Abstract 1472 Reconstitution and characterization of in vitro ribosome biogenesis

Background: Ribosome biogenesis is a recurrent process in which nascent ribosomes are synthesized by pre-existing ribosomes and is essential for the self-replication of life. Hence, the reconstitution of ribosome biogenesis is important for understanding the self-replication of life and creating self-replicating artificial cells. Previously, we have successfully reconstituted E. coli ribosome biogenesis in vitro[1]. In this study, we tried to analyze the characteristics of in vitro ribosome biogenesis. Method: For the reconstitution of ribosome biogenesis in vitro, we expressed a ribosomal RNA (rRNA) operon gene and ribosomal protein (r-protein) genes in a cell-free transcription and translation system based on an optimized S150 E. coli cell extract. To identify the optimal condition, we comprehensively explored the concentration ratio of rRNA operon and r-protein genes. To investigate whether alleviating the imbalance of ribosome small subunits (SSU) and large subunits (LSU) improves in vitro ribosome biogenesis, we added extra LSU or SSU to the in vitro SSU or LSU biogenesis. To analyze the post-transcriptional modifications of nascent rRNA, we purified nascent ribosome using 16S and 23S rRNA with streptavidin-binding aptamer (Sb-aptamer)[2][3] and performed mass spectrometric analysis. To analyze the post-translational modifications of the nascent r-proteins, we labeled nascent r-proteins with stable isotope-labeled l-arginine (13C6, 15N4) and l-lysine (13C6, 15N2) and performed mass spectrometric analysis. Results and Discussion: Comprehensive exploration of the expression patterns of the rRNA operon gene and r-protein genes in an optimized S150 E. coli cell extract resulted in the identification of optimal conditions which enable in vitro ribosome biogenesis. We analyzed the characteristics of the in vitro ribosome biogenesis. First, we confirmed that alleviating the imbalance between SSU and LSU by adding extra LSU or SSU did not improve the efficiency of in vitro ribosome biogenesis. Second, we detected some of the post-transcriptional modifications that native ribosomes have in nascent rRNA. Specifically, we detected post-transcriptional modifications including m62A, m1G, Ψ, and m5C in the nascent 16S and 23S rRNA. We also detected some of the post-translational modifications that native ribosomes have in nascent r-proteins including N-terminal methionine excision in the uS8, uS9, and uS12 and methylation of glutamine in the uL3. This research will facilitate the understanding of the self-replication of life and the bottom-up creation of self-replicating artificial cells. References: [1] Y. Kosaka, et al. bioRxiv, 505692 (2022), [2] A. Golovina et al. Biochimie, 92(7):914–7 (2010), [3] A. Leonov et al. J. Biol. Chem., 11;278(28):25664–70 (2003) This study was supported by JSPS KAKENHI (grant number 19K16109 and 26830139 to W.A.), JSPS Research Fellowship for Young Scientists (grant number 22J22251 to Y.K.), JST FOREST (grant number JPMJFR204K to W.A.), Sugiyama Chemical & Industrial Laboratory (http://www.sugiyama-c-i-l.or.jp/ to W.A.), and the JGC-S ScholarshipFoundation (https://www.jgcs.or.jp/en/ to W.A.).