Light-controlled twister ribozyme with single-molecule detection resolves RNA function in time and space.

Small ribozymes such as twister spontaneously cleave their own RNA when the ribozyme folds into its active conformation. The coupling between twister folding and self-cleavage has been difficult to study, however, because the active ribozyme rapidly converts to product. Here, we describe the synthesis of a photocaged nucleotide that releases guanosine within microseconds upon photosolvolysis with blue light.

Quantitative Analysis of RNA Chaperone Activity by Native Gel Electrophoresis and Fluorescence Spectroscopy.

Diverse types of RNA-binding proteins chaperone the interactions of noncoding RNAs by increasing the rate of RNA base pairing and by stabilizing the final RNA duplex. The E. coli protein Hfq facilitates interactions between small noncoding RNAs and their target mRNAs.

Monitoring co-transcriptional folding of riboswitches through helicase unwinding.

In the cell, RNAs fold and begin to function as they are being transcribed. In contrast, in the laboratory, RNAs are typically studied after transcription is completed. Co-transcriptional folding can regulate the function of riboswitches and ribozymes and dictate the order of ribonucleoprotein assembly.

Mimicking Co-Transcriptional RNA Folding Using a Superhelicase.

Vectorial folding of RNA during transcription can produce intermediates with distinct biochemical activities. Here, we design an artificial minimal system to mimic cotranscriptional RNA folding in vitro. In this system, a presynthesized RNA molecule begins to fold from its 5'-end, as it is released from a heteroduplex by an engineered helicase that translocates on the complementary DNA strand in the 3'-to-5' direction.

Proteins That Chaperone RNA Regulation.

RNA-binding proteins chaperone the biological functions of noncoding RNA by reducing RNA misfolding, improving matchmaking between regulatory RNA and targets, and exerting quality control over RNP biogenesis. Recent studies of CspA, HIV NCp, and Hfq are beginning to show how RNA-binding proteins remodel RNA structures. These different protein families use common strategies for disrupting or annealing RNA double helices, which can be used to understand the mechanisms by which proteins chaperone RNA-dependent regulation in bacteria.

The Pseudomonas aeruginosa PrrF1 and PrrF2 Small Regulatory RNAs Promote 2-Alkyl-4-Quinolone Production through Redundant Regulation of the mRNA.

is an opportunistic Gram-negative pathogen that requires iron for growth and virulence. Under low-iron conditions, transcribes two highly identical (95%) small regulatory RNAs (sRNAs), PrrF1 and PrrF2, which are required for virulence in acute murine lung infection models. The PrrF sRNAs promote the production of 2-akyl-4(1)-quinolone metabolites (AQs) that mediate a range of biological activities, including quorum sensing and polymicrobial interactions.

Metals induce transient folding and activation of the twister ribozyme.

Twister is a small ribozyme present in almost all kingdoms of life that rapidly self-cleaves in variety of divalent metal ions. We used activity assays, bulk FRET and single-molecule FRET (smFRET) to understand how different metal ions promote folding and self-cleavage of the Oryza sativa twister ribozyme. Although most ribozymes require additional Mg for catalysis, twister inverts this expectation, requiring 20-30 times less Mg to self-cleave than to fold.

Arginine Patch Predicts the RNA Annealing Activity of Hfq from Gram-Negative and Gram-Positive Bacteria.

The Sm-protein Hfq facilitates interactions between small non-coding RNA (sRNA) and target mRNAs. In enteric Gram-negative bacteria, Hfq is required for sRNA regulation, and hfq deletion results in stress intolerance and reduced virulence. By contrast, the role of Hfq in Gram-positive is less established and varies among species.

Acidic Residues in the Hfq Chaperone Increase the Selectivity of sRNA Binding and Annealing.

Hfq facilitates gene regulation by small non-coding RNAs (sRNAs), thereby affecting bacterial attributes such as biofilm formation and virulence. Escherichia coli Hfq recognizes specific U-rich and AAN motifs in sRNAs and target mRNAs, after which an arginine patch on the rim promotes base pairing between their complementary sequences. In the cell, Hfq must discriminate between many similar RNAs.

Light-Triggered RNA Annealing by an RNA Chaperone.

Non-coding antisense RNAs regulate bacterial genes in response to nutrition or environmental stress, and can be engineered for artificial gene control. The RNA chaperone Hfq accelerates antisense pairing between non-coding RNAs and their mRNA targets, by a mechanism still unknown. We used a photocaged guanosine derivative in an RNA oligonucleotide to temporally control Hfq catalyzed annealing.

Fluorescence reporters for Hfq oligomerization and RNA annealing.

Fluorescence spectroscopy is a sensitive technique for detecting protein-protein, protein-RNA, and RNA-RNA interactions, requiring only nanomolar concentrations of labeled components. Fluorescence anisotropy provides information about the assembly of multi-subunit proteins, while molecular beacons provide a sensitive and quantitative reporter for base pairing between complementary RNAs. Here we present a detailed protocol for labeling Hfq protein with cyanine 3-maleimide and dansyl chloride to study the protein oligomerization and RNA binding by semi-native polyacrylamide gel electrophoresis (PAGE) and fluorescence anisotropy.

Conserved arginines on the rim of Hfq catalyze base pair formation and exchange.

The Sm-like protein Hfq is required for gene regulation by small RNAs (sRNAs) in bacteria and facilitates base pairing between sRNAs and their mRNA targets. The proximal and distal faces of the Hfq hexamer specifically bind sRNA and mRNA targets, but they do not explain how Hfq accelerates the formation and exchange of RNA base pairs. Here, we show that conserved arginines on the outer rim of the hexamer that are known to interact with sRNA bodies are required for Hfq's chaperone activity.

Hfq proximity and orientation controls RNA annealing.

Regulation of bacterial gene networks by small non-coding RNAs (sRNAs) requires base pairing with messenger RNA (mRNA) targets, which is facilitated by Hfq protein. Hfq is recruited to sRNAs and mRNAs through U-rich- and A-rich-binding sites, respectively, but their distance from the sRNA-mRNA complementary region varies widely among different genes. To determine whether distance and binding orientation affect Hfq's chaperone function, we engineered 'toy' RNAs containing strong Hfq-binding sites at defined distances from the complementary target site.

Hexamer to monomer equilibrium of E. coli Hfq in solution and its impact on RNA annealing.

The bacterial Sm-like protein Hfq forms a ring-shaped homo-hexamer that is necessary for Hfq to bind nucleic acids and to act in small noncoding RNA regulation. Using semi-native gels and fluorescence anisotropy, we show that Hfq undergoes a cooperative conformational change from monomer to hexamer around 1 μM protein, which is comparable to the in vivo concentration of Hfq and above the dissociation constant of the Hfq hexamer from many RNA substrates. Above 2 μM protein, Hfq hexamers associate in high-molecular-weight complexes.

Rapid binding and release of Hfq from ternary complexes during RNA annealing.

The Sm protein Hfq binds small non-coding RNA (sRNAs) in bacteria and facilitates their base pairing with mRNA targets. Molecular beacons and a 16 nt RNA derived from the Hfq binding site in DsrA sRNA were used to investigate how Hfq accelerates base pairing between complementary strands of RNA. Stopped-flow fluorescence experiments showed that annealing became faster with Hfq concentration but was impaired by mutations in RNA binding sites on either face of the Hfq ring or by competition with excess RNA substrate.

Effect of salt and RNA structure on annealing and strand displacement by Hfq.

The Sm-like protein Hfq promotes the association of small antisense RNAs (sRNAs) with their mRNA targets, but the mechanism of Hfq's RNA chaperone activity is unknown. To investigate RNA annealing and strand displacement by Hfq, we used oligonucleotides that mimic functional sequences within DsrA sRNA and the complementary rpoS mRNA. Hfq accelerated at least 100-fold the annealing of a fluorescently labeled molecular beacon to a 16-nt RNA.

Mechanism of protonophores-mediated induction of heat-shock response in Escherichia coli.

Background: Protonophores are the agents that dissipate the proton-motive-force (PMF) across E. coli plasma membrane. As the PMF is known to be an energy source for the translocation of membrane and periplasmic proteins after their initial syntheses in cell cytoplasm, protonophores therefore inhibit the translocation phenomenon.

Plasmid DNA binds to the core oligosaccharide domain of LPS molecules of E. coli cell surface in the CaCl2-mediated transformation process.

In the standard procedure for artificial transformation of E. coli by plasmid DNA, cellular competence for DNA uptake is developed by suspending the cells in ice-cold CaCl2 (50-100 mM). It is believed that CaCl2 helps DNA adsorption to the lipopolysaccharide (LPS) molecules on E.

How does plasmid DNA penetrate cell membranes in artificial transformation process of Escherichia coli?

Artificial transformation of Escherichia coli with plasmid DNA in presence of CaCl2 is a widely used technique in recombinant DNA technology. However, exact mechanism of DNA transfer across cell membranes is largely obscure. In this study, measurements of both steady state and time-resolved anisotropies of fluorescent dye trimethyl ammonium diphenyl hexatriene (TMA-DPH), bound to cellular outer membrane, indicated heat-pulse (0 degrees C42 degrees C) step of the standard transformation procedure had lowered considerably outer membrane fluidity of cells.

In vitro interaction between calf thymus DNA and Escherichia coli LPS in the presence of divalent cation Ca2+.

With increasing addition of Escherichia coli LPS to calf thymus DNA, both dissolved in CaCl2, absorption maxima of DNA at 260 nm decreased gradually with the appearance of isosbastic points at both ends of spectra, which implied some binding between DNA and LPS. Hill plot of absorbance data showed that the binding interaction was positive cooperative in nature. For any fixed concentration of DNA and LPS, extent of interaction increased as concentration of CaCl2 was raised from 1.

Role of membrane potential on artificial transformation of E. coli with plasmid DNA.

The standard method of transformation of Escherichea coli with plasmid DNA involves two important steps: cells are first suspended in 100mM CaCl(2) at 0 degrees C (in which DNA is added), followed by the administration of a heat-pulse from 0 to 42 degrees C for 90s [Cohen, S., Chang, A., Hsu, L.