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

Era, an essential GTPase, plays a regulatory role in several cellular proce sses. The Era protein of Streptococcus pneumoniae has recently been shown t o, 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 p art, responsible for the GTPase activity of the protein, was kept intact. T he resulting truncated Era proteins were purified and characterized. The C- terminal deletion of 9 or 19 amino-acid residues did not affect 16S rRNA-bi nding activity while further deletions of the C-terminus (29-114 amino-acid residues) abolished the activity. These results indicate; that the integri ty of the putative KH domain of Era, spanning the amino-acid residues betwe en approximate to 22-83 from the C-terminus, is required for 16S rRNA-bindi ng. Furthermore, the Era proteins with a deletion up to 45 residues from th e C-terminus retained membrane-binding activity, but longer deletions signi ficantly reduced the activity. These results indicate that part of the puta tive KH domain is also required for membrane-binding. Thus, these results i ndicate 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' terminus complemented an Escherishia coli mutan t strain deficient in Era production whereas the genes with longer deletion s failed to do so, thereby indicating that the KH domain is essential for E ra function. Taken together, the results of this study indicate that the pu tative 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.