Search

Numerous classes of riboswitches have been found to regulate bacterial gene expression in response to physiological cues, offering new paths to antibacterial drugs. As common studies of isolated riboswitches lack the functional context of the transcription machinery, we here combine single-molecule, biochemical, and simulation approaches to investigate the coupling between co-transcriptional folding of the pseudoknot-structured preQ1 riboswitch and RNA polymerase (RNAP) pausing. We show that pausing at a site immediately downstream of the riboswitch requires a ligand-free pseudoknot in the nascent RNA, a precisely spaced sequence resembling the pause consensus, and electrostatic and steric interactions with the RNAP exit channel. While interactions with RNAP stabilize the native fold of the riboswitch, binding of the ligand signals RNAP release from the pause. Our results demonstrate that the nascent riboswitch and its ligand actively modulate the function of RNAP and vice versa, a paradigm likely to apply to other cellular RNA transcripts.

Riboswitches are mRNA segments that regulate gene expression in response to ligand binding. The Class I preQ1 riboswitch consists of a stem‐loop and an adenine‐rich single‐stranded tail ("L3"), which adopt a pseudoknot structure upon binding of the ligand preQ1. We inserted 2‐aminopurine (2‐AP), a fluorescent analogue of adenine (A), into the riboswitch at six different positions within L3. Here, 2‐AP functions both as a spectroscopic probe and as a "mutation" that reveals how alteration of specific A residues impacts the riboswitch. Using fluorescence and circular dichroism spectroscopy, we found that 2‐AP decreases the affinity of the riboswitch for preQ1 at all labeling positions tested, although modified and unmodified variants undergo the same global conformational changes at sufficiently high preQ1 concentration. 2‐AP substitution is most detrimental to ligand binding at sites proximal to the ligand‐binding pocket, while distal labeling sites exhibit the largest impacts on the stability of the L3 domain in the absence of ligand. Insertion of multiple 2‐AP residues does not induce significant additional disruptions. Our results show that interactions involving the A residues in L3 play a critical role in ligand recognition by the preQ1 riboswitch and that 2‐AP substitution exerts complex and varied impacts on this riboswitch. [ABSTRACT FROM AUTHOR]

Fluorescent analogues of the indoleside chain of tryptophan canbe useful spectroscopic probes of protein–protein and protein–DNAinteractions. Here we present linear dichroism and solvent-dependentspectroscopic studies of two fluorescent analogues of indole, in whichthe organicCC unit is substituted with theisosteric inorganicB–N unit. We studied theso-called “external” BN indole, which has C2vsymmetry, and the “fused”BN indole with Cssymmetry.We performed a combination of absorption and fluorescence spectroscopy,ultraviolet linear dichroism (UV-LD) in stretched poly(ethylene) (PE)films, and quantum chemical calculations on both BN indole compounds.Our measurements allowed us to characterize the degree of alignmentfor both molecules in stretched PE films. We thus determined the orientationsand magnitudes of the two lowest energy electric dipole transitionmoments (EDTMs) for external BN indole, and the two lowest energyEDTMs for fused BN indole within the 30 000–45 000cm–1spectral range. We compared our experimentalresults to those of quantum chemical calculations using standard densityfunctional theory (DFT). Our theoretical predictions for the low-energyEDTMs are in good agreement with our experimental data. The absorptionand fluorescence spectra of the external and the fused BN indolesare sensitive to solvent polarity. Our results indicate that the fusedBN indole experiences much greater solvation interactions with polarsolvents than does the external BN indole. [ABSTRACT FROM AUTHOR]

Westudied the equilibrium conformations of a zinc porphyrin tweezercomposed of two carboxylphenyl-functionalized zinc tetraphenyl porphyrinsubunits connected by a 1,4-butyndiol spacer, which was suspendedinside the amphiphilic regions of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) liposomes. By combining phase-modulationtwo-dimensional fluorescence spectroscopy (2D FS) with linear absorbanceand fluorimetry, we determined that the zinc porphyrin tweezer adoptsa mixture of folded and extended conformations in the membrane. Byfitting an exciton-coupling model to a series of data sets recordedover a range of temperatures (17–85 °C) and at differentlaser center wavelengths, we determined that the folded form of thetweezer is stabilized by a favorable change in the entropy of thelocal membrane environment. Our results provide insights toward understandingthe balance of thermodynamic factors that govern molecular assemblyin membranes. [ABSTRACT FROM AUTHOR]

We have observed the conformation-dependent electronic coupling between the monomeric subunits of a dinucleotide of 2-aminopurine (2-AP), a fluorescent analog of the nucleic acid base adenine. This was accomplished by extending two-dimensional fluorescence spectroscopy (2D FS) - a fluorescence-detected variation of 2D electronic spectroscopy - to excite molecular transitions in the ultraviolet (UV) regime. A collinear sequence of four ultrafast laser pulses centered at 323 nm was used to resonantly excite the coupled transitions of 2-AP dinucleotide. The phases of the optical pulses were continuously swept at kilohertz frequencies, and the ensuing nonlinear fluorescence was phase-synchronously detected at 370 nm. Upon optimization of a point-dipole coupling model to our data, we found that in aqueous buffer the 2-AP dinucleotide adopts an average conformation in which the purine bases are non-helically stacked (center-to-center distance R 12 = 3.5 Å ± 0.5 Å, twist angle θ 12 = 5° ± 5°), which differs from the conformation of such adjacent bases in duplex DNA. These experiments establish UV-2D FS as a method for examining the local conformations of an adjacent pair of fluorescent nucleotides substituted into specific DNA or RNA constructs, which will serve as a powerful probe to interpret, in structural terms, biologically significant local conformational changes within the nucleic acid framework of protein-nucleic acid complexes.