Role of Escherichia coli YbeY, a highly conserved protein, in rRNA processing

Abstract

The UPF0054 protein family is highly conserved with homologues present in nearly every sequenced bacterium. In some bacteria, the respective gene is essential, while in others its loss results in a highly pleiotropic phenotype. Despite detailed structural studies, a cellular role for this protein family has remained unknown. We report here that deletion of the Escherichia coli homologue, YbeY, causes striking defects that affect ribosome activity, translational fidelity and ribosome assembly. Mapping of 16S, 23S and 5S rRNA termini reveals that YbeY influences the maturation of all three rRNAs, with a particularly strong effect on maturation at both the 5'- and 3'-ends of 16S rRNA as well as maturation of the 5'-termini of 23S and 5S rRNAs. Furthermore, we demonstrate strong genetic interactions between ybeY and rnc (encoding RNase III), ybeY and rnr (encoding RNase R), and ybeY and pnp (encoding PNPase), further suggesting a role for YbeY in rRNA maturation. Mutation of highly conserved amino acids in YbeY, allowed the identification of two residues (H114, R59) that were found to have a significant effect in vivo. We discuss the implications of these findings for rRNA maturation and ribosome assembly in bacteria.

Figure 1

Steps in the processing of rRNA in E. coli. All enzymes indicated are endonucleases with the exception of RNase T that generates mature 3' termini by trimming the rRNA precursors.

Figure 2

Phenotypic analysis of the E. coli ΔybeY mutant. (A) Growth curves of MC4100 (WT) and the ΔybeY mutant complemented strains in LB at 37 °C. Doubling times: 40 ± 2 min (ΔybeY mutant) vs. 28 ± 3 min (MC4100). Sensitivity of the ΔybeY mutant to stresses (B) cefotaxim and (C) temperature. The ΔybeY mutant with empty vector only (ΔybeY+vector) is shown on each plot for clarity. UPF0054 homologs: ybeY (E. coli), yqfG (B. subtilis) and SMc01113 (S. meliloti). WT+vector (□), ΔybeY+vector (●), ΔybeY+pybeY (▲), ΔybeY+pyqfG (▼) and ΔybeY+pSMc01113 (◀). “p” indicates that the gene indicated is expressed from a plasmid. In panel (C), the pairs of samples show a ten-fold dilution each. (D) Polysome profiles for MC4100 and the ΔybeY mutant. The positions of polysomes, 70S ribosomes and 50S and 30S ribosomal subunits are indicated. (E) In vitro translation assay under saturating substrate conditions. MC4100 S100 fractions were mixed with a polyU template and equal amounts of MC4100 or ΔybeY mutant 70S ribosomes in an in vitro translation reaction as described in the Materials and Methods section. Translational activity is normalized to MC4100 70S ribosome reactions. MC4100 and the ΔybeY mutant were transformed with plasmids expressing lacZ containing (F) frameshift mutations (+1 or −1) or (G) nonsense codons. LacZ activity was assayed as described in the Materials and Methods section. The 33 percent LacZ activity (in Miller units) of each mutant lacZ allele relative to the wild type lacZ allele for MC4100 or the ΔybeY mutant is reported. The value of LacZ activity from the wild type allele (18921 Miller units in the ΔybeY mutant and 12247 Miller units in MC4100; respectively) was set to as 100% activity. For clarity, wild type lacZ activity has been omitted from the plots. Each assay was performed in triplicate. (H) Immunoblots identifying IF2 and IF3 in whole cell lysates, 30S, 50S and 70S ribosome fractions from MC4100 and the ΔybeY mutant. Immunoblotting for OmpA is used as a loading control. Equal A₂₆₀ amounts were loaded for the 30S, 50S and 70S ribosome fractions. The experiment was repeated 3 times and a representative result is shown.

Figure 3

Analysis of rRNA from E. coli MC4100 and the ΔybeY mutant. (A) Total RNA isolated from whole cells, 30S ribosomal subunits and 70S ribosomes from MC4100 and the ΔybeY mutant. The positions of 23S, 17S, 16S and 16S* rRNAs are indicated based on their mobility. (B) Northern blot analysis using probes directed against the 5′- and 3′-termini of 17S rRNA. Equal amounts of total RNA from MC4100 and the ΔybeY mutant were used. The locations of the probes are shown in the diagram below the blots. (C, D) Primer extension and site-specific RNase H cleavage assays to map the 5′- and 3′-termini of 16S, 23S and 5S rRNAs from MC4100 and the ΔybeY mutant. “P” and “M” indicate the location of bands corresponding to precursor and mature forms of the rRNA, respectively. Annotation of the positions of the 5′ and 3′-termini of 16S, 23S and 5S rRNAs, mature and precursor species, were based on previous observations (Li et al., 1999b). Total RNA was prepared from MC4100 and the ΔybeY mutant strains as described in the Methods section. 34

Figure 4

Analysis of rRNA from the ΔybeY mutant and seven well characterized E. coli RNase mutant strains. The relevant genotype from which the rRNA was extracted is indicated under each lane. The parental strain MC4100 rRNA is shown in each case as a control. (A–B) Agarose gel electrophoresis of total rRNA from single and double RNase mutant strains. The positions of 23S, 18S, 17S, 16S and 16S* rRNAs are indicated. (CH) Primer extension and site-specific RNase H cleavage assays to map the 5′- and 3′-termini of 16S, 23S and 5S rRNAs from single and double RNase mutant strains. “P” and “M” indicate the position of bands corresponding to the precursor and mature form for each rRNA. Annotation of the positions of the 5′ and 3′-termini of 16S, 23S and 5S rRNAs, mature and precursor species, were based on previous observations (Li et al., 1999b).

Figure 5

Growth of MC4100, ΔybeY mutant and several ΔybeY double mutants in rich medium at 37 °C. Most ΔybeY double mutants did not show a growth defect (data not shown); the ΔybeY Δpnp double mutant is shown as an example. In contrast, the ΔybeY Δrnc and ΔybeY Δrnr mutants showed a significant decrease in growth rate.

Figure 6

(A) Sequence alignment of UPF0054 homologs from bacteria and eukaryotes. Alignments were performed using T-coffee (Poirot et al., 2003). The red bar underlines the conserved H3XH5XH motif that is used to classify members of this family. Red asterisks indicate amino acid residues that were analyzed in this study by Ala 35 mutagenesis. (B) Sensitivity of the ΔybeY mutant strain expressing mutant ybeY alleles to high temperature. Strains were serially diluted (1:10), plated on LB plates and incubated at 45 °C. (C) rRNA profiles of the ΔybeY mutant expressing the mutant ybeY alleles H114A and R59A. The positions of 23S, 17S, 16S and 16S* rRNAs are indicated.