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Review
. 2013 Dec;8(12):1379-95.
doi: 10.1002/biot.201300018. Epub 2013 Oct 4.

The centrality of RNA for engineering gene expression

James Chappell  1 , Melissa K Takahashi, Sarai Meyer, David Loughrey, Kyle E Watters, Julius Lucks
Affiliations
Free PMC article
Review

The centrality of RNA for engineering gene expression

James Chappell et al. Biotechnol J. 2013 Dec.
Free PMC article

Abstract

Synthetic biology holds promise as both a framework for rationally engineering biological systems and a way to revolutionize how we fundamentally understand them. Essential to realizing this promise is the development of strategies and tools to reliably and predictably control and characterize sophisticated patterns of gene expression. Here we review the role that RNA can play towards this goal and make a case for why this versatile, designable, and increasingly characterizable molecule is one of the most powerful substrates for engineering gene expression at our disposal. We discuss current natural and synthetic RNA regulators of gene expression acting at key points of control--transcription, mRNA degradation, and translation. We also consider RNA structural probing and computational RNA structure predication tools as a way to study RNA structure and ultimately function. Finally, we discuss how next-generation sequencing methods are being applied to the study of RNA and to the characterization of RNA's many properties throughout the cell.

Keywords: Gene regulation; Next-generation sequencing; Non-coding RNA; RNA structure; Synthetic biology.

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Figures

Figure 1
Figure 1
The central synthesis and degradation steps of prokaryotic gene expression. The points of control where cis- and trans-acting RNA regulators exert their regulation are highlighted. Pointed arrows indicate activation while blunt arrows indicate repression. Dashed arrow indicates a gap in regulatory mechanisms. RBS, ribosome binding site; UTR, untranslated region; sRNA, small RNA.
Figure 2
Figure 2
The major classes of natural and synthetic RNA regulators, organized into (A) transcriptional and translational regulators and (B) regulators of mRNA degradation.
Figure 3
Figure 3
Chemical probing of RNA structure. Specific chemicals covalently modify folded RNAs at unpaired and flexible nucleotide positions. These modified RNAs are converted into DNA via reverse transcription, which is blocked by the modifications, thus creating truncated single-stranded DNA (ssDNA) species. These ssDNA species are then sequenced, and locations of chemical modifications identified to derive a measure of nucleotide “reactivity” to the chemical. Reactivities are then used to infer structural properties of the input RNAs.
Figure 4
Figure 4
Next-generation sequencing can characterize RNA abundances, structures and interactions across the cell. Names of established NGS-techniques that characterize these features are highlighted next to the interactions examined.
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