Dual-acting riboswitch control of translation initiation and mRNA decay

Abstract

Riboswitches are mRNA regulatory elements that control gene expression by altering their structure in response to specific metabolite binding. In bacteria, riboswitches consist of an aptamer that performs ligand recognition and an expression platform that regulates either transcription termination or translation initiation. Here, we describe a dual-acting riboswitch from Escherichia coli that, in addition to modulating translation initiation, also is directly involved in the control of initial mRNA decay. Upon lysine binding, the lysC riboswitch adopts a conformation that not only inhibits translation initiation but also exposes RNase E cleavage sites located in the riboswitch expression platform. However, in the absence of lysine, the riboswitch folds into an alternative conformation that simultaneously allows translation initiation and sequesters RNase E cleavage sites. Both regulatory activities can be individually inhibited, indicating that translation initiation and mRNA decay can be modulated independently using the same conformational switch. Because RNase E cleavage sites are located in the riboswitch sequence, this riboswitch provides a unique means for the riboswitch to modulate RNase E cleavage activity directly as a function of lysine. This dual inhibition is in contrast to other riboswitches, such as the thiamin pyrophosphate-sensing thiM riboswitch, which triggers mRNA decay only as a consequence of translation inhibition. The riboswitch control of RNase E cleavage activity is an example of a mechanism by which metabolite sensing is used to regulate gene expression of single genes or even large polycistronic mRNAs as a function of environmental changes.gene regulation | RNA degradosome | translational control S ince the first demonstration that translation attenuation regulates the expression of the tryptophan operon (1), accumulating evidence has revealed the importance of posttranscriptional regulation in prokaryotes and eukaryotes alike. Posttranscriptional regulators include RNA molecules that operate through several mechanisms to control a wide range of physiological responses (2). Among newly identified RNA regulators are riboswitches, which are located in untranslated regions of several mRNAs and that modulate gene expression at the level of transcription, translation, or splicing (3). Riboswitches are highly structured regulatory domains that directly sense cellular metabolites such as amino acids, carbohydrates, coenzymes, and nucleobases. These genetic switches are composed of two modular domains consisting of an aptamer and an expression platform. The aptamer is involved in the specific recognition of the metabolite, and the expression platform is used to control gene expression by altering the structure of the mRNA. Recent bioinformatic analyses have reported the existence of several new RNA motifs exhibiting unconventional expression platforms (4, 5), suggesting that riboswitches using different regulation mechanisms are still likely to be discovered (6).The lysine riboswitch was first...

A Kissing Loop Is Important for btuB Riboswitch Ligand Sensing and Regulatory Control

“…2B). These findings are in contrast to other riboswitches where mutations introduced in the 5Ј side of the P1 stem usually result in the derepression of gene expression (27,32). Given that M1.2 and M1.4 mutants directly weaken base pair formation of the AЈ-RBS/A-RBS interaction, the A-RBS/RBS helical domain (P14) is readily favored in such a context, thereby resulting in low ␤-galactosidase activities.…”

“…was radiolabeled using a polynucleotide kinase, T4 (Thermo Scientific). Briefly, 5 pmol of trinucleotide was labeled according to the manufacturer's protocol using [␥- 32 P]ATP at 37°C for 1 h and directly added to the transcription mixture without further purification.…”

“…N-Methylisatoic anhydride-treated RNA was precipitated, resuspended, and reverse transcribed using the oligonucleotide BtuB202 (Table 3) as described previously (31). ␤-Galactosidase Assays-␤-Galactosidase experiments were performed as described previously (32). Briefly, the bacterial culture was grown overnight in M63 0.2% glycerol minimal medium and diluted 50ϫ into fresh medium.…”

Hierarchy of RNA Functional Dynamics

“…These structural elements can be either a contiguous stretch of nucleotides that are either single-stranded and exposed, or sequestered into hairpins via base-pairing, or be an entire hairpin that is either present or absent from the secondary structure. For example, single stranded mRNA ribosome binding sites (24), degradative endonuclease cleavage sites (25), and splicing sites (26), as well transcription terminator hairpins (27), among others, have all been shown to be exposed or sequestered by secondary structure changes as part of regulatory processes (Figure 2). …”

Common themes and differences in SAM recognition among SAM riboswitches

“…They regulate gene expression mainly by premature transcription termination or inhibition of translation initiation. Other regulatory mechanisms have been demonstrated, including the control of mRNA degradation or alternative splicing[2, 3]. Canonical riboswitches typically consist of two domains: an upstream aptamer domain responsible for ligand recognition and a downstream expression platform.…”