Abstract
We describe a simple strategy to control mRNA translation in both prokaryotic and eukaryotic cells which relies on a unique protein–RNA interaction. Specifically, we used the Pumilio/FBF (PUF) protein to repress translation by binding in between the ribosome binding site (RBS) and the start codon (in Escherichia coli), or by binding to the 5′ untranslated region of target mRNAs (in mammalian cells). The design principle is straightforward, the extent of translational repression can be tuned and the regulator is genetically encoded, enabling the construction of artificial signal cascades. We demonstrate that this approach can also be used to regulate polycistronic mRNAs; such regulation has rarely been achieved in previous reports. Since the regulator used in this study is a modular RNA-binding protein, which can be engineered to target different 8-nucleotide RNA sequences, our strategy could be used in the future to target endogenous mRNAs for regulating metabolic flows and signaling pathways in both prokaryotic and eukaryotic cells.
Highlights
Advances in synthetic biology, especially in gene regulation, provide powerful tools to study biological systems as well as to develop complex artificial genetic processes [1,2,3,4,5,6,7,8]
Repression of prokaryotic mRNA translation relying on PUF–Nanos response element (NRE) interaction in E. coli
Translation is initiated through an interaction between the 16S ribosomal RNA and the ribosome binding site (RBS) that lies in the 5 UTR of the mRNA
Summary
Especially in gene regulation, provide powerful tools to study biological systems as well as to develop complex artificial genetic processes [1,2,3,4,5,6,7,8]. In recent years, the advantages of translational regulation over transcriptional regulation and developments in RNA technology and nucleic acid engineering have accelerated the design and construction of RNA-based gene regulatory systems [6,11,12,13,14,15,16,17]. In eukaryotic cells, such a regulatory approach may allow the synthesis of proteins at specific locations in cells. Despite these advantages, there are numerous challenges associated with engineering RNA-based regulators. The mechanisms of translation initiation are different in prokaryotic and eukaryotic cells, making it difficult to develop universal approaches for translational regulation
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