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

Abstract

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.

In vitrotranscription-based biosensing of glycolate for prototyping of a complex enzyme cascade

“…Key to establish our biosensor was the finding that IVT-based sensing is highly sensitive to components that are commonly used in in vitro systems, including ATP, NADPH, and other nucleotide-based and phosphorylated cofactors, which strongly inhibit IVT activity. These results are in line with recent studies that investigated the inhibition of reconstituted transcription, translation and DNA replication systems 30 . In our study, we extend these findings by providing a fitness landscape of T7 RNA polymerase-based transcription in the presence of common metabolic cofactors.…”

“…ATP inhibition was partially due to an increased competition between ATP and the other three rNTPs (Supplementary Figure 8). However, the majority of ATP inhibition seemed to be caused by chelation of free magnesium ions, which are essential for T7 RNA polymerase activity, similar to the inhibition of phi29 DNA polymerase by rNTPs 30 . We further speculated that NADPH, creatine phosphate (CP), CoA and FAD, followed a similar inhibitory mechanism.…”

Leveraging Active Learning to Establish Efficient In Vitro Transcription and Translation from Bacterial Chromosomal DNA

“…In our situation, instead, the reaction conditions suggested by active learning seem to reduce potassium in order to improve transcription in a system with a low template DNA concentration. Recent work reported a similar behavior when in vitro systems were optimized for different reactions, showing that translation was favored by higher potassium concentrations compared to transcription from T7 RNA polymerase …”

“…Recent work reported a similar behavior when in vitro systems were optimized for different reactions, showing that translation was favored by higher potassium concentrations compared to transcription from T7 RNA polymerase. 41 …”

Slow Freeze‐Thaw Cycles Enhanced Hybridization of Kilobase DNA with Long Complementary Sticky Ends

“…Mg 2 + is a well-known divalent cation that interacts with nucleic acids and affects their physicochemical behavior and genomic function. [32][33][34][35] We thus varied the MgCl 2 concentration ([MgCl 2 ]) while maintaining other ingredients at concentrations the same as those of 0.1×buffer (Figure 2). Notably, omitting adenosine triphosphate (ATP) or dithiothreitol (DTT) did not affect the hybridized fractions in C F/T(+) (Supplementary Figure S3); therefore, neither seemed to be an essential component for hybridization.…”