Subribosomal particle analysis reveals the stages of bacterial ribosome assembly at which rRNA nucleotides are modified

Abstract

Modified nucleosides of ribosomal RNA are synthesized during ribosome assembly. In bacteria, each modification is made by a specialized enzyme. In vitro studies have shown that some enzymes need the presence of ribosomal proteins while other enzymes can modify only protein-free rRNA. We have analyzed the addition of modified nucleosides to rRNA during ribosome assembly. Accumulation of incompletely assembled ribosomal particles (25S, 35S, and 45S) was induced by chloramphenicol or erythromycin in an exponentially growing Escherichia coli culture. Incompletely assembled ribosomal particles were isolated from drug-treated and free 30S and 50S subunits and mature 70S ribosomes from untreated cells. Nucleosides of 16S and 23S rRNA were prepared and analyzed by reverse-phase, high-performance liquid chromatography (HPLC). Pseudouridines were identified by the chemical modification/primer extension method. Based on the results, the rRNA modifications were divided into three major groups: early, intermediate, and late assembly specific modifications. Seven out of 11 modified nucleosides of 16S rRNA were late assembly specific. In contrast, 16 out of 25 modified nucleosides of 23S rRNA were made during early steps of ribosome assembly. Free subunits of exponentially growing bacteria contain undermodified rRNA, indicating that a specific set of modifications is synthesized during very late steps of ribosome subunit assembly.

FIGURE 1.

Isolation of ribosomal particles from E. coli grown in the presence of chloramphenicol or erythromycin or in the absence of a drug. Exponentially growing bacterial cells without the drug (A), or treated with chloramphenicol (B), or erythromycin (C) were lysed and centrifuged in 10%–25% sucrose. 45S, 35S, and 25S fractions of chloramphenicol and erythromycin treated cells were combined (only chloramphenicol particles are indicated by gray zones in B). Ribosomal particles were purified by a second sucrose gradient centrifugation (D–F). Indicated fractions were collected.

FIGURE 2.

Purification of ribosomal RNA by sucrose gradient centrifugation. Ribosomal RNA from 70S ribosomes (A) and 35S particles (B) was deproteinized by phenol extraction and centrifuged in 5%–20% (w/w) sucrose gradient (buffer 20 mM Na-acetate, 100 mM NaCl, 1 mM EDTA) at 25,700 rpm in an SW28 rotor (Beckman) for 16 h. 16S and 23S rRNA were collected as indicated by the gray zones.

FIGURE 3.

HPLC analysis of 16S rRNA. 16S rRNA was prepared from mature 70S ribosomes, free 30S subunits, and Cam 25S particles. Nucleoside composition was determined by RP-HPLC on a Supelcosil LC-18-S. Peaks corresponding to three standard nucleosides (U, G, and A) and m⁵C, m⁷G, m³U, m⁴Cm, m²G, and m²₂A are indicated. X corresponds to an unknown compound.

FIGURE 4.

HPLC analysis of 23S rRNA. 23S rRNA was isolated from mature 70S ribosomes, free 50S subunits, Cam 45S, and Cam 35S particles. Peaks corresponding to four standard nucleosides (C, U, G, and A) and m⁵C, Cm, m⁷G, m⁵U, Um, Gm, m¹G, m²G, m²A, and m⁶A are indicated. Peak corresponding to the m⁵C contains also m³Ψ. X corresponds to an unknown compound.

FIGURE 5.

Primer extension analysis of the pseudouridines in helix–loop 69 of 23S rRNA. 23S rRNA was isolated from mature 70S ribosomes, free 50S subunits, and Cam 45S, Ery 45S, Cam 35S, and Ery 35S particles and analyzed for pseudouridines by CMCT/alkali and reverse transcriptase directed primer extension. +, CMCT/alkali treatment; −, untreated RNA. Sequence of the 23S rRNA around helix–loop 69 is shown in sequencing lanes (A, C, G, T). Note that the CMCT induced stop site is one nucleotide below the actual site.

FIGURE 6.

Summary of the specificity of the rRNA modification enzymes with respect to ribosome subunit assembly. Ribosome assembly is divided into three stages (early, intermediate, and late), which are shown by white and gray zones. Activity of rRNA modification enzymes is shown by black bars. (A) Modification of 16S rRNA. (B) Modification of 23S rRNA.