In vitro reconstitution of functional small ribosomal subunit assembly for comprehensive analysis of ribosomal elements in E. coli

Masaru Shimojo,Takashi Kanamori,Takuya Ueda,Yoshihiro Shimizu,Keiko Masuda,Kazuaki Amikura

doi:10.1038/s42003-020-0874-8

Abstract

In vitro reconstitution is a powerful tool for investigating ribosome functions and biogenesis, as well as discovering new ribosomal features. In this study, we integrated all of the processes required for Escherichia coli small ribosomal subunit assembly. In our method, termed fully Recombinant-based integrated Synthesis, Assembly, and Translation (R-iSAT), assembly and evaluation of the small ribosomal subunits are coupled with ribosomal RNA (rRNA) synthesis in a reconstituted cell-free protein synthesis system. By changing the components of R-iSAT, including recombinant ribosomal protein composition, we coupled ribosomal assembly with ribosomal protein synthesis, enabling functional synthesis of ribosomal proteins and subsequent subunit assembly. In addition, we assembled and evaluated subunits with mutations in both rRNA and ribosomal proteins. The study demonstrated that our scheme provides new ways to comprehensively analyze any elements of the small ribosomal subunit, with the goal of improving our understanding of ribosomal biogenesis, function, and engineering.

Highlights

In vitro reconstitution is a powerful tool for investigating ribosome functions and biogenesis, as well as discovering new ribosomal features
We recently showed that active 30S subunits can be reconstituted from individually prepared recombinant ribosomal proteins under low-salt conditions using ribosome biogenesis factors[16]
These studies prompted us to develop an iSAT-like method based on individually prepared recombinant ribosomal proteins within the protein synthesis using recombinant elements (PURE) system

Summary

Introduction

In vitro reconstitution is a powerful tool for investigating ribosome functions and biogenesis, as well as discovering new ribosomal features. In our method, termed fully Recombinant-based integrated Synthesis, Assembly, and Translation (R-iSAT), assembly and evaluation of the small ribosomal subunits are coupled with ribosomal RNA (rRNA) synthesis in a reconstituted cell-free protein synthesis system. Ribosomes are large macromolecular complexes that play central roles in cellular protein synthesis, the final step of gene expression To elucidate their biogenesis and translational function, extensive in vitro reconstitution studies have been performed on the Escherichia coli ribosome, dating back to the early stages of the field of biochemistry[1,2]. These studies revealed the hierarchical assembly map of both 30S and 50S subunits[3,4]. On the basis of this technology, a unique selection system using a liposome-sorting technique, which integrates iSAT with the protein synthesis using recombinant elements (PURE) reconstituted cell-free protein synthesis system[13], has been developed for the in vitro evolution of 16S rRNA7

Methods

Results

Conclusion