Macromolecular crowding effects on transcription and translation are regulated by free magnesium ion

Abstract

Cell‐free metabolic engineering is an emerging and promising alternative platform for the production of fuels and chemicals. In recent years, macromolecular crowding effect, which is an important function in living cells but ignored in cell‐free systems, has been transferred to cell‐free protein synthesis (CFPS). However, inhibitory effects of crowding agents on CFPS were frequently observed, and the mechanism is unclear. In this study, free Mg2+ was found to be a key factor that can regulate the macromolecular crowding effect on in vitro transcription, in vitro translation, and coupled transcript/translation. Addition of crowding agents (20% of Ficoll‐70 or Ficoll‐400) enhanced in vitro transcription at an index of free Mg2+ concentration (IFMC) below 2 mM but inhibited the transcription when the IFMC was higher than 2 mM. Similarly, Ficoll‐400 enhanced in vitro translation and coupled transcription/translation at a lower IFMC (0.1–2 mM) and inhibited the reactions at higher IFMC (>2 mM). Based on the results, CFPS systems could be further optimized by adjusting the content of crowding agents and the IFMC. Besides, the results also indicate that macromolecular crowding effect is important for maintaining the efficiency of in vivo transcription and translation which occur at a low intracellular IFMC (<1 mM).

Programmable protein topology via SpyCatcher‐SpyTag chemistry in one‐pot cell‐free expression system

“…Magnesium ion is one of the key factors affecting protein synthesis (Figure 5a). It was found that Mg 2+ as a cofactor was involved in the transcription and translation processes 45 . So first of all, the selected four combinations (dimer I + H, trimer K + I and L + H, and multimer K + L) (Figure 5b) were coexpressed in the cell‐free system containing 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, or 200 mM Mg ion concentration.…”

Design Approaches to Expand the Toolkit for Building Cotranscriptionally Encoded RNA Strand Displacement Circuits

“…Such conditions are expected to alter many properties of ctRSD circuits. For example, ion availability and molecular crowding influence transcription, − RNA folding and ribozyme catalysis, − and strand displacement. ,, Further, encapsulation and localization of RNA can influence RNA folding and ribozyme catalysis, , and strand displacement reactions . Strand displacement reactions are also influenced by the presence of non-target nucleic acids, and the presence of RNases will introduce sequence and structure specific degradation rates .…”

A Comprehensive Comparison of Rapid RNA Extraction Methods for Detection of SARS-CoV-2 as the Infectious Agent of the Upper Respiratory Tract using Direct RT-LAMP Assay

“…This may be due to the enhancing effects of BSA as a crowding agent, which reduces the inhibitory effect on the reaction, stabilizes the enzymes, and prevents nucleic acids from binding to the reaction containers. [ 29 ] Perhaps the enhancer effect of BSA along with the specific effects of proteinase K removes the inhibitory effect of interferer proteins and helps improve the efficacy of the LAMP reactions that do not occur with BSA or Triton X-100 alone (methods 4 and 6). However, the combination of BSA and proteinase K with Triton X-100 detergent may help better RNA release, which in turn gives us the possibility of increasing dilution factor.…”