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Open AccessArticle

Observation of SAM-VI Riboswitch Dynamics Using Single-Molecule FRET

by Yanyan Xue, Yi Sun, Yichun Xia, Xiuming Liu and Hua Dai

Biomolecules 2025, 15(6), 841; https://doi.org/10.3390/biom15060841 - 9 Jun 2025

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Riboswitches regulate gene expression through intricate dynamic conformational transitions, with divalent cation Mg²⁺ and their ligands playing pivotal roles in this process. The dynamic structural mechanism by which the S-adenosyl-L-methionine (SAM) responsive SAM-VI riboswitch (riboSAM) regulates the downstream SAM synthase gene translation remains unclear. In this study, we employed position-selective labeling of RNA (PLOR) to incorporate Cy3-Cy5 into designated positions of riboSAM, applying single-molecule Förster resonance energy transfer (smFRET) method to track its conformational switches in response to Mg²⁺ and SAM. smFRET analysis revealed that in the absence of Mg²⁺ and ligand, riboSAM predominantly adopted a translation-activating apo conformation. Physiological concentrations of Mg²⁺ induced riboSAM to fold into dynamic transit-p and holo-p states, creating a transient and structurally pliable binding pocket for ligand binding. SAM binding locks the dynamic transit-p and holo-p states into their final stable transit and holo conformations through conformational selection, turning off downstream cis-gene expression and completing feedback regulation of cellular SAM concentration. The observed synergistic regulatory effect of Mg²⁺ ions and ligand on riboSAM’s conformational dynamics at single-molecule resolution provides new mechanistic insights into gene regulation by diverse riboswitch classes. Full article

(This article belongs to the Collection Feature Papers in Biomacromolecules: Nucleic Acids)

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11 pages, 1326 KB

Open AccessArticle

Integrating PLOR and SPAAC Click Chemistry for Efficient Site-Specific Fluorescent Labeling of RNA

by Yanyan Xue, Xiao Si, Daxu Yin, Shengzhe Zhang and Hua Dai

Int. J. Mol. Sci. 2025, 26(6), 2601; https://doi.org/10.3390/ijms26062601 - 13 Mar 2025

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Abstract

Precisely fluorescently labeling specific nucleotide sites of RNA is critical for gaining insights into the structure and function of RNA through multiple fluorescence detection techniques. The position-selective labeling of RNA (PLOR) method provides a promising strategy to achieve this, wherein the fluorophore-modified NTPs can be co-transcriptionally introduced to specific sites of nascent RNA by using T7 RNA polymerase (T7 RNAP). However, due to steric hindrance limitations, the efficiency of T7 RNAP in recognizing and incorporating large fluorophore-modified NTPs into RNA is far from satisfactory. To overcome this challenge, in this work, we developed an efficient PLOR variant (ePLOR) for the site-specific fluorescent labeling of RNA by integrating PLOR with a post-transcriptional SPAAC (strain-promoted azido-alkyne cycloaddition) click chemistry reaction. The efficiency of the SPAAC reaction occurring on RNA is nearly 100%. Consequently, ePLOR enables the precise fluorescent labeling of designated sites across various structural regions of SAM-VI riboswitch and adenine riboswitch RNA, with labeling and synthesis efficiencies that are 2–2.5 times higher than those of PLOR. The strategy developed in this work can be used for the efficient synthesis of a broader spectrum of long-strand RNAs with site-specific fluorescent labeling and greatly facilitate the detection of the structure and function of these RNAs. Full article

(This article belongs to the Special Issue RNA Function and Structure)

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11 pages, 2068 KB

Open AccessArticle

Quantitative Analysis of Transcription Termination via Position-Selective Labeling of RNA (PLOR) Method

by Ping-Yi Chien, Lingzhi Gao and Yu Liu

Int. J. Mol. Sci. 2023, 24(5), 4934; https://doi.org/10.3390/ijms24054934 - 3 Mar 2023

Cited by 1 | Viewed by 2975

Abstract

T7 RNA polymerase is the most widely used enzyme in RNA synthesis, and it is also used for RNA labeling in position-selective labeling of RNA (PLOR). PLOR is a liquid–solid hybrid phase method that has been developed to introduce labels to specific positions of RNA. Here, we applied PLOR as a single-round transcription method to quantify the terminated and read-through products in transcription for the first time. Various factors, including pausing strategies, Mg²⁺, ligand and the NTP concentration at the transcriptional termination of adenine riboswitch RNA have been characterized. This helps to understand transcription termination, which is one of the least understood processes in transcription. Additionally, our strategy can potentially be used to study the co-transcription behavior of general RNA, especially when continuous transcription is not desired. Full article

(This article belongs to the Collection Feature Papers in Molecular Genetics and Genomics)

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