HflX is a GTP binding protein of unknown function. Based on the presence of the hflX gene in hflA operon, HflX was believed to be involved in the lytic-lysogenic decision during phage infection in Escherichia coli. We find that E. coli HflX binds 16S and 23S rRNA – the RNA components of 30S and 50S ribosomal subunits. Here, using purified ribosomal subunits, we show that HflX specifically interacts with the 50S. This finding is in line with the homology of HflX to GTPases involved in ribosome biogenesis. However, HflX-50S interaction is not limited to a specific nucleotide-bound state of the protein, and the presence of any of the nucleotides GTP/GDP/ATP/ADP is sufficient. In this respect, HflX is different from other GTPases. While E. coli HflX binds and hydrolyses both ATP and GTP, only the GTP hydrolysis activity is stimulated by 50S binding. This work uncovers interesting attributes of HflX in ribosome binding.
The Escherichia coli gene hflX was first identified as part of the hflA operon, mutations in which led to an increased frequency of lysogenization upon infection of the bacterium by the temperate coliphage lambda. Independent mutational studies have also indicated that the HflX protein has a role in transposition. Based on the sequence of its gene, HflX is predicted to be a GTP-binding protein, very likely a GTPase. We report here purification and characterization of the HflX protein. We also specifically examined its suggested functional roles mentioned above. Our results show that HflX is a monomeric protein with a high (30% to 40%) content of helices. It exhibits GTPase as well as ATPase activities, but it has no role in lambda lysogeny or in transposition.
The gal operon of Escherichia coli is negatively regulated by repressor binding to bipartite operators separated by 11 helical turns of DNA. Synergistic binding of repressor to separate sites on DNA results in looping, with the intervening DNA as a topologically closed domain containing the two promoters. A closed DNA loop of 11 helical turns, which is in-flexible to torsional changes, disables the promoters either by resisting DNA unwinding needed for open complex formation or by impeding the processive DNA contacts by an RNA polymerase in flux during transcription initiation. Interaction between two proteins bound to different sites on DNA modulating the activity of the intervening segment toward other proteins by allostery may be a common mechanism of regulation in DNA-multiprotein complexes.Communication between proteins bound to spatially separated sites on DNA appears to be a ubiquitous mechanism governing regulation of cellular macromolecular processes, such as transcription, recombination, and DNA replication in both prokaryotes and eukaryotes (1-7). It involves a direct contact between homologous or heterologous proteins, giving rise to cooperative binding. In such mechanisms, DNA usually facilitates interaction between the DNA-bound proteins, providing a tether to increase the effective local concentration of the proteins. The DNA part of the looped complex has been viewed traditionally as a passive participant of regulation, merely aiding the formation of the correct DNA-multiprotein complexes.The gal operon of Escherichia coli contains two promoters, P1 and P2, separated by only 5 bp, and two operators, OE and O0, which are centered at -60.5 and +53.5 bp, respectively, from the transcription start site of P1 (refs. 8,and 9; Fig. 1). Gal repressor (GalR) negatively regulates transcription from P1 and P2 by repressing transcription from each promoter. For complete repression, GalR must bind to both OE and O,. A chimeric gal operon carrying a lac operator, which interacts with Lac repressor (Lacd), in the place of either one of the gal operators cannot be repressed when provided with both Gal and Lac repressors (10). However, when bothgal operators are replaced by lac operators, the operon can be repressed by LacI, suggesting that an interaction between the DNA-bound proteins may be essential in establishing repression and such interaction only happens between homologous repressors. In vitro transcription studies have shown that a complete repression of both gal promoters can be obtained only if the two operator-bound repressors associate, thereby forming a loop of the intervening DNA (11). Such association occurs with wild-type Lacd, which is a tetramer and capable of binding to two operators simultaneously (11-16). Since wild-type GalR is a dimer, it requires another factor for DNA looping and the associated repression of both promoters (refs. 11 and 17; unpublished data). Wild-type GalR without the looping factor and a mutant Lacd (Lacladi), which is defective in tetramerization but binds t...
The cyclic AMP receptor protein activates transcription in Escherichia coli, only when complexed with cyclic AMP. The cyclic AMP receptor protein-cyclic AMP complex formed at low concentrations of cyclic AMP has a different conformation from either cyclic AMP receptor protein alone or its complex with cyclic AMP formed at high cyclic AMP concentrations. Various biophysical data suggest that the latter complex resembles free cyclic AMP receptor protein. We have examined the conformational and biological properties of cyclic AMP receptor protein as a function of cyclic AMP concentrations, using the gal operon of E. coli. A biphasic behavior is observed. It is shown that only the complex formed at lower concentrations of cyclic AMP is the transcriptionally active form. This difference between the complexes at different levels of cyclic AMP arises from a decreased ability of the cyclic AMP receptor protein-cyclic AMP complex at high cyclic AMP concentrations to bind to DNA at specific sites.z 1999 Federation of European Biochemical Societies.
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