Microsecond Folding of preQ₁ Riboswitch and Its Biological Significance Revealed by Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy

Abstract

Riboswitches are regulatory elements of bacterial mRNA which function with conformational switching upon binding of specific cellular metabolites. In particular, transcriptional riboswitches regulate gene expression kinetically through the conformational change of the aptamer domain. In this study, we investigate the conformational dynamics and ligand binding mechanisms of the aptamer domain of a transcriptional prequeuosine (preQ 1 ) riboswitch from Bacillus subtilis using two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS) with microsecond time resolution. The obtained time-resolved single-molecule data indicate that the aptamer domain undergoes folding/ unfolding including three forms, which are attributed to hairpin (O), pseudoknot-like (pF), and H-type pseudoknot (fF) structures. It is found that a cofactor, Mg 2+ , binds only to the fF form with the conformational selection mechanism. In contrast, it is indicated that the ligand, preQ 1 , binds to the O form with the induced-fit mechanism and significantly accelerates the microsecond O → pF folding process. It is also shown that the binding with preQ 1 substantially stabilizes the fF form that is generated from the pF form with a long time constant (>10 ms). Combining these results with the results of a former smFRET study on the slower time scale, we obtain an overall picture of the folding/unfolding dynamics of the aptamer domain as well as its energy landscape. On the basis of the picture obtained, we discuss the significance of the microsecond folding/ unfolding of the aptamer domain for biological function of the riboswitch and propose the molecular mechanism of the gene expression controlled by the structural dynamics of the aptamer domain.

Scanning Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy: Conformational Dynamics of DNA Holliday Junction from Microsecond to Subsecond

“…To date, 2D FLCS has successfully been applied to reveal microsecond structural dynamics of protein, 19,20 DNA, 21,22 and RNA. 22,23 However, there has been a crucial constraint: the observation time window is limited up to several hundreds of microseconds because of a relatively short residence time of the target molecule in the confocal volume due to diffusion.…”

“…Unlike FCS, however, 2D FLCS can clearly distinguish multiple conformational species based on their fluorescence lifetimes and determine their interconversion rates with high accuracy. To date, 2D FLCS has successfully been applied to reveal microsecond structural dynamics of protein, , DNA, , and RNA. , However, there has been a crucial constraint: the observation time window is limited up to several hundreds of microseconds because of a relatively short residence time of the target molecule in the confocal volume due to diffusion.…”

“…In the following, we show that these quantities can be determined by utilizing 2D FLCS. We employ a recently developed extension of 2D FLCS that incorporates the analysis of the acceptor fluorescence, i.e., two-color 2D FLCS. , In two-color 2D FLCS, the states exhibiting different FRET efficiencies are determined as independent fluorescence components that are required to reproduce the 2D correlation maps that are constructed from the donor and acceptor fluorescence photons recorded with time-tagged TCSPC. Detailed procedures and interpretations of two-color 2D FLCS are described in our previous paper − as well as in the Supporting Information.…”

Transcriptome analysis and prediction of the metabolic state of stress-induced viable but non-culturable Bacillus subtilis cells

“…The second most signi cantly upregulated genes in VBNC cells belong to the queosine biosynthesis pathway, particularly the queC-queD-queE-queF operon (yvkI), regulated by the preQ-riboswitch 34,35 (Fig. 3).…”

Affinity-Based Profiling of the Flavin Mononucleotide Riboswitch

“…Mechanistic data of riboswitches are often obtained with synthetic RNA, using a combination of physical methods, such as nuclear magnetic resonance (NMR) studies, − X-ray diffraction crystallography, , fluorescence spectroscopy, , in vitro profiling methods, ,,, and genetic approaches. , Though biologically vital, the structural features of riboswitches can prove challenging to study and remain of continued interest. , …”