Ribosomal RNA modification enzymes stimulate large ribosome subunit assembly in E. coli

Abstract Ribosomal RNA modifications are introduced by specific enzymes during ribosome assembly in bacteria. Deletion of individual modification enzymes has a minor effect on bacterial growth, ribosome biogenesis, and translation, which has complicated the definition of the function of the enzymes and their products. We have constructed an Escherichia coli strain lacking 10 genes encoding enzymes that modify 23S rRNA around the peptidyl-transferase center. This strain exhibits severely compromised growth and ribosome assembly, especially at lower temperatures. Re-introduction of the individual modification enzymes allows for the definition of their functions. The results demonstrate that in addition to previously known RlmE, also RlmB, RlmKL, RlmN and RluC facilitate large ribosome subunit assembly. RlmB and RlmKL have functions in ribosome assembly independent of their modification activities. While the assembly stage specificity of rRNA modification enzymes is well established, this study demonstrates that there is a mutual interdependence between the rRNA modification process and large ribosome subunit assembly.

50S subunits were purified from mature 70S ribosomes, rRNAs (23S and 5S) were extracted, digested with nuclease P1, and treated with alkaline phosphatase.Nucleosides were separated by high-performance liquid chromatography (HPLC) A was monitored and nucleosides were iden fied according to their retension mes 260 (see Supplementary Methods).Representa ve comparisons with WT are shown for Δ10.Modifica on peak areas were compared with WT controls normalized to 1. Quan fica ons of HPLC peak areas are included in Fig. 1B.E. coli WT (MG1655), Δ10, as well as Δ10 strain expressing plasmid-borne modifica on enzymes RlmM, RluB, RluE, and RluF were grown at 37°C.Cells were lysed and ribosome profiles were analyzed by sucrose gradient ultracentrifuga on.Peak areas corresponding to 70S ribosomes and free 50S subunits were quan fied, and 50S to 70S ra os are shown.At least two biological replicates with two technical replicates were used for calcula ons with standard devia ons shown.

Figure S1 .
Figure S1.Nucleoside composi on of 23S rRNA of WT and Δ0 strain.

Figure S3 .
Figure S3.50S subunit nucleoside composi on of Δ10 strain expressing na ve or mutant RlmB, RlmK, RlmL, RlmM, RlmN, or RluC.50S subunits were purified from mature 70S ribosomes, rRNAs (23S and 5S) were extracted and nucleosides were analyzed as specified above by high-performance liquid chromatography (HPLC).Nucleoside peaks are shown on the le side.Corresponding peak areas of add-back strains rela ve to the WT 23S rRNA control are shown as blue columns.Rela ve nucleoside content of nonfunc onal ME expression strains are as grey columns.Nucleoside corresponding to the respec ve enzyme is marked by red asterisk.

Figure S4 .Figure S5 .
Figure S4.Ribosome profiles of WT and Δ9 strains in the late-log growth phase.E. coli WT (panel A) and Δ9 (panel B) were grown into late-log phase (OD 1.4 -1.5) at 37°C, 30°C, and 25°C.Cells 580 were lysed and ribosome par cles analyzed by sucrose gradient centrifuga on.

Figure
Figure S6.R-protein composi on of free 50S subunits.Free 50S subunits from 1:1 MG1655, Δ9, Δ10, or Δ10 strains expressing RlmE (Δ10+RlmE) were mixed in a ra o with "medium" labeled reference 70S ribosomes (from E. coli MG1655-SILAC strain grown in MOPS medium supplemented with "medium"-labeled arginine and lysine) for r-protein quan fica on using LC-MS/MS.Proteins bL31A, bL31B, bL36A, and bL36B were not quan fied due to insufficient number of unique pep des.The rela ve quan es of 31 r-proteins are presented as the L/M ra o (L = sample; M = reference).L/M ra os are normalized against the average L/M ra o of all 50S r-proteins.The blue box marks the ±10% range of the L/M ra o.The values shown in the figure are the means of two independent biological experiments with a standard devia on.

Figure S7 .Figure S8 .
Figure S7.Effect of na ve RlmKL and individual K and L domains on the ribosome biogenesis phenotype of the Δ10 stain.E. coli WT (MG1655), Δ10, as well as Δ10 strains expressing plasmid-borne na ve RlmKL, individual RlmK, RlmL or mutant RlmL (N309A) were grown into late log phase (OD 1.4 -1.6) at 37°C and were analyzed as 600 described in the legend of Figure 5. Representa ve ribosome profiles of at least three independent samples are shown in panel A. Peak areas corresponding to 70S ribosomes and free 50S subunits were quan fied, and 50S to 70S ra os are shown in panel B. 5´ends of rRNA from the 70S and 50S frac ons were mapped by primer extension analysis.(panel C). +3 and +7 refer to extra nucleo des at the 23S rRNA 5` end.