Boundary-Free Ribosome Compartmentalization by Gene Expression on a Surface.

Michael Levy,Reuven Falkovich,Alexandra M Tayar,Shirley S Daube,Yoshihiro Shimizu,Ohad Vonshak,Dan Bracha,Roy H Bar-Ziv

doi:10.1021/acssynbio.0c00613

Abstract

The design of artificial cell models based on minimal surface-bound transcription–translation reactions aims to mimic the compartmentalization facilitated by organelles and inner interfaces in living cells. Dense DNA brushes as localized sources of RNA and proteins serve as synthetic operons that have recently proven useful for the autonomous synthesis and assembly of cellular machines. Here, we studied ribosome compartmentalization in a minimal gene-expression reaction on a surface in contact with a macroscopic reservoir. We first observed the accumulation and colocalization of RNA polymerases, ribosomes, nascent RNAs and proteins, in dense DNA brushes using evanescent field fluorescence, showing transcription–translation coupling in the brush. Fluorescence recovery after photobleaching showed that ribosomes engaged in translation in the brush had a 4-fold slower diffusion constant. In addition, ribosomes in the brush had over a 10-fold higher local concentration relative to free ribosomes, creating a boundary-free functional ribosome-rich compartment. To decouple translation from transcription, we immobilized dense phases of ribosomes next to DNA brushes. We demonstrated that immobilized ribosomes were capable of protein synthesis, forming 2D subcompartments of active ribosome patterns induced and regulated by DNA brush layout of coding and inhibitory genes. Localizing additional molecular components on the surface will further compartmentalize gene-expression reactions.

Highlights

The design of artificial cell models based on minimal surface-bound transcription−translation reactions aims to mimic the compartmentalization facilitated by organelles and inner interfaces in living cells
Cell-free systems that support gene expression reactions are becoming increasingly versatile and efficient for emulating and simplifying cellular processes.[1−5] Especially powerful is the PURE system made of purified components providing a minimal and controlled environment for RNA transcription and protein translation.[6]
By anchoring coding genes as DNA brushes and surface traps for assembly intermediates and final products, we recently demonstrated in a PURE gene expression reaction the synthesis and assembly of the fiveprotein enzyme E. coli RNA polymerase (RNAP)[16] and the small ribosomal subunit (SSU),[10] composed of 20 ribosomal proteins (r-proteins) and one ribosomal RNA (r-RNA)

Summary

Introduction

The design of artificial cell models based on minimal surface-bound transcription−translation reactions aims to mimic the compartmentalization facilitated by organelles and inner interfaces in living cells. We studied the effect of DNA immobilization on gene expression reactions by patterning fluorescently labeled DNA brushes with a diameter of 60−80 μm on a fused silica surface coated with a photosensitive and biocompatible polymer monolayer (Figure 1A, Methods).

Results

Conclusion