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. 2004 Apr 27;101(17):6421-6.
doi: 10.1073/pnas.0308014101. Epub 2004 Apr 19.

New RNA motifs suggest an expanded scope for riboswitches in bacterial genetic control

Jeffrey E Barrick  1 , Keith A Corbino, Wade C Winkler, Ali Nahvi, Maumita Mandal, Jennifer Collins, Mark Lee, Adam Roth, Narasimhan Sudarsan, Inbal Jona, J Kenneth Wickiser, Ronald R Breaker
Affiliations

New RNA motifs suggest an expanded scope for riboswitches in bacterial genetic control

Jeffrey E Barrick et al. Proc Natl Acad Sci U S A. .

Abstract

The expression of certain genes involved in fundamental metabolism is regulated by metabolite-binding "riboswitch" elements embedded within their corresponding mRNAs. We have identified at least six additional elements within the Bacillus subtilis genome that exhibit characteristics of riboswitch function (glmS, gcvT, ydaO/yuaA, ykkC/yxkD, ykoK, and yybP/ykoY). These motifs exhibit extensive sequence and secondary-structure conservation among many bacterial species and occur upstream of related genes. The element located upstream of the glmS gene in Gram-positive organisms functions as a metabolite-dependent ribozyme that responds to glucosamine-6-phosphate. Other motifs form complex folded structures when transcribed as RNA molecules and carry intrinsic terminator structures. These findings indicate that riboswitches serve as a major genetic regulatory mechanism for the control of metabolic genes in many microbial species.

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Figures

Fig. 1.
Fig. 1.
Consensus sequences and secondary structure models for riboswitch candidates. Two variations of the gcvT element (types I and II) are depicted. Black and red letters identify nucleotides whose sequence identities are conserved in >80% and 95% of the representatives, respectively. Purine (R) or pyrimidine (Y) designations are given when a single nucleotide does not reach 80% conservation threshold.
Fig. 2.
Fig. 2.
The phylogenetic distribution, structural configuration, and function of the glmS RNA element. (A) Sequence alignment of the conserved element residing upstream of glmS gene homologs. Nucleotides shaded with purple, blue, green, and orange identify putative base-paired regions P1 through P4, respectively. Lowercase and uppercase letters of the consensus identify nucleotides that are conserved in 80% and 95% of the representatives, respectively. Nucleotides highlighted in red differ between the database sequence and that determined by the sequencing of clones for Bce and Bsu prepared by our laboratory. The organism and gene name are provided for each representative shown. Organism abbreviations: Bce, Bacillus cereus; Bha, B. halodurans; Bsu, B. subtilis; Cpe, Clostridium perfringens, Lpl, Lactobacillus plantarum; Lin, Listeria innocua; Oih, Oceanobacillus iheyensis; Sau, Staphylococcus aureus; Tte, Thermoanaerobacter tengcongensis; Fnu, Fusobacterium nucleatum. (B) Sequence and predicted secondary structure of the glmS ribozyme that has been engineered to function as a bimolecular construct. (C) Metabolite-dependent ribozyme function of the conserved glmS element. Reactions were conducted in the absence (-) or presence (+) of ribozyme and 100 μM effector as indicated for each lane. Sub and Clv identify the substrate and 5′ cleavage product, respectively. Glc6P, glucose-6-phosphate.
Fig. 3.
Fig. 3.
In-line probing data for the gcvT and ykoK elements from B. subtilis.(A) Sequence and secondary structure model for the 192 gcvT RNA construct. The nucleotides whose 3′ linkage undergoes a high level of spontaneous cleavage relative to most other linkages are identified by shaded circles. Arrowheads demark the boundaries of the RNA region where spontaneous cleavage data are mapped. (B) Sequence and secondary structure model for the 221 ykoK RNA construct. (C) In-line probing of 5′-32P-labeled 192 gcvT RNA. Products resulting from partial digestion with nuclease T1 (T1), partial digestion with alkali (-OH), and spontaneous cleavage during a 40-h incubation were separated by PAGE. NR, no reaction; Pre, the full-length 192 gcvT RNA. Bands corresponding to certain T1-dependent (G-specific) cleavage products are identified. Nucleotide numbering does not include the unnatural G residues (lowercase letters) inserted to improve transcription in vitro. (D) In-line probing analysis of 5′-32P-labeled 221 ykoK RNA.
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