ABSTRACT
Era is a small G-protein widely conserved in eubacteria and eukaryotes. Although essential for bacterial growth and implicated in diverse cellular processes, its actual function remains unclear. Several lines of evidence suggest that Era may be involved in some aspect of RNA biology. The GTPase domain contains features in common with all G-proteins and is required for Era function in vivo. The C-terminal domain (EraCTD) bears scant similarity to proteins outside the Era subfamily. On the basis of sequence comparisons, we argue that the EraCTD is similar to, but distinct from, the KH RNA-binding domain. Although both contain the consensus VIGxxGxxI RNA-binding motif, the protein folds are probably different. We show that bacterial Era binds RNA in vitro and can form higher-order RNA-protein complexes. Mutations in the VIGxxGxxI motif and other conserved residues of the Escherichia coli EraCTD decrease RNA binding in vitro and have corresponding effects on Era function in vivo, including previously described effects on cell division and chromosome partitioning. Importantly, mutations in L-66, located in the predicted switch II region of the E. coli Era GTPase domain, also perturb binding, leading us to propose that the GTPase domain regulates RNA binding in response to unknown cellular cues. The possible biological significance of Era RNA binding is discussed.
Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Escherichia coli Proteins , GTP-Binding Proteins/genetics , GTP-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Amino Acid Motifs , Amino Acid Sequence , Base Sequence , Binding Sites/genetics , Cell Division/genetics , Chromosomes, Bacterial/genetics , Conserved Sequence , DNA Primers/genetics , Escherichia coli/cytology , Escherichia coli/genetics , Escherichia coli/metabolism , Molecular Sequence Data , Mutation , Protein Binding , RNA/metabolism , Ribonucleoproteins/metabolism , Sequence Homology, Amino AcidABSTRACT
Four polar metabolites were isolated from the urine of human subjects orally treated with tripelennamine, and their structures elucidated by various chemical and physical methods. One of the metabolites, which is a minor one, was identified as an N-oxide of tripelennamine, and the other three as glucuronide conjugates. One of the conjugates, which is a major metabolite, has been assigned a unique quaternary ammonium N-glucuronide structure, since it gave tripelennamine and D-glucuronic acid on incubation with beta-glucuronidase. The N-oxide, which has also been prepared synthetically, remained unchanged on similar treatment. The other two conjugates were O-glucuronides of hydroxylated derivatives, the glucuronide of hydroxytripelennamine being the principal metabolite. No desmethyltripelennamine was found in the urine, however. Hydroxylation in both cases had occurred in the pyridine ring.
