The bacterial RNA polymerase (RNAP) is a multi-subunit protein complex (α2ββ’ω σ) containing the smallest subunit, ω. Although identified early in RNAP research, its function remained ambiguous and shrouded with controversy for a considerable period. It was shown before that the protein has a structural role in maintaining the conformation of the largest subunit, β’, and its recruitment in the enzyme assembly. Despite evolutionary conservation of ω and its role in the assembly of RNAP, E. coli mutants lacking rpoZ (codes for ω) are viable due to the association of the global chaperone protein GroEL with RNAP. To get a better insight into the structure and functional role of ω during transcription, several dominant lethal mutants of ω were isolated. The mutants showed higher binding affinity compared to that of native ω to the α2ββ’ subassembly. We observed that the interaction between α2ββ’ and these lethal mutants is driven by mostly favorable enthalpy and a small but unfavorable negative entropy term. However, during the isolation of these mutants we isolated a silent mutant serendipitously, which showed a lethal phenotype. Silent mutant of a given protein is defined as a protein having the same sequence of amino acids as that of wild type but having mutation in the gene with alteration in base sequence from more frequent code to less frequent one due to codon degeneracy. Eventually, many silent mutants were generated to understand the role of rare codons at various positions in rpoZ. We observed that the dominant lethal mutants of ω having either point mutation or silent in nature are more structured in comparison to the native ω. However, the silent code’s position in the reading frame of rpoZ plays a role in the structural alteration of the translated protein. This structural alteration in ω makes it more rigid, which affects the plasticity of the interacting domain formed by ω and α2ββ’. Here, we attempted to describe how the conformational flexibility of the ω helps in maintaining the plasticity of the active site of RNA polymerase. The dominant lethal mutant of ω has a suppressor mapped near the catalytic center of the β’ subunit, and it is the same for both types of mutants.

The bacterial RNA polymerase is a multi-subunit enzyme complex composed of six subunits, α2ββ’σω. The function of this enzyme is to transcribe the DNA base sequence to the RNA intermediate, which is ultimately translated to protein. Though the contribution of each subunit in RNA synthesis has been clearly elucidated, the role of the smallest ω-subunit is still unclear despite several studies. Recently, a study on a dominant negative mutant of rpoZ has been reported in which the mutant was shown to render the RNA polymerase defective in transcription initiation (ω6, N60D) and gave an insight on the function of ω in RNA polymerase. Serendipitously, we also obtained a silent mutant, and the mutant was found to be lethal during the isolation of toxic mutants. The primary focus of this study is to understand the mechanistic details of this lethality. Isolated ω shows a predominantly unstructured circular dichroism profile and becomes α-helical in the enzyme complex. This structural transition is perhaps the reason for this lack of function. Subsequently, we generated several silent mutants of ω to investigate the role of codon bias and the effect of rare codons with respect to their position in rpoZ. Not all silent mutations affect the structure. RNA polymerase when reconstituted with structurally altered silent mutants of ω is transcriptionally inactive. The CodonPlus strain, which has surplus tRNA, was used to assess for the rescue of the phenotype in lethal silent mutants.

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Phosphatidate phosphatases (PAH) play a central role in lipid metabolism and intracellular signaling. Herein, we report the presence of a low-molecular-weight PAH homolog in the single-celled ciliate . phosphatase assay showed that TtPAH2 belongs to the magnesium-dependent phosphatidate phosphatase (PAP1) family. Loss of function of did not affect the growth of. Unlike other known homologs, did not regulate lipid droplet number and ER morphology. did not rescue growth and ER/nuclear membrane defects of theΔ yeast cells, suggesting that the phosphatidate phosphatase activity of the protein is not sufficient to perform these cellular functions. Surprisingly, complemented the respiratory defect in theΔ yeast cells indicating a specific role of in respiration. Overall, our results indicate that TtPAH2 possesses the minimal function of PAH protein family in respiration. We suggest that the amino acid sequences absent from TtPAH2 but present in all other known PAH homologs are critical for lipid homeostasis and membrane biogenesis.

Spread of multidrug resistant infections is a threat to human race and need for new drug development is great. Bark ethanol extract of Cassia fistula inhibited MDR bacteria isolated from Ganga River water, human and animal. On TLC, a gray colour major band ran fast (CU1; 6.6% of bark and 30% of crude extract) which quickly purified on HPLC C18 column at 3 min. Chemical assays suggested a triterpene linked to polyphenol known as saponin. CHN Elements analysis (35.9% C; 5.5% H) did not identified nitrogen suggesting a polyphenol or glycoside. VU and Vis spectra gave high peak at below 200nm with a secondary peak at 275nm with minor hinge at 578nm indicating a fused ring with bromopolyphenol. CU1 Mass (897 Daltons) with fragments of 515, 325, 269, 180 daltons and six halogen substitutions reflected by 82 molecular mass of DBr deviate six larger fragments. FTIR suggested broad band at 3500 to 3000 cm-1 for OH group where as two strong peaks at 1552cm-1 for aromatic C=C and 1408cm-1 for phenol. Proton NMR confirmed polymeric phenol at delta = 4.86 to 4.91 ppm and tetratet at delta = 3.57 to 3.618 ppm with phenolic bromosubstituents. Carbon NMR identified a strong peak at delta = 23.7ppm for many CBr and at 165ppm for a polybenzoid compound. CU1 inhibited the RNA Polymerase of E. coli and M. tuberculosis at low concentration but not DNA polymerase. Gel shift assays demonstrated that CU1 drug interacted with enzyme and inhibited its binding to open promoter complex. Thus, CU1 phytochemical is an alternative safer and low cost drug against MDR TB as well as other MDR pathogens.