Optimization and compartmentalization of a cell-free mixture of DNA amplification and protein translation

Han, Fuhai; Xu, Bo-Ying; Lu, Nan; Caliari, Adriano; Lü, Hui; Xia, Yang; Su’etsugu, Masayuki; Xu, Jian; Yomo, Tetsuya

doi:10.1007/s00253-022-12278-2

Cited by 2 publications

(3 citation statements)

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“…The inhibitory effect of rNTP on DNA replication observed in this study (Figure 2A) was reported in a previous study, 36 in which the collisions of DNA and RNA polymerases at a higher concentration of rNTP were suggested as a possible cause of the inhibitory effect. However, since DNA replication was performed in the absence of transcription machinery in our study, the observed inhibitory effect on DNA replication must be due to a different mechanism.…”

Section: ■ Discussionmentioning

confidence: 99%

“…Other research groups have also reported inhibition of DNA replication when coupled with transcription and translation. ,, Recently, Han et al reported incompatibility between the reconstituted E. coli DNA replication and translation, which was partially resolved by reducing rNTP and tRNA concentrations . These results suggest that DNA replication and transcription/translation have different optimal conditions.…”

Section: Introductionmentioning

confidence: 99%

“…26,27,35 Recently, Han et al reported incompatibility between the reconstituted E. coli DNA replication and translation, which was partially resolved by reducing rNTP and tRNA concentrations. 36 These results suggest that DNA replication and transcription/translation have different optimal conditions. However, the factors that cause this discrepancy and how to address it are not yet fully understood.…”

Section: ■ Introductionmentioning

confidence: 99%

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Investigation of Compatibility between DNA Replication, Transcription, and Translation for in Vitro Central Dogma

Seo

Ichihashi

2023

ACS Synth. Biol.

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Recent advances in in vitro synthetic biology have made it possible to reconstitute various cellular functions in a test tube. However, the integration of these functions remains a major challenge. This study aimed to identify a suitable condition to achieve all three reactions that constitute the central dogma: transcription, translation, and DNA replication. Specifically, we investigated the effect of the concentrations of 11 nonprotein factors required for in vitro transcription, translation, and DNA replication on each of these reactions. Our results indicate that certain factors have opposing effects on the three reactions. For example, while dNTP is necessary for DNA replication, it inhibited translation, and both rNTP and tRNA, which are essential for transcription and translation, inhibited DNA replication with several DNA polymerases. We also found that these opposing effects were partially alleviated by optimizing the magnesium concentration. Using this knowledge, we successfully demonstrated transcription/translation-coupled DNA replication with higher levels of transcription and translation while maintaining a certain level of DNA replication. These findings not only provide useful insights for the development of a complex artificial system with the central dogma but also raise the question of how natural cells overcome the incompatibility between the three reactions.

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Section: ■ Discussionmentioning

confidence: 99%