Assembly of the Bacterial Ribosome with Circularly Permuted rRNA

Abstract

Co-transcriptional assembly is an integral feature of the formation of RNA-protein complexes that mediate translation. For ribosome synthesis, prior studies have indicated that the strict order of transcription of rRNA domains may not be obligatory during bacterial ribosome biogenesis, since a series of circularly permuted rRNAs are viable. In this work, we report the insights into assembly of the bacterial ribosome large subunit (LSU) based on cryo-EM density maps of intermediates that accumulate during in vitro ribosome synthesis using a set of circularly permuted (CiPer) rRNAs. The observed ensemble of twenty-three resolved ribosome large subunit intermediates reveals conserved assembly routes with an underlying hierarchy among cooperative assembly blocks. There are intricate interdependencies for the formation of key structural rRNA helices revealed from the circular permutation of rRNA. While the order of domain synthesis is not obligatory, the order of domain association does appear to proceed with a particular order, likely due to the strong evolutionary pressure on efficient ribosome synthesis. This work reinforces the robustness of the known assembly hierarchy of the bacterial large ribosomal subunit, and offers a coherent view of how efficient assembly of CiPer rRNAs can be understood in that context.

Figure 1.. Circularly permuted rRNAs succeed in biogenesis in iSAT.

(A) Schematics of the wild type and rearranged rRNA operons: CiPer45, CiPer63, CiPer63(+PS), and CiPer78. (B) The iSAT curve of circular permutated rRNA, reported by fluorescence signal of newly translated GFP. The reaction materials for the cryo-EM analysis were collected at the time point indicated by the arrow. (C) Agarose gel electrophoresis for the transcripts extracted from the iSAT reaction at the early time point (30 min) and late time point (120 min), respectively. The RNA product was purified by Direct-zol RNA Miniprep Kits of Zymo Research.

Figure 2.. Density maps for circularly permuted LSU intermediates.

Twenty-three 50S intermediates were obtained by heterogeneous subclassification from the iSAT reaction with circularly permutated rRNAs. The 50S precursors are named according to the major class they belong to, and are generally ordered from immature to mature. The number of particles contributing to each class is given along with the final resolution of the map.

Figure 3.. Circularly permuted LSU intermediates exhibit identified structural features.

Four B classes in four CiPer datasets are shown in back (A) and side view (B) by the semi-transparent mask aligning with the atomic model of 50S subunit (PDB:4YBB). Arrows indicate the helices with extra density due to the circularly permuted rRNA constructs.

Figure 4.. Certain structural elements are absent in circularly permuted LSU intermediates.

(A) Occupancy of rRNA helices and ribosomal proteins. The values were used to analyze the correlation of the 139 assembly elements for each of the circularly permuted LSU intermediates density maps. The blue or white squares represent the presence or absence of the segments in the intermediates. The theoretical densities of the H45 Block (B) and H63 Block (C) are colored purple and green respectively in the 50S crystal structure (PDB: 4YBB). CiPer45_B (D) and CiPer63_D (E) classes are aligned with the 50S atomic model, with the H45 Block and H63 Block covered by a transparent mask, respectively.

Figure 5.. Organization of circularly permuted LSU intermediates into assembly pathways.

The reconstructed 50S intermediates from CiPer45 (A), CiPer63 (B), CiPer63(+PS) (C) and CiPer78 (D) are organized according to the composition analysis. The arrows indicate the minimal folding steps required to connect the set of intermediates. The purple and green arrows indicate the parallel assembly pathways caused by the perturbation of the split helices in CiPer45 and CiPer63 rRNA constructs.

Figure 6.. The docking hierarchy drives rRNA folding predominantly.

(A) Simplified schematic diagram illustrating the assembly process of wild type 23S rRNA. The assembly intermediates at various stages are listed alongside the transcription directionality from the 5’ end to the 3’ end. Consistent color schemes are applied to DNA and RNA strands. Irregular polygons depict the docked assembly blocks composed of folded rRNA and ribosomal proteins. With the state-of-the-art single-particle cryo-EM analysis, only the docked portions are sufficiently rigid to be resolved. (B) Assembly of circularly permuted 23S rRNA with CiPer63 presented as an example, showcasing two mechanisms governed by different logics. In the top mechanism, validated in this study, rRNA can fold and assemble into modules concurrently with transcription but docks hierarchically, beginning with the pink module. In the bottom mechanism, rRNA folds and docks in tandem with transcription from the 5’ to 3’ end, which could begin with any module.