Cis regulation within a cluster of viral microRNAs.

MicroRNAs (miRNAs) are small regulatory RNAs involved in virtually all biological processes. Although many of them are co-expressed from clusters, little is known regarding the impact of this organization on the regulation of their accumulation. In this study, we set to decipher a regulatory mechanism controlling the expression of the ten clustered pre-miRNAs from Kaposi's sarcoma associated herpesvirus (KSHV). We measured in vitro the efficiency of cleavage of each individual pre-miRNA by the Microprocessor and found that pre-miR-K1 and -K3 were the most efficiently cleaved pre-miRNAs. A mutational analysis showed that, in addition to producing mature miRNAs, they are also important for the optimal expression of the whole set of miRNAs. We showed that this feature depends on the presence of a canonical pre-miRNA at this location since we could functionally replace pre-miR-K1 by a heterologous pre-miRNA. Further in vitro processing analysis suggests that the two stem-loops act in cis and that the cluster is cleaved in a sequential manner. Finally, we exploited this characteristic of the cluster to inhibit the expression of the whole set of miRNAs by targeting the pre-miR-K1 with LNA-based antisense oligonucleotides in cells either expressing a synthetic construct or latently infected with KSHV.

Isothermal titration calorimetry in the single-injection mode with imperfect mixing.

Isothermal titration calorimetry (ITC) is now a method of choice to obtain thermodynamic information about the interaction between two molecular partners. Most often, the method in use is the so-called multiple-injection method (MIM) consisting in distinct short-time injections of the titrant separated by sufficient delay to reach equilibrium before each new injection. However, an alternative single-injection method (SIM) exists. It consists in a unique continuous injection and, despite the fact that it is quite simple and generally faster than MIM, it is very little used. The goal of this work is to reconsider its theoretical basis. A new equation taking into account the effect of dilution resulting from the continuous titration process is obtained. It allows to consider efficiently the continuum of possibilities from perfect to imperfect mixing of the cell content. It is shown that, to good approximation, imperfect mixing can be accounted for by considering the cell volume as an adjustable parameter. Most likely, this should lead to an artificial increase of it, although one cannot reject the possibility of a decrease. The processing of experimental data on the interaction of Ba++ with 18-crown-6 from led to an increase by 6.9%, which resulted in a much better fit of the titration curve and improved results on the association constant Ka and enthalpy variation ∆H. A criterion is also obtained on the maximum injection rate to be used for maintaining quasi-equilibrium during the whole titration for the association-dissociation mechanism [Formula: see text].

AFFINImeter: A software to analyze molecular recognition processes from experimental data.

The comprehension of molecular recognition phenomena demands the understanding of the energetic and kinetic processes involved. General equations valid for the thermodynamic analysis of any observable that is assessed as a function of the concentration of the involved compounds are described, together with their implementation in the AFFINImeter software. Here, a maximum of three different molecular species that can interact with each other to form an enormous variety of supramolecular complexes are considered. The corrections currently employed to take into account the effects of dilution, volume displacement, concentration errors and those due to external factors, especially in the case of ITC measurements, are included. The methods used to fit the model parameters to the experimental data, and to generate the uncertainties are described in detail. A simulation tool and the so called kinITC analysis to get kinetic information from calorimetric experiments are also presented. An example of how to take advantage of the AFFINImeter software for the global multi-temperature analysis of a system exhibiting cooperative 1:2 interactions is presented and the results are compared with data previously published. Some useful recommendations for the analysis of experiments aimed at studying molecular interactions are provided.

Multianalytical Study of the Binding between a Small Chiral Molecule and a DNA Aptamer: Evidence for Asymmetric Steric Effect upon 3'- versus 5'-End Sequence Modification.

Nucleic acid aptamers are involved in a broad field of applications ranging from therapeutics to analytics. Deciphering the binding mechanisms between aptamers and small ligands is therefore crucial to improve and optimize existing applications and to develop new ones. Particularly interesting is the enantiospecific binding mechanism involving small molecules with nonprestructured aptamers. One archetypal example is the chiral binding between l-tyrosinamide and its 49-mer aptamer for which neither structural nor mechanistic information is available. In the present work, we have taken advantage of a multiple analytical characterization strategy (i.e., using electroanalytical techniques such as kinetic rotating droplet electrochemistry, fluorescence polarization, isothermal titration calorimetry, and quartz crystal microbalance) for interpreting the nature of binding process. Screening of the binding thermodynamics and kinetics with a wide range of aptamer sequences revealed the lack of symmetry between the two ends of the 23-mer minimal binding sequence, showing an unprecedented influence of the 5' aptamer modification on the bimolecular binding rate constant kon and no significant effect on the dissociation rate constant koff. The results we have obtained lead us to conclude that the enantiospecific binding reaction occurs through an induced-fit mechanism, wherein the ligand promotes a primary nucleation binding step near the 5'-end of the aptamer followed by a directional folding of the aptamer around its target from 5'-end to 3'-end. Functionalization of the 5'-end position by a chemical label, a polydA tail, a protein, or a surface influences the kinetic/thermodynamic constants up to 2 orders of magnitude in the extreme case of a surface immobilized aptamer, while significantly weaker effect is observed for a 3'-end modification. The reason is that steric hindrance must be overcome to nucleate the binding complex in the presence of a modification near the nucleation site.

Quantitative and predictive model of kinetic regulation by E. coli TPP riboswitches.

Riboswitches are non-coding elements upstream or downstream of mRNAs that, upon binding of a specific ligand, regulate transcription and/or translation initiation in bacteria, or alternative splicing in plants and fungi. We have studied thiamine pyrophosphate (TPP) riboswitches regulating translation of thiM operon and transcription and translation of thiC operon in E. coli, and that of THIC in the plant A. thaliana. For all, we ascertained an induced-fit mechanism involving initial binding of the TPP followed by a conformational change leading to a higher-affinity complex. The experimental values obtained for all kinetic and thermodynamic parameters of TPP binding imply that the regulation by A. thaliana riboswitch is governed by mass-action law, whereas it is of kinetic nature for the two bacterial riboswitches. Kinetic regulation requires that the RNA polymerase pauses after synthesis of each riboswitch aptamer to leave time for TPP binding, but only when its concentration is sufficient. A quantitative model of regulation highlighted how the pausing time has to be linked to the kinetic rates of initial TPP binding to obtain an ON/OFF switch in the correct concentration range of TPP. We verified the existence of these pauses and the model prediction on their duration. Our analysis also led to quantitative estimates of the respective efficiency of kinetic and thermodynamic regulations, which shows that kinetically regulated riboswitches react more sharply to concentration variation of their ligand than thermodynamically regulated riboswitches. This rationalizes the interest of kinetic regulation and confirms empirical observations that were obtained by numerical simulations.

Peptide-Based Covalent Inhibitors Bearing Mild Electrophiles to Target a Conserved His Residue of the Bacterial Sliding Clamp.

Peptide-based covalent inhibitors targeted to nucleophilic protein residues have recently emerged as new modalities to target protein-protein interactions (PPIs) as they may provide some benefits over more classic competitive inhibitors. Covalent inhibitors are generally targeted to cysteine, the most intrinsically reactive amino acid residue, and to lysine, which is more abundant at the surface of proteins but much less frequently to histidine. Herein, we report the structure-guided design of targeted covalent inhibitors (TCIs) able to bind covalently and selectively to the bacterial sliding clamp (SC), by reacting with a well-conserved histidine residue located on the edge of the peptide-binding pocket. SC is an essential component of the bacterial DNA replication machinery, identified as a promising target for the development of new antibacterial compounds. Thermodynamic and kinetic analyses of ligands bearing different mild electrophilic warheads confirmed the higher efficiency of the chloroacetamide compared to Michael acceptors. Two high-resolution X-ray structures of covalent inhibitor-SC adducts were obtained, revealing the canonical orientation of the ligand and details of covalent bond formation with histidine. Proteomic studies were consistent with a selective SC engagement by the chloroacetamide-based TCI. Finally, the TCI of SC was substantially more active than the parent noncovalent inhibitor in an in vitro SC-dependent DNA synthesis assay, validating the potential of the approach to design covalent inhibitors of protein-protein interactions targeted to histidine.

Thermodynamics of HIV-1 reverse transcriptase in action elucidates the mechanism of action of non-nucleoside inhibitors.

HIV-1 reverse transcriptase (RT) is a heterodimeric enzyme that converts the genomic viral RNA into proviral DNA. Despite intensive biochemical and structural studies, direct thermodynamic data regarding RT interactions with its substrates are still lacking. Here we addressed the mechanism of action of RT and of non-nucleoside RT inhibitors (NNRTIs) by isothermal titration calorimetry (ITC). Using a new incremental-ITC approach, a step-by-step thermodynamic dissection of the RT polymerization activity showed that most of the driving force for DNA synthesis is provided by initial dNTP binding. Surprisingly, thermodynamic and kinetic data led to a reinterpretation of the mechanism of inhibition of NNRTIs. Binding of NNRTIs to preformed RT/DNA complexes is hindered by a kinetic barrier and NNRTIs mostly interact with free RT. Once formed, RT/NNRTI complexes bind DNA either in a seemingly polymerase-competent orientation or form high-affinity dead-end complexes, both RT/NNRTI/DNA complexes being unable to bind the incoming nucleotide substrate.

kinITC: a new method for obtaining joint thermodynamic and kinetic data by isothermal titration calorimetry.

Isothermal titration calorimetry (ITC) is the method of choice for obtaining thermodynamic data on a great variety of systems. Here we show that modern ITC apparatus and new processing methods allow researchers to obtain a complete kinetic description of systems more diverse than previously thought, ranging from simple ligand binding to complex RNA folding. We illustrate these new features with a simple case (HIV-1 reverse transcriptase/inhibitor interaction) and with the more complex case of the folding of a riboswitch triggered by the binding of its ligand. The originality of the new kinITC method lies in its ability to dissect, both thermodynamically and kinetically, the two components: primary ligand binding and subsequent RNA folding. We are not aware of another single method that can yield, in a simple way, such deep insight into a composite process. Our study also rationalizes common observations from daily ITC use.

Conductance fluctuations in gold point contacts: an atomistic picture.

This paper concerns an experimental and theoretical study of the transition between two consecutive conductance plateaus as obtained in breaking gold contact experiments. The experimental measurements performed at 100 K with a scanning tunneling microscope and variable elongation speeds show that the transitions between consecutive plateaus can appear in the conductance traces as an abrupt conductance step, a smooth quasicontinuous change or as large amplitude conductance fluctuations. The theoretical calculations based on a non-orthogonal tight-binding Hamiltonian have shown that for a given deformation there are several structures having close and competing energies.We discuss the relation between the temperature, sampling frequency, stretching speed and energy barriers which can explain the conditions for the observation of the three kinds of conductance traces.

Sequence and structure requirements for specific recognition of HIV-1 TAR and DIS RNA by the HIV-1 Vif protein.

The HIV-1 Vif protein plays an essential role in the regulation of the infectivity of HIV-1 virion and in vivo pathogenesis. Vif neutralizes the human DNA-editing enzyme APOBEC3 protein, an antiretroviral cellular factor from the innate immune system, allowing the virus to escape the host defence system. It was shown that Vif is packaged into viral particles through specific interactions with the viral genomic RNA. Conserved and structured sequences from the 5'-noncoding region, such as the Tat-responsive element (TAR) or the genomic RNA dimerization initiation site (DIS), are primary binding sites for Vif. In the present study we used isothermal titration calorimetry to investigate sequence and structure determinants important for Vif binding to short viral RNA corresponding to TAR and DIS stem-loops. We showed that Vif specifically binds TAR and DIS in the low nanomolar range. In addition, Vif primarily binds the TAR UCU bulge, but not the apical loop. Determinants for Vif binding to the DIS loop-loop complex are likely more complex and involve the self-complementary loop together with the upper part of the stem. These results suggest that Tat-TAR inhibitors or DIS small molecule binders might be also effective to disturb Vif-TAR and Vif-DIS binding in order to reduce Vif packaging into virions.

Cation-dependent cleavage of the duplex form of the subtype-B HIV-1 RNA dimerization initiation site.

The crystal structure of subtype-B HIV-1 genomic RNA Dimerization Initiation Site duplex revealed chain cleavage at a specific position resulting in 3'-phosphate and 5'-hydroxyl termini. A crystallographic analysis showed that Ba(2+), Mn(2+), Co(2+) and Zn(2+) bind specifically on a guanine base close to the cleaved position. The crystal structures also point to a necessary conformational change to induce an 'in-line' geometry at the cleavage site. In solution, divalent cations increased the rate of cleavage with pH/pKa compensation, indicating that a cation-bound hydroxide anion is responsible for the cleavage. We propose a 'Trojan horse' mechanism, possibly of general interest, wherein a doubly charged cation hosted near the cleavage site as a 'harmless' species is further transformed in situ into an 'aggressive' species carrying a hydroxide anion.

Unsupervised bubble calorimetry analysis: Surface tension from isothermal titration calorimetry.

HYPOTHESIS: The injection of air into the sample cell of an isothermal titration calorimeter containing a liquid provides a rich-in-information signal, with a periodic contribution arising from the creation, growing and release of bubbles. The identification and analysis of such contributions allow the accurate determination of the surface tension of the target liquid. EXPERIMENTS: Air is introduced at a constant rate into the sample cell of the calorimeter containing either a pure liquid or a solution. The resulting calorimetric signal is analyzed by a new algorithm, which is implemented into a computational code. FINDINGS: The thermal power generated by our experiments is often noisy, thus hiding the periodic signal arising from the bubbles' formation and release. The new algorithm was tested with a range of different types of calorimetric raw data, some of them apparently being just noise. In all cases, the contribution of the bubbles to the signal was isolated and the corresponding period was successfully determined in an automated way. It is also shown that two reference measurements suffice to calibrate the instrument at a given temperature, regardless the injection rate, allowing the direct determination of surface tension values for the liquid contained in the sample cell.

Transcription factors modulate RNA polymerase conformational equilibrium.

RNA polymerase (RNAP) frequently pauses during the transcription of DNA to RNA to regulate gene expression. Transcription factors NusA and NusG modulate pausing, have opposing roles, but can bind RNAP simultaneously. Here we report cryo-EM reconstructions of Escherichia coli RNAP bound to NusG, or NusA, or both. RNAP conformational changes, referred to as swivelling, correlate with transcriptional pausing. NusA facilitates RNAP swivelling to further increase pausing, while NusG counteracts this role. Their structural effects are consistent with biochemical results on two categories of transcriptional pauses. In addition, the structures suggest a cooperative mechanism of NusA and NusG during Rho-mediated transcription termination. Our results provide a structural rationale for the stochastic nature of pausing and termination and how NusA and NusG can modulate it.

A fast selenium derivatization strategy for crystallization and phasing of RNA structures.

Site-specific 2'-methylseleno RNA labeling is a promising tool for tackling the phase problem in RNA crystallography. We have developed an efficient strategy for crystallization and structure determination of RNA and RNA/protein complexes based on preliminary crystallization screening of 2'-OCH(3)-modified RNA sequences, prior to the replacement of 2'-OCH(3) groups with their 2'-SeCH(3) counterparts. The method exploits the similar crystallization properties of 2'-OCH(3)- and 2'-SeCH(3)-modified RNAs and has been successfully validated for two test cases. In addition, our data show that 2'-SeCH(3)-modified RNA have an increased resistance to X-ray radiolysis in comparison with commonly used 5-halogen-modified RNA, which permits collection of experimental electron density maps of remarkable quality.

Fluid interface calorimetry.

HYPOTHESIS: Amphiphilic molecules spontaneously adsorb to fluid polar-nonpolar interfaces. The timescale of such adsorption depends on the molecular size and structure of the solute. This process should be accompanied by a power heat exchange that could be detected by commercial isothermal calorimeters. EXPERIMENTS: Air is injected in the bulk of different aqueous solutions contained in the sample cell of an isothermal titration calorimeter. The formation of the resulting bubbles leads to a liquid/air interface to which the solute molecules spontaneously adsorb. Continuous injection experiments to produce multiple bubbles as well as experiments with static bubbles stand from the capillary tip, aiming to observe slow adsorption processes, were performed. FINDINGS: The power associated with the formation, growth and release of air bubbles in different liquids was measured. Different independent contributions that can be associated to the pressure change in the gas phase, the evaporation-condensation of the solvent, the increase of interfacial area, the change in the heat capacity of the sample cell content, and the release of the bubble were observed. The periodic pattern produced by the continuous injection of air at a constant rate is used to determine the surface tension of different liquids, including solutions of different molecules and (bio)macromolecules.

Aminoglycoside binding to the HIV-1 RNA dimerization initiation site: thermodynamics and effect on the kissing-loop to duplex conversion.

Owing to a striking, and most likely fortuitous, structural and sequence similarity with the bacterial 16 S ribosomal A site, the RNA kissing-loop complex formed by the HIV-1 genomic RNA dimerization initiation site (DIS) specifically binds 4,5-disubstituted 2-deoxystreptamine (2-DOS) aminoglycoside antibiotics. We used chemical probing, molecular modeling, isothermal titration calorimetry (ITC) and UV melting to investigate aminoglycoside binding to the DIS loop-loop complex. We showed that apramycin, an aminoglycoside containing a bicyclic moiety, also binds the DIS, but in a different way than 4,5-disubstituted 2-DOS aminoglycosides. The determination of thermodynamic parameters for various aminoglycosides revealed the role of the different rings in the drug-RNA interaction. Surprisingly, we found that the affinity of lividomycin and neomycin for the DIS (K(d) approximately 30 nM) is significantly higher than that obtained in the same experimental conditions for their natural target, the bacterial A site (K(d) approximately 1.6 microM). In good agreement with their respective affinity, aminoglycoside increase the melting temperature of the loop-loop interaction and also block the conversion from kissing-loop complex to extended duplex. Taken together, our data might be useful for selecting new molecules with improved specificity and affinity toward the HIV-1 DIS RNA.

Thermodynamics of Molecular Machines Using Incremental ITC.

Molecular biomachines, such as DNA and RNA polymerases or the ribosome, are fascinating biological assemblies able to swiftly perform repeated and highly regulated tasks, with a remarkable accuracy. Significant advances in structural studies during the past 20 years provided a wealth of information regarding their architecture and considerably contributed to a better understanding of their mechanism of action. However, the three-dimensional structure of a biological nanomachine alone does not provide access to its detailed mechanism of action, even when obtained at atomic resolution. When combined with other biophysical approaches, thermodynamic data, together with kinetic data, are essential for a complete description of any binding interaction, revealing forces driving complex formation and providing insights into mechanisms of action. We have developed an incremental ITC approach that is well-suitable for analysis of biomolecular machines. This strategy allows a dissection of molecular biomachine reactions through successive additions in the ITC cell of consecutive substrates.

Thermodynamic and Kinetic Analysis of Isothermal Titration Calorimetry Experiments by Using KinITC in AFFINImeter.

Standard molecular binding isothermal titration calorimetric (ITC) experiments are designed to get thermodynamic information: changes in Gibbs energy, enthalpy, and entropy associated to the studied process. Traditionally, the kinetic information contained in the ITC raw signal has been ignored. For a usual one-step process, this corresponds to the rate constants for the association and the dissociation of the complex (kon and koff). The availability of highly sensitive ITC instruments with low response time, together with the development of theoretical methods and of public software for the proper analysis of the signal, cancels any reason for not retrieving this kinetic information. Here we describe how to further exploit ITC experiments of simple one-step interactions by using the software AFFINImeter.The method is exemplified using a standard reference system for thermodynamic and kinetic molecular binding analysis: the interaction of carbonic anhydrase (CA) with its inhibitor 4-carboxybenzenesulfonamide (4-CBS) at several temperatures. It is to be emphasized that old experiments initially designed and executed just for thermodynamic analysis can be readily recycled by using AFFINImeter to retrieve the previously ignored kinetic information.

Interaction of a Model Peptide on Gram Negative and Gram Positive Bacterial Sliding Clamps.

Bacterial sliding clamps control the access of DNA polymerases to the replication fork and are appealing targets for antibacterial drug development. It is therefore essential to decipher the polymerase-clamp binding mode across various bacterial species. Here, two residues of the E. coli clamp binding pocket, EcS346 and EcM362, and their cognate residues in M. tuberculosis and B. subtilis clamps, were mutated. The effects of these mutations on the interaction of a model peptide with these variant clamps were evaluated by thermodynamic, molecular dynamics, X-rays crystallography, and biochemical analyses. EcM362 and corresponding residues in Gram positive clamps occupy a strategic position where a mobile residue is essential for an efficient peptide interaction. EcS346 has a more subtle function that modulates the pocket folding dynamics, while the equivalent residue in B. subtilis is essential for polymerase activity and might therefore be a Gram positive-specific molecular marker. Finally, the peptide binds through an induced-fit process to Gram negative and positive pockets, but the complex stability varies according to a pocket-specific network of interactions.