In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 109, No. 50 ( 2012-12-11)
Abstract:
Here, we describe a regulatory mechanism by which a translationally acting riboswitch directly modulates mRNA decay through the conformation of its expression platform. From an evolutionary perspective, RNase E cleavage sites may have appeared in riboswitches operating at the translational level, suggesting that dual-acting riboswitches, such as lysC , may represent more modern riboregulators than those acting only at the levels of transcription or translation. How does the lysC riboswitch control RNase E cleavage of the lysC mRNA as a function of lysine intracellular levels? To address this question, we devised an in vitro assay to map precisely RNase E cleavage sites, which we expected to be located in the lysC riboswitch sequence. We found that RNase E cleaved in two regions that are located in the riboswitch expression platform. Cleavage sites were found to be accessible by RNase E only when the riboswitch was bound to lysine, allowing a direct mechanism by which the mRNA is targeted only under high cellular concentrations of lysine ( Fig. P1 ). Mutational analysis revealed that both riboswitch regulatory activities, mRNA decay and translation initiation, can be inhibited individually, directing the riboswitch to regulate at either the mRNA or protein level. These results indicate that riboswitches operate at multiple levels, thus providing an additional layer of complexity in gene regulation. Despite the ever-increasing recognition of the importance of riboswitches as genetic regulators, there is still relatively little information regarding their mechanisms of regulation in vivo. To gather key insights into how riboswitches control gene expression, we characterized the lysine-sensing lysC riboswitch of Escherichia coli . The lysC riboswitch controls the expression of the lysC gene, which is involved in the biosynthetic pathway of lysine. When there is a high concentration of lysine, the latter binds to the riboswitch to repress the expression of the lysC . Although this riboswitch had been predicted to regulate translation initiation ( 2 ), no experimental data had been reported to support this mechanism. Thus, we used reporter gene assays and confirmed that the lysC riboswitch indeed is able to modulate translation initiation as a function of lysine. However, using transcriptional reporter fusions, we also found that this riboswitch negatively modulated the level of mRNA upon lysine binding, suggesting an even more complex mechanism of regulation. Moreover, using Escherichia coli mutants, we showed that the RNA degradosome, and more particularly RNase E, is involved in the lysine-dependent decrease of the mRNA level. We found the degradation to be fast and efficient, suggesting the very tight regulation of lysC expression. There are reports that translationally repressed mRNAs are targeted rapidly by cellular ribonucleases ( 3 ). However, in these cases, mRNA degradation occurs as a consequence of translation inhibition, which is part of normal mRNA turnover. In stark contrast, the results of our reporter gene assays suggest a mechanism by which the lysC riboswitch directly modulates mRNA cleavage as a function of lysine binding and not as a consequence of translation inhibition. Riboswitches are composed of two domains: an aptamer and an expression platform. Although the aptamer domain is highly conserved and is involved in the specific recognition of the metabolite, the expression platform is not as well conserved and controls gene expression upon ligand binding. During the last decade, much effort has been devoted to characterizing bacterial riboswitches operating at the levels of transcription and translation. In transcriptionally acting riboswitches, ligand recognition induces the expression platform to fold into a transcription terminator. However, translationally acting riboswitches achieve genetic regulation by modulating the access of ribosomes to the ribosome-binding site (RBS) or initiating codon sequences by using a stem–loop structure. Bioinformatic analyses revealed several RNA motifs exhibiting unconventional expression platforms ( 1 ), thereby suggesting that novel riboswitch regulation mechanisms remain to be discovered. In prokaryotes, an intricate network of biochemical pathways allows adaptation to continuous metabolic changes by using a variety of nutrients. RNA-based regulatory systems, such as riboswitches, regulate essential metabolic processes. Riboswitches are highly structured regulatory elements located in noncoding regions of mRNAs that control gene expression at the levels of transcription, translation, or splicing. These switches specifically recognize cellular metabolites, such as amino acids, and undergo structural changes upon ligand binding that directly affect the expression of the regulated gene(s). Here, we provide strong experimental evidence that riboswitches also can control gene expression directly at an additional level—mRNA decay—through the use of a ubiquitous ribonuclease, RNase E.
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.1214024109
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
2012
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
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