YbxF, a Protein Associated with Exponential-Phase Ribosomes inBacillus subtilis

Abstract

The ybxF gene is a member of the streptomycin operon in a wide range of gram-positive bacteria. In Bacillus subtilis, it codes for a small basic protein (82 amino acids, pI 9.51) of unknown function. We demonstrate that, in B. subtilis, YbxF localizes to the ribosome, primarily to the 50S subunit, with dependence on growth phase. Based on three-dimensional structures of YbxF generated by homology modeling, we identified helix 2 as important for the interaction with the ribosome. Subsequent mutational analysis of helix 2 revealed Lys24 as crucial for the interaction. Neither the B. subtilis ybxF gene nor its paralogue, the ymxC gene, is essential, as shown by probing ⌬ybxF, ⌬ymxC, or ⌬ybxF ⌬ymxC double deletion strains in several functional assays.The streptomycin (str) operon belongs to the most conserved operons in prokaryotic evolution (14, 16). The str operon of gram-negative Escherichia coli, from which most of our knowledge about this operon is derived, is composed of four genes: rpsL, coding for ribosomal protein S12; rpsG, coding for ribosomal protein S7; fus, coding for elongation factor G; and tufA, coding for elongation factor Tu. These proteins are essential components of the protein synthesis machinery of the cell.In contrast to E. coli, we found out in our previous studies that the str operon of two gram-positive organisms, Bacillus stearothermophilus and Bacillus subtilis, is a transcriptional unit composed of five genes (Fig.

YbxF and YlxQ are bacterial homologs of L7Ae and bind K-turns but not K-loops

“…Such regulation of RNA binding by protein-protein interaction has been previously reported, for instance, for the human spliceosomal U2B 00 protein, which only binds its cognate RNA in the presence of U2A9 (Price et al 1998). The genomic location of YlxQ between transcription factor NusA and translational initiation factor 2 (IF2) is consistent with an involvement in transcriptional or translational regulation, in line with proposed role in translation for YbxF (Sojka et al 2007). Curiously, in the E. coli genome, the nusA/infB operon is similarly organized as in B. subtilis but the ylxRylxQ genes are absent.…”

“…This may hint of a YlxQ function that is specific to B. subtilis such as sporulation. Neither YbxF nor YlxQ were found to be essential for B. subtilis under nonsporulating conditions (Sojka et al 2007), but the nusA/infB operon and IF2 are known to be involved with the onset of sporulation (Milon et al 2006). At present, then, the authentic biological targets of YbxF and YlxQ remain unknown, but our work suggests that these will likely be RNAs containing K-turns.…”

“…1B). Sojka et al (2007) demonstrated that neither YbxF nor YlxQ is essential for B. subtilis growth in rich media, and that a YbxF-GFP fusion localizes to ribosomes (primarily the large subunits) in vivo. Although the latter result hints that YbxF might be an rRNA-binding protein, in the absence of structural and biochemical characterization, it remains unknown whether YbxF and YlxQ are authentic bacterial homologs of L7Ae, and what their RNA-binding properties might be.…”

“…Although these experiments suggest that K-turn-binding proteins might modulate the activity of generegulatory RNAs such as riboswitches and T-boxes, the heterologous interaction between the archaeal L7Ae protein and bacterial RNAs is not biologically meaningful. Sequencebased searches indicate the presence of at least two likely L7Ae homologs in Gram-positive bacteria (Koonin et al 1994;Winkler 2002;Sojka et al 2007), YbxF and YlxQ (also called YmxC). Alignment of the sequences of these bacterial proteins with their putative archaeal and eukaryal homologs (Fig.…”

The molecular recognition of kink-turn structure by the L7Ae class of proteins

“…YbxF is a bacterial homolog of L7Ae protein (Sojka et al 2007). Interestingly, this protein binds k-turns but not kloops.…”