Recombinant yeast and human cells as screening tools to search for antibacterial agents targeting the transcription termination factor Rho.

The alarming issue of antibiotic resistance expansion requires a continuous search for new and efficient antibacterial agents. Here we describe the design of new tools to screen for target-specific inhibitors of the bacterial Rho factor directly inside eukaryotic cells. Rho factor is a global regulator of gene expression which is essential to most bacteria, especially Gram-negative. Since Rho has no functional or structural homolog in eukaryotes, it constitutes a valuable and well known bacterial target as evidenced by its inhibition by the natural antibiotic, Bicyclomycin. Our screening tools are based on perturbation of mRNA processing and packaging reactions in the nucleus of eukaryotic cells by the RNA-dependent helicase/translocase activity of bacterial Rho factor leading to a growth defect phenotype. In this approach, any compound that impedes Rho activity should restore growth to yeast or human cells expressing Rho protein, providing valuable means to screen for target-specific antibacterial agents within the environment of a eukaryotic cell. The yeast tool expressing E. coli Rho factor was validated using Bicyclomycin as the control antibacterial agent. The validation of the screening tool was further extended with a stable human cell line expressing Rho factor conditionally. Finally, we show that Rho factors from different bacterial pathogens can also be designed as yeast-based screening tools which can reveal subtle variations in the functional features of the proteins.

Structure of the intrastrand cis-[Pt(NH3)2(d(GpCpG))] adduct in a dodecanucleotide duplex: I. A 1H and 31P n.m.r. study.

The structure of an intrastrand cis-[Pt(NH3)2(d(GpCpG))] adduct in a dodecanucleotide duplex has been investigated by using ultraviolet absorption, circular dichroism, 1H and 31P n.m.r. The binding of cis-DDP does not inhibit the formation of a duplex but it induces a lowering of congruent to 26 degrees C of its melting temperature. A broadening of the 1H spectrum prevents an accurate analysis of the platination site. Nevertheless, by considering its thermal behavior and the number of imino protons a model of structure of the platinated duplex is proposed in which the central C.G. pair is disrupted and a neighboring C.G pair is very accessible or distorted. The environment of two phosphate groups is disturbed by the cis-DDP binding.