Transfer RNA Synthesis-Coupled Translation and DNA Replication in a Reconstituted Transcription/Translation System

Abstract

Transfer RNAs (tRNAs) are key molecules involved in translation. In vitro synthesis of tRNAs and their coupled translation are important challenges in the construction of a self-regenerative molecular system. Here, we first purified EF-Tu and ribosome components in a reconstituted translation system of Escherichia coli to remove residual tRNAs. Next, we expressed 15 types of tRNAs in the repurified translation system and performed translation of the reporter luciferase gene depending on the expression. Furthermore, we demonstrated DNA replication through expression of a tRNA encoded by DNA, mimicking information processing within the cell. Our findings highlight the feasibility of an in vitro selfreproductive system, in which tRNAs can be synthesized from replicating DNA.

Emergence of linkage between cooperative RNA replicators encoding replication and metabolic enzymes thorough experimental evolution

“…All protein components of the translation system were purified by two successive affinity column chromatography in a stringent buffer to further reduce the remaining NDK activity derived from Escherichia coli . For all proteins except ribosomes, the re-purification procedure was the same as that used for removing tRNA from EF-Tu, described in our previous study [33]. Ribosomes were purified as described previously [19] and then washed with another stringent buffer (20 mM Hepes-KOH (pH 7.6), 6 mM magnesium acetate, 7 mM 2-mercaptoethanol, 1% Triton X-100, 1 mM dithiothreitol, and 0.33 M potassium chloride).…”

“…All protein components of the translation system were purified by two successive affinity column chromatography in a stringent buffer to further reduce the remaining NDK activity derived from Escherichia coli. For all proteins except ribosomes, the re-purification procedure was the same as that used for removing tRNA from EF-Tu, described in our previous study [33].…”

The Long Road to a Synthetic Self-Replicating Central Dogma

“…In their work, expression of reporter luciferase was maintained when PURE was depleted both of a single tRNA and of 15 separate tRNAs. In both cases, PURE was supplied with the corresponding linear DNA templates of the missing tRNAs and transcribed by T7 RNAP runoff transcription . However, in addition to synthesizing its own tRNAs, ideally from a single minimal genome, a self-replicating PURE system would need to process transcripts into mature tRNAs to (i) remove excess nucleotides upstream of the 5′ end and (ii) expose or add the 3′-CCA end for aminoacylation.…”

“…As an alternative to RNase P treatment and to couple tRNA synthesis with replication of a circular DNA template encoding the same tRNAs, Miyachi et al generated a single-stranded nick immediately downstream of the 3′-CCA end. With their approach, after treatment with Nt.BspQI, the sense strand encoding the tRNA is transcribed similarly to runoff transcription and is used to synthesize Phi29 for RCA of the encoding plasmid . However, while this approach reduces cumbersome enzymatic treatments for maturation of tRNAs, it cannot be incorporated into a self-replicating PURE without augmenting the PURE proteome with Nt.BspQI.…”

“…In both cases, PURE was supplied with the corresponding linear DNA templates of the missing tRNAs and transcribed by T7 RNAP runoff transcription. 73 However, in addition to synthesizing its own tRNAs, ideally from a single minimal genome, a self-replicating PURE system would need to process transcripts into mature tRNAs to (i) remove excess nucleotides upstream of the 5′ end and (ii) expose or add the 3′-CCA end for aminoacylation. In vivo , these steps are carried out post-transcriptionally by (i) RNase P and (ii) RNase Z, RNase E, and CCase, respectively; 74 therefore, Hibi et al incubated runoff transcription products with 3′-CCA with RNase P before supplementing them to PURE.…”