Comprehensive Functional Analysis of Escherichia coli Ribosomal RNA Methyltransferases

Abstract

Ribosomal RNAs in all organisms are methylated. The functional role of the majority of modified nucleotides is unknown. We systematically questioned the influence of rRNA methylation in Escherichia coli on a number of characteristics of bacterial cells with the help of a set of rRNA methyltransferase (MT) gene knockout strains from the Keio collection. Analysis of ribosomal subunits sedimentation profiles of the knockout strains revealed a surprisingly small number of rRNA MT that significantly affected ribosome assembly. Accumulation of the assembly intermediates was observed only for the rlmE knockout strain whose growth was retarded most significantly among other rRNA MT knockout strains. Accumulation of the 17S rRNA precursor was observed for rsmA (ksgA) knockout cells as well as for cells devoid of functional rsmB and rlmC genes. Significant differences were found among the WT and the majority of rRNA MT knockout strains in their ability to sustain exogenous protein overexpression. While the majority of the rRNA MT knockout strains supported suboptimal reporter gene expression, the strain devoid of the rsmF gene demonstrated a moderate increase in the yield of ectopic gene expression. Comparative 2D protein gel analysis of rRNA MT knockout strains revealed only minor perturbations of the proteome.

Cryo‐EM structure of the ribosome functional complex of the human pathogen Staphylococcus aureus at 3.2 Å resolution

“…In bacteria, almost all rRNA modifications are made by site‐specific enzymes [22]. Studies of methylation enzymes showed that deletion of a modification enzyme is rarely accompanied by a noticeable fitness loss for bacteria in non‐native environments [28,29]. However, several studies have also shown that loss of some particular rRNA modifications leads to diverse negative effects: severe growth retardation [30], slower rates and accuracy of translation [31], alterations of active sites [32], and a decrease in the fidelity of translation initiation [33].…”

Epitranscriptomics of Mammalian Mitochondrial Ribosomal RNA

“…This result leads to an idea that RsmA acts as a quality control switch releasing the subunit after completion of late assembly events [ 57 ]. In concert with this idea, rsmA deletion excessively retarded cell growth at 25 °C, when assembly defects are exacerbated [ 57 ], and caused accumulation of the small subunit rRNA precursor [ 57 , 58 ]. Apart from RsmA role in ribosome assembly, the methylation itself is also essential for the proper rRNA folding.…”

“…In bacteria equivalent nucleotide Gm2251 is modified by RlmB. However, despite a universal conservation and location in the functionally important region, lack of G2251 modification in E. coli due to a deletion of rlmB gene doesn’t result in any observable difference from wild type cells [ 73 ] except for the mild reduction of ectopic expression of foreign gene introduced on a plasmid [ 58 ].…”

“…In gram negative bacteria, nucleotide Um2552 is methylated by RlmE (FtsJ) [ 84 ], which is co-expressed with a number of heat shock genes. Inactivation of bacterial rlmE gene results in growth retardation [ 85 ], which is most severe among all knockouts of rRNA methyltransferases in E. coli [ 58 ]. Without RlmE, the amount of 70S ribosomes decreases, while the 45S precursor of the large ribosomal subunit accumulates.…”

“…Most likely RsmF is active at late stage of SSU assembly because in vitro it can methylate 30S subunit but not naked 16S rRNA [ 96 ]. Remarkably, inactivation of rsmF gene leads to an increased ectopic expression of exogenous gene introduced on a plasmid [ 58 ] suggesting a role for this protein in control of gene expression. While in E. coli RsmF methyltransferase modifies only a single 16S rRNA nucleotide, in T. thermophilus its homologue modifies three nucleotides, equivalent to the E. coli 16S rRNA residues C1400, C1404 and C1407.…”