(15a) Use of 23S Ribosomal RNA Circular Permutants to Investigate E. Coli Ribosome Biogenesis in Vivo

The ribosome is an amazingly complex and intricate molecular machine, responsible for producing the cell’s proteome. Escherichia coli ribosome synthesis and assembly (biogenesis) is a tightly regulated and ordered process with complex—and not fully understood—folding and assembly steps. Understanding the intricacies and regulation of this process is key for ribosome engineering efforts. During rRNA transcription, co-transcriptional folding occurs as local secondary structure begins to emerge. Additionally, ribosomal proteins interact with the nescient rRNA as binding domains are transcribed. As different regions of the rRNA are exposed, tertiary interactions form as well. One aspect of understanding is the order in which the rRNA 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. By observing effects on cell growth, a more complete understanding of transcription order effects on functional ribosome formation can be obtained. The current data set in this space is severely limited; CPs at only two of the over 100 23S helices are reported. Towards testing the remaining circular permutations, we utilize a parallel and efficient construction method to synthesize CPs of 23S rRNA at 90 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. In preliminary work, 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. In this presentation, I will discuss our efforts to catalogue all possible 23S rRNA CPs. This work will provide a more thorough understanding of ribosome biogenesis in vivo and inform ongoing efforts to control gene regulation with orthogonal ribosomes.