Era and RbfA Have Overlapping Function in Ribosome Biogenesis in Escherichia coli

Abstract

A cold-shock protein, RbfA (ribosome-binding factor A), is essential for cell growth at low temperature. In an rbfA-deletion strain, 30S and 50S ribosomal subunits increase relative to 70S monosomes with concomitant accumulation of a precursor 16S rRNA (17S rRNA). Recently, we have reported that overexpression of Era, an essential GTP-binding protein, suppresses not only the cold-sensitive cell growth but also defective ribosome biogenesis in the rbfA-deletion strain. Here, in order to elucidate how RbfA and Era functionally overlap, we characterized a cold-sensitive Era mutant (a point mutation at the Glu-200 to Lys; E200K) which shows a similar phenotype as the rbfA-deletion strain; accumulation of free ribosome subunits and 17S rRNA. To examine the effect of E200K in the rbfA-deletion strain, we constructed an E200K-inducible expression system. Interestingly, unlike wild-type Era, overexpression of Era(E200K) protein in the rbfA-deletion strain severely inhibited cell growth even at permissive temperature with further concomitant reduction of 16S rRNA. Purified Era(E200K) protein binds to 30S ribosomal subunits in a nucleotide-dependent manner like wild-type Era and retains both GTPase and autophosphorylation activities. Furthermore, we isolated spontaneous revertants of the E200K mutant. These revertants partially suppressed the accumulation of 17S rRNA. All the spontaneous mutations were found to result in higher Era(E200K) expression. These results suggest that the Era(E200K) protein has an impaired function in ribosome biogenesis without losing its ribosome binding activity. The severe growth defect caused by E200K in the rbfA-deletion strain may be due to competition between intrinsic wild-type Era and overexpressed Era(E200K) for binding to 30S ribosomal subunits. We propose that Era and RbfA have an overlapping function that is essential for ribosome biogenesis, and that RbfA becomes dispensable only at high temperatures because Era can complement its function only at higher temperatures.