Use of 23S Ribosomal RNA Circular Permutants to Study E. coli Ribosome Biogenesis

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Erik Carlson, Cedric Orelle, Teresa Szal, Alexander Mankin, and Michael Jewett

Jewett lab, United States

Escherichia coli ribosome synthesis and assembly (biogenesis) is a tightly regulated and ordered process with complex folding and assembly steps. Understanding the complexities of this process is key for ribosome engineering efforts. One aspect of understanding is the order in which the ribosomal (r)RNA is transcribed. To study this, a plasmid based ribosomal operon is mutated such that the native 5’ and 3’ ends of the 23S rRNA are covalently linked, and a circular permutant (CP) is created by opening up new 5’ and 3’ ends at an internal helix. CPs are then evaluated on the ability to support cell growth in E. coli without genomic copies of rRNA. The current data set in this space is severely limited; CPs at only two of the over 100 23S helices are known to be viable. Towards testing the remaining circular permutations, we utilize a parallel and efficient construction method to synthesize CPs of 23S rRNA at all helices on a plasmid based operon. Then CP23S constructs are evaluated on their ability to support cell growth in a strain lacking all 7 genomic copies of ribosomal operons. We identified several new mutations that support cell growth with limited reduction in growth rate. Several mutants cannot support cell growth alone, but cells are viable in their presence. Finally, several mutations are toxic when expressed in E. coli. This work provides a more thorough understanding of ribosome biogenesis in vivo, and informs ongoing ribosomal engineering efforts.