), a member of CDC family, is one of the important virulence factors produced by many strains of S. pneumoniae. The sequence type 306 (ST306) of serotype-1 is highly pathogenic and globally disseminated strain. Ply from ST306 (Ply-306) is a non-haemolytic variant with six amino acids substitutions compared to the widely studied hemolytic Ply from TIGR4 strain (Ply-4). The electron microscopy studies suggested similar pore architectures formed by these two Ply variants which corroborated with the SDS-agarose experiment using liposomes implying that Ply-306 and Ply-4 both forms higher order oligomers. Calcein leakage assay revealed that Ply-306 is unable to permeabilise the calcein molecule from calcein encapsulated liposomes.Structural analysis of Ply-306 reveals that H150 is not engaged in cation-pi interactions with K268 residues from β5 of domain 3. While Ply-4 structure shows that Y150 is stabilized by a cation-pi interaction. The pore formation process involves the large domain movement and hence, the hydrophobic interactions around Y150 (Ply-4) favours proper domain movement over H150 . The second important substitution in Ply-306 is T172 to I172; which is found to be stabilized by hydrophobic interaction from other D3 residues. On contrary in case of Ply-4 the T172 being a polar residue is unstable in hydrophobic pocket and favourable for domain motion. Structurally, Y150 and T172 are found to be essential for pore formation. Mutations of these residues on Ply-306 to wild type amino acids such as H150Y and I172T must regain the haemolytic activity. Interestingly, the double mutant (H150Y and I172T) of ST306 shows approximately 80 % gain in haemolytic activity (Figure-1). These two mutations are found to control the haemolytic activity in Ply-306. Further spectroscopic experiments are in progress to identify the conformational changes (disengagement of D3 from D2 and β5 from β4) that are essential steps in pore formation.

Homeotic selector (HOX) transcription factors (TFs) regulate gene expression that determines the identity of Drosophila body segments along the anterior-posterior (A-P) axis. Besides a highly conserved DNA-binding homeodomain (HD), HOX proteins also contain functionally important, evolutionarily conserved small motifs, which may be Short Linear Motifs (SLiMs). SLiMs are proposed to be sites of phosphorylation and this may regulate the activity of HOX proteins. Sex combs reduced protein fused to a triple tag (SCRTT) was extracted from developing embryos that express SCRTT from a heat-shock promoter fusion gene and purified using Ni-NTA beads under denaturing conditions. Multiple sites of post-translational modification (PTM) were identified in purified SCRTT