Protein-nucleic Acid Interactions Research Articles

Flipping by DNA Bound Proteins Occurs through Rapid Rebinding

Flipping transitions are rapid rearrangements of protein bound to nucleic acid substrates. Flipping allows an enzyme to switch to a complimentary strand in a nucleic acid duplex and thereby facilitates the search for specific targets in a DNA or RNA sequence. However, in previous studies it has not been clear how a protein is able to rapidly flip its orientation. Here we have chosen HIV-1 reverse transcriptase as a model system to probe the mechanism of flipping. We directly observed flipping transitions on DNA using single-molecule FRET and we studied the effects of varied ionic strength and macromolecular crowding of the buffer to alter the strength and the range of protein-nucleic acid interactions. Our single molecule results reveal that the increased ionic strength of the buffer weakens the binding of RT to DNA, but increased macromolecular crowding strengthened the binding affinity.With a high spatio-temporal resolution (2nm and 100 msec, respectively) of single molecule FRET, we analysed the flipping kinetics such as rate of flipping and probability of flipping at each ionic and crowding concentration. Based on the kinetic data, we derived two possible models, “tumbling mechanism” and “hopping mechanism”. Interpreting the data using the tumbling mechanism forces us to adopt an unphysical transition state, while the hopping mechanism characterized by rapid rebinding from a pseudo-bound intermediate state predicted the results correctly. Our data support a view that proteins that bound on DNA undergo many rapid re-bindings, allowing the macromolecules to reorient themselves in different configurations and engage in different catalytic activities before complete dissociation.

Removing nucleic acids from nucleoid-associated proteins purified by affinity column.

In bacteria, DNA is tightly compacted in a supercoiled organization, which is mediated in part by nucleoid-associated proteins (NAPs). NAPs are well characterized for their ability to bind nucleic acids and for their involvement in gene regulation. A method commonly used to study protein-nucleic acid interactions involves immunoprecipitation of the protein of interest which is subsequently incubated with nucleic acids. A common cause of artifact is due to nucleic acids that remains bound to the protein of interest during the whole purification process. We developed an optimized method for the purification of tagged NAPs on affinity columns. The combination of three known methods allows removal of most of the nucleic acids bound to proteins during the purification process. This protocol is designed to improve the quality and specificity of results of in vitro experiments involving nucleic acid binding tests on purified NAPs. It can be used for in vitro studies of other RNA/DNA binding proteins.

Single-Molecule Imaging With One Color Fluorescence.

Single-molecule fluorescence imaging is a powerful tool that enables real-time observation of DNA-protein or RNA-protein interactions with a nanometer precision. Here, we provide a detailed procedure for a previously developed single-molecule fluorescence method, termed "single-molecule protein-induced fluorescence enhancement" (smPIFE). While smFRET (Förster resonance energy transfer) requires both donor and acceptor, protein-induced fluorescence enhancement (PIFE) employs a single dye and measures the increase in fluorescence intensity induced by protein binding near the dye. PIFE displays distance sensitivity within 0-4nm, making it a powerful complementary or alternative tool to FRET method. In this chapter, we will discuss the various ways that PIFE has been utilized to study protein-nucleic acid interactions.

Cyanotryptophans as Novel Fluorescent Probes for Studying Protein Conformational Changes and DNA-Protein Interaction.

Described herein are the syntheses and photophysical characterization of three novel cyanotryptophans, and their efficient incorporation into proteins as fluorescent probes. Photophysical characteristics indicated that each was significantly brighter and red-shifted in fluorescence emission relative to tryptophan. Each analogue was used to activate a suppressor tRNA transcript and was incorporated with good efficiency into two different positions (Trp22 and Trp74) of Escherichia coli dihydrofolate reductase (ecDHFR). The Trp analogues could be monitored selectively in the presence of multiple native Trp residues in DHFR. 6-CNTrp (A) formed an efficient Förster resonance energy transfer (FRET) pair with l-(7-hydroxycoumarin-4-yl)ethylglycine (HCO, D) at position 17. Further, 6-CNTrp (A) was incorporated into two DNA binding proteins, including the Klenow fragment of DNA polymerase I and an RNA recognition motif (RRM2) of heterogeneous nuclear ribonucleoprotein L-like (hnRNP LL). Using these proteins, we demonstrated the use of FRET involving A as a fluorescence donor and benzo[g]quinazoline-2,4-(1H,3H)-dione 2'-deoxyriboside (Tf) or 4-aminobenzo[g]quinazoline-2-one 2'-deoxyriboside (Cf) as fluorescent acceptors to study the binding interaction of the Klenow fragment with duplex DNA oligomers (labeled with Tf), or the domain-specific association between hnRNP LL and the BCL2 i-motif DNA (labeled with Cf). Thus, the non-natural amino acid could be used as a FRET partner for studying protein-nucleic acid interactions. Together, these findings demonstrate the potential utility of 6-CNTrp (A) as a fluorescence donor for the study of protein conformational events.

Structural Basis for Dimerization and DNA Binding of Transcription Factor FLI1.

FLI1 (Friend leukemia integration 1) is a metazoan transcription factor that is upregulated in a number of cancers. In addition, rearrangements of the fli1 gene cause sarcomas, leukemias, and lymphomas. These rearrangements encode oncogenic transcription factors, in which the DNA binding domain (DBD or ETS domain) of FLI1 on the C-terminal side is fused to a part of an another protein on the N-terminal side. Such abnormal cancer cell-specific fusions retain the DNA binding properties of FLI1 and acquire non-native protein-protein or protein-nucleic acid interactions of the substituted region. As a result, these fusions trigger oncogenic transcriptional reprogramming of the host cell. Interactions of FLI1 fusions with other proteins and with itself play a critical role in the oncogenic regulatory functions, and they are currently under intense scrutiny, mechanistically and as potential novel anticancer drug targets. We report elusive crystal structures of the FLI1 DBD, alone and in complex with cognate DNA containing a GGAA recognition sequence. Both structures reveal a previously unrecognized dimer of this domain, consistent with its dimerization in solution. The homodimerization interface is helix-swapped and dominated by hydrophobic interactions, including those between two interlocking Phe362 residues. A mutation of Phe362 to an alanine disrupted the propensity of this domain to dimerize without perturbing its structure or the DNA binding function, consistent with the structural observations. We propose that FLI1 DBD dimerization plays a role in transcriptional activation and repression by FLI1 and its fusions at promoters containing multiple FLI1 binding sites.

An updated version of NPIDB includes new classifications of DNA-protein complexes and their families.

The recent upgrade of nucleic acid–protein interaction database (NPIDB, http://npidb.belozersky.msu.ru/) includes a newly elaborated classification of complexes of protein domains with double-stranded DNA and a classification of families of related complexes. Our classifications are based on contacting structural elements of both DNA: the major groove, the minor groove and the backbone; and protein: helices, beta-strands and unstructured segments. We took into account both hydrogen bonds and hydrophobic interaction. The analyzed material contains 1942 structures of protein domains from 748 PDB entries. We have identified 97 interaction modes of individual protein domain–DNA complexes and 17 DNA–protein interaction classes of protein domain families. We analyzed the sources of diversity of DNA–protein interaction modes in different complexes of one protein domain family. The observed interaction mode is sometimes influenced by artifacts of crystallization or diversity in secondary structure assignment. The interaction classes of domain families are more stable and thus possess more biological sense than a classification of single complexes. Integration of the classification into NPIDB allows the user to browse the database according to the interacting structural elements of DNA and protein molecules. For each family, we present average DNA shape parameters in contact zones with domains of the family.

Translation elongation factor eEF1A1 is a novel partner of a multifunctional protein Sgt1.

Mammalian translation elongation factor eEF1A is involved in ribosomal polypeptide synthesis. Also, the protein fulfills many additional duties in an eukaryotic cell. Here, we identified a novel partner of the eEF1A1 isoform, namely Sgt1, a protein that possesses co-chaperon properties and participates in antiviral defense processes. By applying different methods, we demonstrated the interaction between eEF1A1 and Sgt1 using both purified proteins and cell lysates. We also found that the D2 and D3 domains of eEF1A1 and the TPR domain of Sgt1 are involved in complex formation. Modeling of the Sgt1-eEF1A1 complex suggested both shape and charge complementarities of the eEF1A1-Sgt1 interface stabilized by a number of salt bridges. As long as such interaction mode is typical more for protein-nucleic acid interaction we suggested a possibility that Sgt1 competes with viral RNA for binding to eEF1A and obtained invitro evidence to this effect.

RNA structure: merging chemistry and genomics for a holistic perspective.

The advent of deep sequencing technology has unexpectedly advanced our structural understanding of molecules composed of nucleic acids. A significant amount of progress has been made recently extrapolating the chemical methods to probe RNA structure into sequencing methods. Herein we review some of the canonical methods to analyze RNA structure, and then we outline how these have been used to probe the structure of many RNAs in parallel. The key is the transformation of structural biology problems into sequencing problems, whereby sequencing power can be interpreted to understand nucleic acid proximity, nucleic acid conformation, or nucleic acid-protein interactions. Utilizing such technologies in this way has the promise to provide novel structural insights into the mechanisms that control normal cellular physiology and provide insight into how structure could be perturbed in disease.

Paraformaldehyde Fixation May Lead to Misinterpretation of the Subcellular Localization of Plant High Mobility Group Box Proteins.

Arabidopsis High Mobility Group Box (HMBG) proteins were previously found associated with the interphase chromatin but not the metaphase chromosome. However, these studies are usually based on immunolocalization analysis involving paraformaldehyde fixation. Paraformaldehyde fixation has been widely adapted to preserved cell morphology before immunofluorescence staining. On one hand, the processed cells are no longer living. On the other hand, the processing may lead to misinterpretation of localization. HMGBs from Arabidopsis were fused with enhanced green fluorescence protein (EGFP) and transformed into tobacco BY-2 cells. Basically, the localization of these HMGB proteins detected with EGFP fluorescence in interphase agreed with previous publications. Upon 4% paraformaldehyde fixation, AtHMGB1 was found associated with interphase but not the metaphase chromosomes as previously reported. However, when EGFP fluorescence signal was directly observed under confocal microscope without fixation, association of AtHMGB1 with metaphase chromosomes can be detected. Paraformaldehyde fixation led to dissociation of EGFP tagged AtHMBG1 protein from metaphase chromosomes. This kind of pre-processing of live specimen may lead to dissociation of protein-protein or protein-nucleic acid interaction. Therefore, using of EGFP fusion proteins in live specimen is a better way to determine the correct localization and interaction of proteins.

Evolution of Macromolecular Docking Techniques: The Case Study of Nickel and Iron Metabolism in Pathogenic Bacteria.

The interaction between macromolecules is a fundamental aspect of most biological processes. The computational techniques used to study protein-protein and protein-nucleic acid interactions have evolved in the last few years because of the development of new algorithms that allow the a priori incorporation, in the docking process, of experimentally derived information, together with the possibility of accounting for the flexibility of the interacting molecules. Here we review the results and the evolution of the techniques used to study the interaction between metallo-proteins and DNA operators, all involved in the nickel and iron metabolism of pathogenic bacteria, focusing in particular on Helicobacter pylori (Hp). In the first part of the article we discuss the methods used to calculate the structure of complexes of proteins involved in the activation of the nickel-dependent enzyme urease. In the second part of the article, we concentrate on two applications of protein-DNA docking conducted on the transcription factors HpFur (ferric uptake regulator) and HpNikR (nickel regulator). In both cases we discuss the technical expedients used to take into account the conformational variability of the multi-domain proteins involved in the calculations.

Bioconjugation of Oligodeoxynucleotides Carrying 1,4-Dicarbonyl Groups via Reductive Amination with Lysine Residues.

We evaluated the efficacy of bioconjugation of oligodeoxynucleotides (ODNs) containing 1,4-dicarbonyl groups, a C4'-oxidized abasic site (OAS), and a newly designed 2'-methoxy analogue, via reductive amination with lysine residues. Dicarbonyls, aldehyde and ketone at C1- and C4-positions of deoxyribose in the ring-opened form of OAS allowed efficient reaction with amines. Kinetic studies indicated that reductive amination of OAS-containing ODNs with a proximal amine on the complementary strand proceeded 10 times faster than the corresponding reaction of an ODN containing an abasic site with C1-aldehyde. Efficient reductive amination between the DNA-binding domain of Escherichia coli DnaA protein and ODNs carrying OAS in the DnaA-binding sequence proceeded at the lysine residue in proximity to the phosphate group at the 5'-position of the OAS, in contrast to unsuccessful conjugation with abasic site ODNs, even though they have similar aldehydes. Theoretical calculation indicated that the C1-aldehyde of OAS was more accessible to the target lysine than that of the abasic site. These results demonstrate the potential utility of cross-linking strategies that use dicarbonyl-containing ODNs for the study of protein-nucleic acid interactions. Conjugation with a lysine-containing peptide that lacked specific affinity for ODN was also successful, further highlighting the advantages of 1,4-dicarbonyls.

Abstract 5120: Using flow-proteometric platform to analyze individual signaling complexes in tumor tissue

Abstract Cellular signaling complexes, which are made up of mostly proteins and nucleic acids, play a major role in signal transduction by carrying and delivering messages that coordinate basic biological functions. These processes are mainly relayed through protein-protein and protein-nucleic acid interactions. Delineating the underlying mechanisms of cancer signal transduction, which is often deregulated, relies highly on the analysis of specific protein-protein and protein-nucleic acid interactions. Conventional methods for detecting protein-protein and protein-nucleic acid interactions mostly rely on cells collected from two-dimensional cell cultures, which are very different from the natural cellular environment in organs or tissues. Therefore, we developed a multiplex flow-proteometric platform that can analyze individual signaling complexes directly from tissue to enable us to accurately acquire the information on in vivo signal transduction. To demonstrate that single signaling complexes can be directly detected in lysates from fluorescent-labeled frozen tissue sections, we selected STAT3, p300, and genomic DNA as targets to detect and quantify individual complexes in xenograft tumor tissues. Endogenous STAT3 and p300 in frozen tumor tissue were immunolabeled with A488 and QD605, respectively, and with TOTO3 to label genomic DNA. All the detected events were presented in a 3D fluorescence plot, revealing seven different types of events. A minimal number of non-specific control IgG events were detected, and no interactions events were observed, indicating low background noise. Among all of the detected STAT3 events, on average 7.04% interacted with both p300 and genomic DNA in the same complex, 2.99% interacted with p300 only, and 15.23% interacted with DNA only. Thus, in the frozen xenograft tissue section, around 22% of STAT3 (15.23% STAT3−DNA + 7.04% STAT3−p300 −DNA) interacted with DNA and 7.04% of STAT3 interacted with p300 when bound on DNA to activate gene transcription. Looking at the data from the standpoint of p300, on average 5.88% of total p300 protein molecules interacted with STAT3 and genomic DNA, 2.5% with STAT3 only, and 3.06% with DNA only. All these data simultaneously obtained in each experiment not only identified STAT3, p300, and DNA in a single complex but also quantified the distribution of lone proteins and population complexes, which cannot be analyzed by conventional methods. Thus, we expect that this technique may reveal new aspects of molecular interaction in tissue. Citation Format: Chao-Kai Chou, Heng-Huan Lee, Pei-Hsiang Tsou, Chun-Te Chen, Jung-Mao Hsu, Hirohito Yamaguchi, Ying-Nai Wang, Jennifer L. Hsu, Jin-Fong Lee, Jun Kameoka, Mien-Chie Hung. Using flow-proteometric platform to analyze individual signaling complexes in tumor tissue. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5120. doi:10.1158/1538-7445.AM2015-5120

SNBRFinder: A Sequence-Based Hybrid Algorithm for Enhanced Prediction of Nucleic Acid-Binding Residues.

Protein-nucleic acid interactions are central to various fundamental biological processes. Automated methods capable of reliably identifying DNA- and RNA-binding residues in protein sequence are assuming ever-increasing importance. The majority of current algorithms rely on feature-based prediction, but their accuracy remains to be further improved. Here we propose a sequence-based hybrid algorithm SNBRFinder (Sequence-based Nucleic acid-Binding Residue Finder) by merging a feature predictor SNBRFinderF and a template predictor SNBRFinderT. SNBRFinderF was established using the support vector machine whose inputs include sequence profile and other complementary sequence descriptors, while SNBRFinderT was implemented with the sequence alignment algorithm based on profile hidden Markov models to capture the weakly homologous template of query sequence. Experimental results show that SNBRFinderF was clearly superior to the commonly used sequence profile-based predictor and SNBRFinderT can achieve comparable performance to the structure-based template methods. Leveraging the complementary relationship between these two predictors, SNBRFinder reasonably improved the performance of both DNA- and RNA-binding residue predictions. More importantly, the sequence-based hybrid prediction reached competitive performance relative to our previous structure-based counterpart. Our extensive and stringent comparisons show that SNBRFinder has obvious advantages over the existing sequence-based prediction algorithms. The value of our algorithm is highlighted by establishing an easy-to-use web server that is freely accessible at http://ibi.hzau.edu.cn/SNBRFinder.

Cooperativity in biological systems

Living organisms can sense and respond to external and internal stimuli. Response isdemonstrated in many forms including modulation of gene expression profiles, motility,secretion, cell death, etc. Nevertheless, all forms share a basic property: they depend on sensingsmall changes in the concentration of an effector molecule or subtle conformational changes ina protein and invoking the appropriate molecular response by the relevant signaling pathways.Sensing, transduction, and response to signals may be directly carried out by controlled changesin the conformation or the assembly of pre-existing components(1,2)or may involve changes ingene expression patterns (as in cell differentiation and development), which in turn is carriedout by protein-nucleic acid interactions and complex formation. Hence, understandingconformational changes in proteins and nucleic acids, ligand binding, and complex formationplay acentral role in advancing our knowledge of cellular dynamics. Large-scale interactionmapping projects continue to provide detailed (though approximate) interaction networksbetween pairs of proteins (3–6), but fall short of capturing the stability or dynamics of theinteractions. Integration of these maps with thermodynamic and kinetic information aboutconformational changes and binding events in proteins and nucleic acids holds the promise ofdiscovering simple universal mechanisms that explain and relate seemingly disparate biologicalphenomena at many levels of complexity. In this article, I will explore ‘cooperativity’, one ofthe most ubiquitous features in molecular biology and discuss how it impacts macromolecularfolding, complex assembly, formation of biological networks, and eventually cellular functionand pathology.

Solvent accessible surface area-based hot-spot detection methods for protein-protein and protein-nucleic acid interfaces.

Due to the importance of hot-spots (HS) detection and the efficiency of computational methodologies, several HS detecting approaches have been developed. The current paper presents new models to predict HS for protein-protein and protein-nucleic acid interactions with better statistics compared with the ones currently reported in literature. These models are based on solvent accessible surface area (SASA) and genetic conservation features subjected to simple Bayes networks (protein-protein systems) and a more complex multi-objective genetic algorithm-support vector machine algorithms (protein-nucleic acid systems). The best models for these interactions have been implemented in two free Web tools.

My 65 years in protein chemistry.

This is a tour of a physical chemist through 65 years of protein chemistry from the time when emphasis was placed on the determination of the size and shape of the protein molecule as a colloidal particle, with an early breakthrough by James Sumner, followed by Linus Pauling and Fred Sanger, that a protein was a real molecule, albeit a macromolecule. It deals with the recognition of the nature and importance of hydrogen bonds and hydrophobic interactions in determining the structure, properties, and biological function of proteins until the present acquisition of an understanding of the structure, thermodynamics, and folding pathways from a linear array of amino acids to a biological entity. Along the way, with a combination of experiment and theoretical interpretation, a mechanism was elucidated for the thrombin-induced conversion of fibrinogen to a fibrin blood clot and for the oxidative-folding pathways of ribonuclease A. Before the atomic structure of a protein molecule was determined by x-ray diffraction or nuclear magnetic resonance spectroscopy, experimental studies of the fundamental interactions underlying protein structure led to several distance constraints which motivated the theoretical approach to determine protein structure, and culminated in the Empirical Conformational Energy Program for Peptides (ECEPP), an all-atom force field, with which the structures of fibrous collagen-like proteins and the 46-residue globular staphylococcal protein A were determined. To undertake the study of larger globular proteins, a physics-based coarse-grained UNited-RESidue (UNRES) force field was developed, and applied to the protein-folding problem in terms of structure, thermodynamics, dynamics, and folding pathways. Initially, single-chain and, ultimately, multiple-chain proteins were examined, and the methodology was extended to protein-protein interactions and to nucleic acids and to protein-nucleic acid interactions. The ultimate results led to an understanding of a variety of biological processes underlying natural and disease phenomena.

Synthesis of Fluorescently‐Labeled Tobacco Etch Virus (TEV) RNA and Its Interactions with Pokeweed Antiviral Protein (PAP)

Fluorescence spectroscopy has been extensively used to investigate protein‐protein and protein‐nucleic acid interactions. Changes in fluorescence intensity provide vital information about these macromolecular interactions. Availability of extrinsic fluorophores became invaluable for researchers monitoring these interactions. Here, we successfully synthesize a fluorescent anthranioyl‐m7GpppG‐capped tobacco etch virus (TEV) RNA, and examine its properties pertaining to interaction with pokeweed antiviral protein (PAP). PAP is a ribosome inactivating protein (RIP) and is an RNA N‐glycosidase that removes purines from the sarcin/ricin loop of large rRNA. This leads to the translational termination. PAP possesses antiviral properties, and it is thought to bind to the 5'‐cap of viral RNA and depurinate it down the stream of the cap. RIPs are thought to play an important role in plant defense mechanisms. We employ fluorescence spectroscopy and HPLC techniques to investigate and characterize Ant‐m7GpppG‐capped TEV RNA with PAP.

A Non-Invasive NMR Method Based on Histidine Imidazoles to Analyze the pH-Modulation of Protein-Nucleic Acid Interfaces.

A useful (2) J(N-H) coupling-based NMR spectroscopic approach is proposed to unveil, at the molecular level, the contribution of the imidazole groups of histidines from RNA/DNA-binding proteins on the modulation of binding to nucleic acids by pH. Such protonation/deprotonation events have been monitored on the single His96 located at the second RNA/DNA recognition motif (RRM2) of T-cell intracellular antigen-1 (TIA-1) protein. The pKa values of the His96 ionizable groups were substantially higher in the complexes with short U-rich RNA and T-rich DNA oligonucleotides than those of the isolated TIA-1 RRM2. Herein, the methodology applied to determine changes in pKa of histidine side chains upon DNA/RNA binding, gives valuable information to understand the pH effect on multidomain DNA/RNA-binding proteins that shuttle among different cellular compartments.

Mass Spectrometric Determination of Zn<sup>2+</sup> Binding/Dissociation Constant for Zinc Finger Peptides

Abstract : In the present study, we proposed a simple ESI-MS model for determining Zn 2+ binding (or dissociation) constants forzinc finger peptides (ZFPs) with a unique ββα fold consensus. The ionization efficiency (response) factors for this model, i.e., α andβ, could be determined for ZiCo ZFP with a known Zn 2+ binding constant. We could determine the binding constants for other ZFPsassuming those with a ββα consensus conformation have the same α/β response ratio. In general, the ZPF dissociation constantsexhibited K d values of 10 -7 ~10 -9 M, while K d values for a negative control non-specific Zn 2+ peptides were high, e.g., 5.5 ×10 -6 M and4.3×10 -4 M for BBA1 and melittin, respectively.Key words : zinc finger, binding constant, electrospray-mass spectrometry, zinc ion Introduction Since its discovery in transcription factor IIIA (TFIIIA)from Xenopuslaevis, zinc finger proteins (ZFPs) have beenextensively researched due to their implications foreukaryotic protein-nucleic acid interactions.

NuProPlot: nucleic acid and protein interaction analysis and plotting program.

Growing numbers of protein and nucleic acid complex structures are being determined and deposited in the Protein Data Bank and the Nucleic Acid Database. With the increasing complexity of these structures, it is challenging to analyse and visualize the three-dimensional interactions. The currently available programs for such analysis and visualization are limited in their applications. They can only analyse a subset of protein-nucleic acid complexes and require multiple iterations before obtaining plots that are suitable for presentation. An interactive web-based program, NuProPlot (http://www.nuproplot.com), has been developed which can automatically identify hydrogen, electrostatic and van der Waals interactions between proteins and nucleic acids and generate a plot showing all of the interactions. Protein-DNA and protein-RNA interactions can be visualized in simple two-dimensional schematics. Interactive schematic drawing options allow selection of the plotted area and repositioning of the individual interactions for better legibility. NuProPlot is a fully automated and user-friendly program providing various custom options. NuProPlot represents a greatly improved option for analysis and presentation of protein-nucleic acid interactions.