Analysis of the interaction of 16S rRNA and cytoplasmic membrane with the C-terminal part of the Streptococcus pneumoniae Era GTPase

Abstract

Era, an essential GTPase, plays a regulatory role in several cellular processes. The Era protein of Streptococcus pneumoniae has recently been shown to bind to 16S rRNA and the cytoplasmic membrane. However, exact locations of Era responsible for RNA-and membrane-binding were unknown. To identify the regions in Era that interact with the RNA and membrane, the C-terminal part of S. pneumoniae Era was systematically deleted while the N-terminal part, responsible for the GTPase activity of the protein, was kept intact. The resulting truncated Era proteins were purified and characterized. The C-terminal deletion of 9 or 19 amino-acid residues did not affect 16S rRNA-binding activity while further deletions of the C-terminus (29-114 amino-acid residues) abolished the activity. These results indicate that the integrity of the putative KH domain of Era, spanning the amino-acid residues between < 22-83 from the C-terminus, is required for 16S rRNA-binding. Furthermore, the Era proteins with a deletion up to 45 residues from the C-terminus retained membrane-binding activity, but longer deletions significantly reduced the activity. These results indicate that part of the putative KH domain is also required for membrane-binding. Thus, these results indicate for the first time that the regions critical for the membrane-and 16S rRNA-binding activities of Era overlap. The era gene with a deletion of 9 or 19 codons from its 3 0 terminus complemented an Escherishia coli mutant strain deficient in Era production whereas the genes with longer deletions failed to do so, thereby indicating that the KH domain is essential for Era function. Taken together, the results of this study indicate that the putative KH domain is required for 16S rRNA-binding activity and that part of the KH domain is also required for membrane-binding activity. The results also suggest that the interaction between Era and 16S rRNA is essential for bacterial growth.

MazG, a Nucleoside Triphosphate Pyrophosphohydrolase, Interacts with Era, an Essential GTPase inEscherichia coli

“…It has been suggested that Era might play a role in the control of the cell cycle (8,9,12). It has also been shown that Era interacts with 16S rRNA and the 30S ribosomal subunit (13,24,29). Furthermore, 16S rRNA dimethyltransferase has been demonstrated to suppress a cold-sensitive mutant of Era E200K when expressed in a multicopy plasmid (19).…”

“…Era consists of an N-terminal GTP-binding domain and a conserved C-terminal domain that contains a putative KH domain (10) capable of binding RNA (13,17,24). The cellular function of Era still remains elusive.…”

Elimination of the vertebrate Escherichia coli Ras-like protein homologue leads to cell cycle arrest at G1 phase and apoptosis

“…Indeed, RNA-binding activity of bacterial ERA has previously been reported (Meier et al, 1999(Meier et al, , 2000Sayed et al, 1999;Hang et al, 2001). We have also demonstrated that mammalian ERA has RNA-binding activity, which is contributed by the KH-like domain (Akiyama et al, 2001).…”

“…Possible requirement of RNA-binding activity for the regulation of cell cycle by vertebrate ERA It has been reported that eukaryotic ERA, as well as prokaryotic ERA, is an RNA-binding protein (Meier et al, 1999;Sayed et al, 1999;Akiyama et al, 2001;Hang et al, 2001). As shown in Figure 4d, the phenotype of the ERA-null cells was not reversed by the expression of the Hs1-375 mutant, which possesses no RNA-binding activity.…”

The C‐terminal α‐helix of YsxC is essential for its binding to 50S ribosome and rRNAs

“…The importance of the KH domain has been demonstrated for Era, since truncated Era genes were unable to complement an E. coli mutant strain defective in Era expression [29]. Likewise, the KH domain of EngA has been shown to be involved in the binding of EngA to the 30S ribosomal subunit [30].…”