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The genome of Escherichia coli K-12 is transcribed by a single species of RNA polymerase. The selectivity of transcriptional targets is determined via interaction with one of seven species of the sigma subunit and a total of approximately 300 species of transcription factor (TFs). For comprehensive identification of the regulatory targets of these two groups of regulatory proteins on the genome, we developed an in vitro approach, "Genomic SELEX" (gSELEX) screening. Here we describe a detailed protocol of the gSELEX screening system, which uses purified regulatory proteins and fragments of genomic DNA from E. coli. Moreover, we describe methods and examples of results using cell-free synthetic proteins.

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Viruses exploit the host cell machinery to enable infection and propagation. This review discusses the complex landscape of DNA virus-host interactions, focusing primarily on herpesviruses and adenoviruses, which replicate in the nucleus of infected cells, and vaccinia virus, which replicates in the cytoplasm. We discuss experimental approaches used to discover and validate interactions of host proteins with viral genomes and how these interactions impact processes that occur during infection, including the host DNA damage response and viral genome replication, repair, and transcription. We highlight the current state of knowledge regarding virus-host protein interactions and also outline emerging areas and future directions for research.

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In Escherichia coli, the master transcription regulator catabolite repressor activator (Cra) regulates >100 genes in central metabolism. Cra binding to DNA is allosterically regulated by binding to fructose-1-phosphate (F-1-P), but the only documented source of F-1-P is from the concurrent import and phosphorylation of exogenous fructose. Thus, many have proposed that fructose-1,6-bisphosphate (F-1,6-BP) is also a physiological regulatory ligand. However, the role of F-1,6-BP has been widely debated. Here, we report that the E. coli enzyme fructose-1-kinase (FruK) can carry out its "reverse" reaction under physiological substrate concentrations to generate F-1-P from F-1,6-BP. We further show that FruK directly binds Cra with nanomolar affinity and forms higher order, heterocomplexes. Growth assays with a ΔfruK strain and fruK complementation show that FruK has a broader role in metabolism than fructose catabolism. Since fruK itself is repressed by Cra, these newly-reported events add layers to the dynamic regulation of E. coli's central metabolism that occur in response to changing nutrients. These findings might have wide-spread relevance to other γ-proteobacteria, which conserve both Cra and FruK.

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BACKGROUND: The electrophoretic mobility shift assay (EMSA) is a common technology to detect DNA-protein interactions. However, in most cases, the protein used in EMSA is obtained via prokaryotic expression, and rarely from plants. At the same time, the proteins expressed from prokaryotic systems usually cannot fold naturally and have no post translationally modification, which may affect the binding of proteins to DNA. RESULTS: Here, we develop a technique to quickly isolate proteins of interest from host plants and then analyze them using fluorescent EMSA. This technology system is called: protein from plants fluorescent EMSA method (PPF-EMSA). In PPF-EMSA, a special transient transformation method is employed to transiently deliver genes into the plant, enabling efficient synthesis the encoded proteins. Then, the target protein is isolated using immunoprecipitation, and the DNA probes were labeled with cyanine 3 (Cy3). Both fluorescent EMSA and super-shift fluorescent EMSA can be performed using the proteins from plants. Three kinds of plants, Betula platyphylla, Populus. davidiana×P. bolleana and Arabidopsis thaliana, are used in this study. The proteins isolated from plants are in a natural state, can fold naturally and are posttranslationally modified, enabling true binding to their cognate DNAs. CONCLUSION: As transient transformation can be performed quickly and not depended on whether stable transformation is available or not, we believe this method will have a wide application, enabling isolation of proteins from host plant conveniently.

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MTS1338, a distinctive small RNA in pathogenic mycobacteria, plays a crucial role in host-pathogen interactions during infection. Mycobacterial cells encounter heterogeneous stresses in macrophages, which highly upregulate MTS1338. A dormancy regulatory factor DosR regulates the intracellular abundance of MTS1338. Herein, we investigated the interplay of DosR and a low pH-inducible gene regulator PhoP binding to the MTS1338 promoter. We identified that DosR strongly binds to two regions upstream of the MTS1338 gene. The proximal region possesses a threefold higher affinity than the distal site, but the presence of both regions increased the affinity for DosR by > 10-fold. PhoP did not bind to the MTS1338 gene but binds to the DosR-bound MTS1338 gene, suggesting a concerted mechanism for MTS1338 expression.

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Translesion DNA synthesis pathways are necessary to ensure bacterial replication in the presence of DNA damage. Translesion DNA synthesis carried out by the PolV mutasome is well-studied in Escherichia coli, but ~one third of bacteria use a functionally homologous protein complex, consisting of ImuA, ImuB, and ImuC (also called DnaE2). Numerous in vivo studies have shown that all three proteins are required for translesion DNA synthesis and that ImuC is the error-prone polymerase, but the roles of ImuA and ImuB are unclear. Here we carry out biochemical characterization of ImuA and a truncation of ImuB from Myxococcus xanthus. We find that ImuA is an ATPase, with ATPase activity enhanced in the presence of DNA. The ATPase activity is likely regulated by the C-terminus, as loss of the ImuA C-terminus results in DNA-independent ATP hydrolysis. We also find that ImuA binds a variety of DNA substrates, with DNA binding affinity affected by the addition of ADP or adenylyl-imidodiphosphate. An ImuB truncation also binds DNA, with lower affinity than ImuA. In the absence of DNA, ImuA directly binds ImuB with moderate affinity. Finally, we show that ImuA and ImuB self-interact, but that ImuA is predominantly a monomer, while truncated ImuB is a trimer in vitro. Together, with our findings and the current literature in the field, we suggest a model for translesion DNA synthesis, where a trimeric ImuB would provide sufficient binding sites for DNA, the ß-clamp, ImuC, and ImuA, and where ImuA ATPase activity may regulate assembly and disassembly of the translesion DNA synthesis complex.

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"Allosteric" was first introduced to mean the other site (i.e., a site distinct from the active or orthosteric site), an adjective for "regulation" to imply a regulatory outcome resulting from ligand binding at another site. That original idea outlines a system with two ligand-binding events at two distinct locations on a macromolecule (originally a protein system), which defines a four-state energy cycle. An allosteric energy cycle provides a quantifiable allosteric coupling constant and focuses our attention on the unique properties of the four equilibrated protein complexes that constitute the energy cycle. Because many observed phenomena have been referenced as "allosteric regulation" in the literature, the goal of this work is to use literature examples to explore which systems are and are not consistent with the two-ligand thermodynamic energy cycle-based definition of allosteric regulation. We emphasize the need for consistent language so comparisons can be made among the ever-increasing number of allosteric systems. Building on the mutually exclusive natures of an energy cycle definition of allosteric regulation versus classic two-state models, we conclude our discussion by outlining how the often-proposed Rube-Goldberg-like mechanisms are likely inconsistent with an energy cycle definition of allosteric regulation.

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Coordination of enzymatic activities in the course of base excision repair (BER) is essential to ensure complete repair of damaged bases. Two major mechanisms underlying the coordination of BER are known today: the "passing the baton" model and a model of preassembled stable multiprotein repair complexes called "repairosomes." In this work, we aimed to elucidate the coordination between human apurinic/apyrimidinic (AP) endonuclease APE1 and DNA polymerase Polß in BER through studying an impact of APE1 on Polß-catalyzed nucleotide incorporation into different model substrates that mimic different single-strand break (SSB) intermediates arising along the BER pathway. It was found that APE1's impact on separate stages of Polß's catalysis depends on the nature of a DNA substrate. In this complex, APE1 removed 3' blocking groups and corrected Polß-catalyzed DNA synthesis in a coordinated manner. Our findings support the hypothesis that Polß not only can displace APE1 from damaged DNA within the "passing the baton" model but also performs the gap-filling reaction in the ternary complex with APE1 according to the "repairosome" model. Taken together, our results provide new insights into coordination between APE1 and Polß during the BER process.

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Nucleoid-associated proteins (NAPs) regulate multiple cellular processes such as gene expression, virulence, and dormancy throughout bacterial species. NAPs help in the survival and adaptation of Mycobacterium tuberculosis (Mtb) within the host. Fourteen NAPs have been identified in Escherichia coli; however, only seven NAPs are documented in Mtb. Given its complex lifestyle, it is reasonable to assume that Mtb would encode for more NAPs. Using bioinformatics tools and biochemical experiments, we have identified the heparin-binding hemagglutinin (HbhA) protein of Mtb as a novel sequence-independent DNA-binding protein which has previously been characterized as an adhesion molecule required for extrapulmonary dissemination. Deleting the carboxy-terminal domain of HbhA resulted in a complete loss of its DNA-binding activity. Atomic force microscopy showed HbhA-mediated architectural modulations in the DNA, which may play a regulatory role in transcription and genome organization. Our results showed that HbhA colocalizes with the nucleoid region of Mtb. Transcriptomics analyses of a hbhA KO strain revealed that it regulates the expression of ∼36% of total and ∼29% of essential genes. Deletion of hbhA resulted in the upregulation of ∼73% of all differentially expressed genes, belonging to multiple pathways suggesting it to be a global repressor. The results show that HbhA is a nonessential NAP regulating gene expression globally and acting as a plausible transcriptional repressor.

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The paper describes the development of a novel DNA oligonucleotide-based affinity bioreceptor that binds to lactoferrin, a glycoprotein-type immunomodulator. The research was performed using surface plasmon resonance method to investigate affinity of various types of oligonucleotides to the target protein. The 72 base pair-long 5'[(TAGAGGATCAAA)AAA]4TAGAGGATCAAA3' sequence with the highest affinity to lactoferrin was selected for further investigations. Kinetic analysis of the interaction between selected DNA and lactoferrin provided rate and equilibrium constants: ka = (2.49 ± 0.03)∙104 M-1∙s-1, kd = (1.89 ± 0.02)∙10-3 s-1, KA = (0.13 ± 0.05)∙108 M-1, and KD = (7.61 ± 0.18)∙10-8 M. Thermodynamic study conducted to determine the ΔH0, ΔS0, and ΔG0 for van't Hoff characteristic in the temperature range of 291.15-305.15 K, revealed the complex formation as endothermic and entropically driven. The chosen DNA sequence's selectivity towards lactoferrin was confirmed with interferents' response constituting <3 % of the response to lactoferrin. SPR analysis justified utility of the designed DNA oligonucleotide for Lf determination, with LOD of 4.42∙10-9 M. Finally, the interaction between lactoferrin and DNA was confirmed by electrochemical impedance spectroscopy, providing the basis for further quantitative assay of lactoferrin using the developed DNA-based bioreceptor. The interactions were performed under immobilized DNA ligand conditions, thus reflecting the sensor's surface, which facilitates their transfer to other label-free biosensor technologies.

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The gram-positive bacterium Bacillus velezensis strain DMW1 produces a high level of antimicrobial metabolites that can suppress the growth of phytopathogens. We investigated the mechanism used by degQ and the degS/degU two-component system to regulate the biocontrol characteristics of DMW1. When degQ and degU were deleted, the biofilm formation, cell motility, colonization activities, and antifungal abilities of ΔdegQ and ΔdegU were significantly reduced compared to wild-type DMW1. The expression levels of biofilm-related genes (epsA, epsB, epsC, and tasA) and swarming-related genes (swrA and swrB) were all down-regulated. We also evaluated the impact on secondary metabolites of these two genes. The degQ and degU genes reduced surfactin and macrolactin production and up-regulated the production of fengycin, iturin, bacillaene, and difficidin metabolites. The reverse transcription-quantitative PCR results were consistent with these observations. Electrophoretic mobility shift assay and microscale thermophoresis revealed that DegU can bind to the promoter regions of these six antimicrobial metabolite genes and regulate their synthesis. In conclusion, we provided systematic evidence to demonstrate that the degQ and degU genes are important regulators of multicellular behaviour and antimicrobial metabolic processes in B. velezensis DMW1 and suggested novel amenable strains to be used for the industrial production of antimicrobial metabolites.

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Several experimental studies have shown that Hoogsteen (HG) base pair (bp) stabilizes in the presence of proteins. The molecular mechanism underlying this stabilization is not well known. This leads us to examine the stability of the HG bp in duplex DNA using all-atom molecular dynamics simulation in both the absence and presence of proteins. We use conformational thermodynamics to investigate the stability of a HG bp in duplex DNA at the molecular level. We compute the changes in the conformational free energy and entropy of DNA when DNA adopts a HG bp in its bp sequence rather than a Watson-Crick (WC) bp in both naked DNA and protein-bound DNA complex. We observe that the presence of proteins stabilizes and organizes the HG bp and the entire DNA duplex. Sugar-phosphate, sugar-base, and sugar-pucker torsion angles play key roles in stabilizing and ordering the HG bp in the protein-bound DNA complex.

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Promoter proximal pausing of RNA polymerase II (Pol II) is a critical transcriptional regulatory mechanism in metazoans that requires the transcription factor DRB sensitivity-inducing factor (DSIF) and the inhibitory negative elongation factor (NELF). DSIF, composed of Spt4 and Spt5, establishes the pause by recruiting NELF to the elongation complex. However, the role of DSIF in pausing beyond NELF recruitment remains unclear. We used a highly purified in vitro system and Drosophila nuclear extract to investigate the role of DSIF in promoter proximal pausing. We identified two domains of Spt5, the KOW4 and NGN domains, that facilitate Pol II pausing. The KOW4 domain promotes pausing through its interaction with the nascent RNA while the NGN domain does so through a short helical motif that is in close proximity to the non-transcribed DNA template strand. Removal of this sequence in Drosophila has a male-specific dominant negative effect. The alpha-helical motif is also needed to support fly viability. We also show that the interaction between the Spt5 KOW1 domain and the upstream DNA helix is required for DSIF association with the Pol II elongation complex. Disruption of the KOW1-DNA interaction is dominant lethal in vivo. Finally, we show that the KOW2-3 domain of Spt5 mediates the recruitment of NELF to the elongation complex. In summary, our results reveal additional roles for DSIF in transcription regulation and identify specific domains important for facilitating Pol II pausing.

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Base excision repair (BER) is one of the important systems for the maintenance of genome stability via repair of DNA lesions. BER is a multistep process involving a number of enzymes, including damage-specific DNA glycosylases, apurinic/apyrimidinic (AP) endonuclease 1, DNA polymerase ß, and DNA ligase. Coordination of BER is implemented by multiple protein-protein interactions between BER participants. Nonetheless, mechanisms of these interactions and their roles in the BER coordination are poorly understood. Here, we report a study on Polß's nucleotidyl transferase activity toward different DNA substrates (that mimic DNA intermediates arising during BER) in the presence of various DNA glycosylases (AAG, OGG1, NTHL1, MBD4, UNG, or SMUG1) using rapid-quench-flow and stopped-flow fluorescence approaches. It was shown that Polß efficiently adds a single nucleotide into different types of single-strand breaks either with or without a 5'-dRP-mimicking group. The obtained data indicate that DNA glycosylases AAG, OGG1, NTHL1, MBD4, UNG, and SMUG1, but not NEIL1, enhance Polß's activity toward the model DNA intermediates.

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Initiation of chromosomal replication requires dynamic nucleoprotein complexes. In most eubacteria, the origin oriC contains multiple DnaA box sequences to which the ubiquitous DnaA initiators bind. In Escherichia coli oriC, DnaA boxes sustain construction of higher-order complexes via DnaA-DnaA interactions, promoting the unwinding of the DNA unwinding element (DUE) within oriC and concomitantly binding the single-stranded (ss) DUE to install replication machinery. Despite the significant sequence homologies among DnaA proteins, oriC sequences are highly diverse. The present study investigated the design of oriC (tma-oriC) from Thermotoga maritima, an evolutionarily ancient eubacterium. The minimal tma-oriC sequence includes a DUE and a flanking region containing five DnaA boxes recognized by the cognate DnaA (tmaDnaA). This DUE was comprised of two distinct functional modules, an unwinding module and a tmaDnaA-binding module. Three direct repeats of the trinucleotide TAG within DUE were essential for both unwinding and ssDUE binding by tmaDnaA complexes constructed on the DnaA boxes. Its surrounding AT-rich sequences stimulated only duplex unwinding. Moreover, head-to-tail oligomers of ATP-bound tmaDnaA were constructed within tma-oriC, irrespective of the directions of the DnaA boxes. This binding mode was considered to be induced by flexible swiveling of DnaA domains III and IV, which were responsible for DnaA-DnaA interactions and DnaA box binding, respectively. Phasing of specific tmaDnaA boxes in tma-oriC was also responsible for unwinding. These findings indicate that a ssDUE recruitment mechanism was responsible for unwinding and would enhance understanding of the fundamental molecular nature of the origin sequences present in evolutionarily divergent bacteria.

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BACKGROUND: Identification of the motifs bound by a transcription factor (TF) is important to reveal the function of TF. Previously, we built a transcription factor centered yeast one hybrid (TF-Centered Y1H) that could identify the motifs bound by a target TF. However, that method was difficult to comprehensively identify all the motifs bound by a TF. RESULTS: Here, we build an improved TF-Centered Y1H to comprehensively determine the motifs bound by a target TF. Recombination-mediated cloning in yeast was performed to construct a saturated prey library that contains 7 random base insertions. After TF-Centered Y1H screening, all the positive clones were pooled together to isolate pHIS2 vector. The insertion regions of pHIS2 were PCR amplified and the PCR product was subjected to high-throughput sequencing. The insertion sequences were then retrieved and analyzed using MEME program to identify the potential motifs bound by the TF. Using this technology, we studied the motifs bound by an ethylene-responsive factor (BpERF2) from birch. In total, 22 conserved motifs were identified, and most of them are novel cis-acting elements. Both the yeast one hybrid and electrophoretic mobility shift assay verified that the obtained motifs could be bound by BpERF2. In addition, chromatin immunoprecipitation (ChIP) study further suggested that the identified motifs can be bound by BpERF2 in cells of birch. These results together suggested that this technology is reliable and has biological significance. CONCLUSION: This method will have wide application in DNA-protein interaction studies.

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Integration of retroviral DNA into the host genome involves the formation of integrase (IN)-DNA complexes termed intasomes. Further characterization of these complexes is needed to understand their assembly process. Here, we report the single-particle cryo-EM structure of the Rous sarcoma virus (RSV) strand transfer complex (STC) intasome produced with IN and a preassembled viral/target DNA substrate at 3.36 Å resolution. The conserved intasome core region consisting of IN subunits contributing active sites interacting with viral/target DNA has a resolution of 3 Å. Our structure demonstrated the flexibility of the distal IN subunits relative to the IN subunits in the conserved intasome core, similar to results previously shown with the RSV octameric cleaved synaptic complex intasome produced with IN and viral DNA only. An extensive analysis of higher resolution STC structure helped in the identification of nucleoprotein interactions important for intasome assembly. Using structure-function studies, we determined the mechanisms of several IN-DNA interactions critical for assembly of both RSV intasomes. We determined the role of IN residues R244, Y246, and S124 in cleaved synaptic complex and STC intasome assemblies and their catalytic activities, demonstrating differential effects. Taken together, these studies advance our understanding of different RSV intasome structures and molecular determinants involved in their assembly.

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Chemical shift perturbation (CSP) is a simple NMR technique for studying the DNA binding of proteins. Titration of the unlabeled DNA into the 15N-labeled protein is monitored by acquiring a two-dimensional (2D) heteronuclear single-quantum correlation (HSQC) spectrum at each step of the titration. CSP can also provide information on the DNA-binding dynamics of proteins, as well as protein-induced conformational changes in DNA. Here, we describe the titration of DNA for the 15N-labeled Z-DNA-binding protein, monitored via 2D HSQC spectra. NMR titration data can be analyzed with the active B-Z transition model to provide the protein-induced B-Z transition dynamics of DNA.

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Z-DNA structure is a noncanonical left-handed alternative form of DNA, which has been suggested to be biologically important and is related to several genetic diseases and cancer. Therefore, investigation of Z-DNA structure associated with biological events is of great importance to understanding the functions of these molecules. Here, we described the development of a trifluoromethyl labeled deoxyguanosine derivative and employed it as a 19F NMR probe to study Z-form DNA structure in vitro and in living cells.

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A novel approach to treat the highly virulent and infectious enteric pathogen Shigella flexneri, with the potential for reduced resistance development, is to target virulence pathways. One promising such target is the AraC-family transcription factor VirF, which activates downstream virulence factors. VirF harbors a conserved C-terminal DNA-binding domain (DBD) and an N-terminal dimerization domain (NTD). Previously, we studied the wild type (WT) and seven alanine DBD mutants of VirF binding to the virB promoter (N. J. Ragazzone, G. T. Dow, and A. Garcia, J Bacteriol 204:e00143-22, 2022, https://doi.org/10.1128/jb.00143-22). Here, we report studies of VirF binding to the icsA and rnaG promoters. Gel shift assays (electrophoretic mobility shift assays [EMSAs]) of WT VirF binding to these promoters revealed multiple bands at higher apparent molecular weights, indicating the likelihood of VirF dimerization when bound to DNA. For three of the mutants, we observed consistent effects on binding to the three promoters. For the four other mutants, we observed differential effects on promoter binding. Results of a cell-based, LexA monohybrid ß-galactosidase reporter assay [D. A. Daines, M. Granger-Schnarr, M. Dimitrova, and R. P. Silver, Methods Enzymol 358:153-161, 2002, https://doi.org/10.1016/s0076-6879(02)58087-3] indicated that WT VirF dimerizes in vivo and that alanine mutations to Y132, L137, and L147 significantly reduced dimerization. However, these mutations negatively impacted protein stability. We did purify enough of the Y132A mutant to determine that it binds in vitro to the virB and rnaG promoters, albeit with weaker affinities. Full-length VirF model structures, generated with I-TASSER, predict that these three amino acids are in a "dimerization" helix in the NTD, consistent with our results. IMPORTANCE Antimicrobial-resistant (AMR) infections continue to rise dramatically, and the lack of new approved antibiotics is not ameliorating this crisis. A promising route to reduce AMR is by targeting virulence. The Shigella flexneri virulence pathway is a valuable source for potential therapeutic targets useful to treat this infection. VirF, an AraC-family virulence transcription factor, is responsible for activating necessary downstream virulence genes that allow the bacteria to invade and spread within the human colon. Previous studies have identified how VirF interacts with the virB promoter and have even developed a lead DNA-binding inhibitor, but not much is known about VirF dimerization or binding to the icsA and rnaG promoters. Fully characterizing VirF can be a valuable resource for inhibitor discovery/design.

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