Formation of SRP-like particle induces a conformational change in E. coli 4.5S RNA

E. coli P48 protein is homologous to the SRP54 component of the eukaryotic signal recognition particle. In vivo, P48 is associated with 4.5S RNA which shares a homology with eukaryotic SRP RNA. To study the interaction between P48 and 4.5S RNA in vitro, we used 4.5S RNA with fluorescein coupled to the 3'-terminal ribose. Upon binding of P48, the fluorescent 4.5S RNA shows a substantial decrease in fluorescence. Fluorescence quenching as well as anisotropy measurements reveal that the effect is not due to a direct interaction of P48 with the dye. This suggests that the binding of P48 induces a conformational change in 4.5S RNA which affects the structure at the 3' end of the RNA. From equilibrium titrations with fluorescent 4.5S RNA, a dissociation constant of 0.15 microns is obtained for the RNA.protein complex. The formation of the complex is not affected by GTP binding to or hydrolysis by P48

Colicins and their potential in cancer treatment

Colicins are a family of antibacterial cytotoxins produced by Escherichia coli and released into the environment to reduce competition from other bacterial strains. Colicins kill the target cell by a variety of effects that include depolarisation of the cytoplasmic membrane, a non-specific DNase activity, a highly specific RNase activity or by inhibition of murein synthesis. This review summarises some important findings that implicate colicins as potential anti-tumor agents. Colicins appear to inhibit proliferation of tumor cell lines in a colicin-type--and cell line-dependent fashion and are more toxic to tumor cells than to normal cells within the body. This opens a potential for using bacterial colicins in combating cancer and raises a number of questions concerning the mechanism of action of colicins in targeting tumor cells, their specificity and applicability as anti-tumor therapeutics

Arginines 29 and 59 of elongation factor G are important for GTP hydrolysis or translocation on the ribosome

GTP hydrolysis by elongation factor G (EF-G) is essential for the translocation step in protein elongation. The low intrinsic GTPase activity of EF-G is strongly stimulated by the ribosome. Here we show that a conserved arginine, R29, of Escherichia coli EF-G is crucial for GTP hydrolysis on the ribosome, but not for GTP binding or ribosome interaction, suggesting that it may be directly involved in catalysis. Another conserved arginine, R59, which is homologous to the catalytic arginine of G(α) proteins, is not essential for GTP hydrolysis, but influences ribosome binding and translocation. These results indicate that EF-G is similar to other GTPases in that an arginine residue is required for GTP hydrolysis, although the structural changes leading to GTPase activation are different

Distinct functions of elongation factor G in ribosome recycling and translocation

Elongation factor G (EF-G) promotes the translocation step in bacterial protein synthesis and, together with ribosome recycling factor (RRF), the disassembly of the post-termination ribosome. Unlike translocation, ribosome disassembly strictly requires GTP hydrolysis by EF-G. Here we report that ribosome disassembly is strongly inhibited by vanadate, an analog of inorganic phosphate (Pi), indicating that Pi release is required for ribosome disassembly. In contrast, the function of EF-G in single-round translocation is not affected by vanadate, while the turnover reaction is strongly inhibited. We also show that the antibiotic fusidic acid blocks ribosome disassembly by EF-G/RRF at a 1000-fold lower concentration than required for the inhibition of EF-G turnover in vitro and close to the effective inhibitory concentration in vivo, suggesting that the antimicrobial activity of fusidic acid is primarily due to the direct inhibition of ribosome recycling. Our results indicate that conformational coupling between EF-G and the ribosome is principally different in translocation and ribosome disassembly. Pi release is not required for the mechanochemical function of EF-G in translocation, whereas the interactions between RRF and EF-G introduce tight coupling between the conformational change of EF-G induced by Pi release and ribosome disassembly