Physiological Assembly Of Functionally Active 30S Ribosomal Subunits From In Vitro Synthesized Parts

Abstract

Synthetic ribosomes in vitro can facilitate engineering translation of novel polymers, identifying ribosome biogenesis central components, and paving the road to constructing replicating systems from defined biochemical components. Here, we report functional synthetic Escherichia coli 30S ribosomal subunits constructed using a defined, purified cell free system under physiological conditions. We test hypotheses about key components of natural ribosome biogenesis pathway as required for efficient function - including integration of 16S rRNA modification, cofactors facilitated ribosome assembly and protein synthesis in the same compartment in vitro. We observe ~17% efficiency for fully synthetic 30S and ~70% efficiency from in vitro transcribed 16S rRNA assembled with natural proteins. We observe up to 5 fold improvement over previous crude extracts. We suggest extending the minimal list of components required for central-dogma replication from the 151 gene products previously reported to at least 180 to allow the speed and accuracy of macromolecular synthesis to approach native E. coli values.