| Reference | [1]. Nucleic Acids Res. 2020 Aug 20;48(14):8146-8164. doi: 10.1093/nar/gkaa546.<br />
Analysis of a preQ1-I riboswitch in effector-free and bound states reveals a metabolite-programmed nucleobase-stacking spine that controls gene regulation.<br />
Schroeder GM(1)(2), Dutta D(1)(2), Cavender CE(1)(2), Jenkins JL(1)(2), Pritchett EM(3), Baker CD(3), Ashton JM(3), Mathews DH(1)(2), Wedekind JE(1)(2).<br />
Author information: (1)Department of Biochemistry & Biophysics, University of Rochester School of Medicine & Dentistry, Rochester, NY 14642, USA. (2)Center for RNA Biology, University of Rochester School of Medicine & Dentistry, Rochester, NY 14642, USA. (3)Genomics Research Center, University of Rochester School of Medicine & Dentistry, Rochester, NY 14642, USA.<br />
Riboswitches are structured RNA motifs that recognize metabolites to alter the conformations of downstream sequences, leading to gene regulation. To investigate this molecular framework, we determined crystal structures of a preQ1-I riboswitch in effector-free and bound states at 2.00 Å and 2.65 Å-resolution. Both pseudoknots exhibited the elusive L2 loop, which displayed distinct conformations. Conversely, the Shine-Dalgarno sequence (SDS) in the S2 helix of each structure remained unbroken. The expectation that the effector-free state should expose the SDS prompted us to conduct solution experiments to delineate environmental changes to specific nucleobases in response to preQ1. We then used nudged elastic band computational methods to derive conformational-change pathways linking the crystallographically-determined effector-free and bound-state structures. Pathways featured: (i) unstacking and unpairing of L2 and S2 nucleobases without preQ1-exposing the SDS for translation and (ii) stacking and pairing L2 and S2 nucleobases with preQ1-sequestering the SDS. Our results reveal how preQ1 binding reorganizes L2 into a nucleobase-stacking spine that sequesters the SDS, linking effector recognition to biological function. The generality of stacking spines as conduits for effector-dependent, interdomain communication is discussed in light of their existence in adenine riboswitches, as well as the turnip yellow mosaic virus ribosome sensor.<br />
DOI: 10.1093/nar/gkaa546 PMCID: PMC7641330 PMID: 32597951 [Indexed for MEDLINE]<br />
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[2]. J Bacteriol. 2017 Feb 28;199(6):e00656-16. doi: 10.1128/JB.00656-16. Print 2017 Mar 15.<br />
Characterization of Engineered PreQ1 Riboswitches for Inducible Gene Regulation in Mycobacteria.<br />
Van Vlack ER(1), Topp S(2), Seeliger JC(3).<br />
Author information: (1)Department of Chemistry, Stony Brook University, Stony Brook, New York, USA. (2)Department of Chemistry, University of California, Berkeley, California, USA. (3)Department of Pharmacological Sciences, Stony Brook University, Stony Brook, New York, USA [email protected].<br />
We report here the behavior of naturally occurring and rationally engineered preQ1 riboswitches and their application to inducible gene regulation in mycobacteria. Because mycobacteria lack preQ1 biosynthetic genes, we hypothesized that preQ1 could be used as an exogenous nonmetabolite ligand to control riboswitches in mycobacteria. Selected naturally occurring preQ1 riboswitches were assayed and successfully drove preQ1-dependent repression of a green fluorescent protein reporter in Mycobacterium smegmatis Using structure-based design, we engineered three preQ1 riboswitches from Thermoanaerobacter tencongensis, Bacillus subtilis, and Lactobacillus rhamnosus toward achieving higher response ratios and increased repression. Assuming a steady-state model, variants of the T. tencongensis riboswitch most closely followed the predicted trends. Unexpectedly, the preQ1 dose response was best described by a model with a second, independent preQ1 binding site. This behavior was general to the preQ1 riboswitch family, since the wild type and rationally designed mutants of riboswitches from all three bacteria behaved analogously. Across all variants, the response ratios, which describe expression in the absence versus the presence of preQ1, ranged from <2 to ∼10, but repression in all cases was incomplete up to 1 mM preQ1. By reducing the transcript expression level, we obtained a preQ1 riboswitch variant appropriate for inducible knockdown applications. We further showed that the preQ1 response is reversible, is titratable, and can be used to control protein expression in mycobacteria within infected macrophages. By engineering naturally occurring preQ1 riboswitches, we have not only extended the tools available for inducible gene regulation in mycobacteria but also uncovered new behavior of these riboswitches.IMPORTANCE Riboswitches are elements found in noncoding regions of mRNA that regulate gene expression, typically in response to an endogenous metabolite. Riboswitches have emerged as important tools for inducible gene expression in diverse organisms. We noted that mycobacteria lack the biosynthesis genes for preQ1, a ligand for riboswitches from diverse bacteria. Predicting that preQ1 is not present in mycobacteria, we showed that it controls optimized riboswitches appropriate for gene knockdown applications. Further, the riboswitch response is subject to a second independent preQ1 binding event that has not been previously documented. By engineering naturally occurring riboswitches, we have uncovered a new behavior, with implications for riboswitch function in its native context, and extended the tools available for inducible gene regulation in mycobacteria.<br />
DOI: 10.1128/JB.00656-16 PMCID: PMC5331669 PMID: 28069821 [Indexed for MEDLINE]<br />
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[3]. Proc Natl Acad Sci U S A. 2014 Feb 11;111(6):E663-71. doi: 10.1073/pnas.1400126111. Epub 2014 Jan 27.<br />
Structural determinants for ligand capture by a class II preQ1 riboswitch.<br />
Kang M(1), Eichhorn CD, Feigon J.<br />
Author information: (1)Department of Chemistry and Biochemistry and University of California Los Angeles-Department of Energy Institute for Genomics and Proteomics, University of California, Los Angeles, CA 90095.<br />
Prequeuosine (preQ1) riboswitches are RNA regulatory elements located in the 5' UTR of genes involved in the biosynthesis and transport of preQ1, a precursor of the modified base queuosine universally found in four tRNAs. The preQ1 class II (preQ1-II) riboswitch regulates preQ1 biosynthesis at the translational level. We present the solution NMR structure and conformational dynamics of the 59 nucleotide Streptococcus pneumoniae preQ1-II riboswitch bound to preQ1. Unlike in the preQ1 class I (preQ1-I) riboswitch, divalent cations are required for high-affinity binding. The solution structure is an unusual H-type pseudoknot featuring a P4 hairpin embedded in loop 3, which forms a three-way junction with the other two stems. (13)C relaxation and residual dipolar coupling experiments revealed interhelical flexibility of P4. We found that the P4 helix and flanking adenine residues play crucial and unexpected roles in controlling pseudoknot formation and, in turn, sequestering the Shine-Dalgarno sequence. Aided by divalent cations, P4 is poised to act as a "screw cap" on preQ1 recognition to block ligand exit and stabilize the binding pocket. Comparison of preQ1-I and preQ1-II riboswitch structures reveals that whereas both form H-type pseudoknots and recognize preQ1 using one A, C, or U nucleotide from each of three loops, these nucleotides interact with preQ1 differently, with preQ1 inserting into different grooves. Our studies show that the preQ1-II riboswitch uses an unusual mechanism to harness exquisite control over queuosine metabolism.<br />
DOI: 10.1073/pnas.1400126111 PMCID: PMC3926045 PMID: 24469808 [Indexed for MEDLINE]<br />
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[4]. Chem Biol. 2014 Jul 17;21(7):880-889. doi: 10.1016/j.chembiol.2014.05.015.<br />
Structural, functional, and taxonomic diversity of three preQ1 riboswitch classes.<br />
McCown PJ(#)(1), Liang JJ(#)(2), Weinberg Z(1)(3), Breaker RR(1)(2)(3).<br />
Author information: (1)Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA. (2)Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA. (3)Howard Hughes Medical Institute, Yale University, New Haven, CT 06520, USA. (#)Contributed equally<br />
Previously, two riboswitch classes have been identified that sense and respond to the hypermodified nucleobase called prequeuosine1 (preQ1). The enormous expansion of available genomic DNA sequence data creates new opportunities to identify additional representatives of the known riboswitch classes and to discover novel classes. We conducted bioinformatics searches on microbial genomic DNA data sets to discover numerous additional examples belonging to the two previously known riboswitch classes for preQ1 (classes preQ1-I and preQ1-II), including some structural variants that further restrict ligand specificity. Additionally, we discovered a third preQ1-binding riboswitch class (preQ1-III) that is structurally distinct from previously known classes. These findings demonstrate that numerous organisms monitor the concentrations of this modified nucleobase by exploiting one or more riboswitch classes for this widespread compound.<br />
DOI: 10.1016/j.chembiol.2014.05.015 PMCID: PMC4145258 PMID: 25036777 [Indexed for MEDLINE]<br />
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[5]. Sci Rep. 2016 Aug 3;6:31005. doi: 10.1038/srep31005.<br />
Dynamics Correlation Network for Allosteric Switching of PreQ1 Riboswitch.<br />
Wang W(1), Jiang C(1), Zhang J(1), Ye W(1), Luo R(2), Chen HF(1)(3).<br />
Author information: (1)State Key Laboratory of Microbial metabolism, Department of Bioinformatics and Biostatistics, College of Life Sciences and Biotechnology, Shanghai Jiaotong University, 800 Dongchuan Road, Shanghai, 200240, China. (2)Departments of Molecular Biology and Biochemistry, Chemical Engineering and Materials Science, Biomedical Engineering, University of California, Irvine, California 92697-3900, USA. (3)Shanghai Center for Bioinformation Technology, 1278 Keyuan Road, Shanghai, 200235, China.<br />
Riboswitches are a class of metabolism control elements mostly found in bacteria. Due to their fundamental importance in bacteria gene regulation, riboswitches have been proposed as antibacterial drug targets. Prequeuosine (preQ1) is the last free precursor in the biosynthetic pathway of queuosine that is crucial for translation efficiency and fidelity. However, the regulation mechanism for the preQ1 riboswitch remains unclear. Here we constructed fluctuation correlation network based on all-atom molecular dynamics simulations to reveal the regulation mechanism. The results suggest that the correlation network in the bound riboswitch is distinctly different from that in the apo riboswitch. The community network indicates that the information freely transfers from the binding site of preQ1 to the expression platform of the P3 helix in the bound riboswitch and the P3 helix is a bottleneck in the apo riboswitch. Thus, a hypothesis of "preQ1-binding induced allosteric switching" is proposed to link riboswitch and translation regulation. The community networks of mutants support this hypothesis. Finally, a possible allosteric pathway of A50-A51-A52-U10-A11-G12-G56 was also identified based on the shortest path algorithm and confirmed by mutations and network perturbation. The novel fluctuation network analysis method can be used as a general strategy in studies of riboswitch structure-function relationship.<br />
DOI: 10.1038/srep31005 PMCID: PMC4971525 PMID: 27484311 [Indexed for MEDLINE]
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