Determination of the DNA-binding kinetics of three related but heteroimmune bacteriophage repressors using EMSA and SPR analysis

Henriksson-Peltola, Petri; Sehlén, Wilhelmina; Haggård‐Ljungquist, Elisabeth

doi:10.1093/nar/gkm172

Cited by 20 publications

(9 citation statements)

References 23 publications

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“…The dissociation constant K D s of lysozyme, peptides A40, B40, and C9 with plasmid DNA were estimated to be in the range of 10 −4 to 10 −6 ( Table 1). These values are comparable to the bindings of histones toward chromosomal DNAs [20], the serum factor to the DNA containing specific binding site [21] and the temperate P2-like bacteriophage repressors toward their operator sequences [22]. The most striking finding is that the K D value (2.35×10 −5 M) of the nonapeptide, C9, is very close to that of the histones [20].…”

Section: Discussionmentioning

confidence: 62%

Characterization of the Interactions of Lysozyme with DNA by Surface Plasmon Resonance and Circular Dichroism Spectroscopy

Lin¹,

Wey

Kan

et al. 2008

Appl Biochem Biotechnol

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Association with nucleic acid has been recognized as a unique role of lysozyme and may explain why lysozyme was called a killer protein against HIV infection. In the present study, we characterized the interactions of lysozyme and its derived peptides with a biotin-labeled pUC19 plasmid DNA. Real-time detection of the macromolecular interaction was performed using the SPR (surface plasmon resonance) spectroscopy. The SPR sensorgrams were analyzed and the association and dissociation rate constants as well as the dissociation equilibrium constant KD were, thus, estimated. The results reveal that other than the electrostatic interactions between the basic protein and the nucleotide sequences carrying negative charges, the specific DNA-binding motifs at the N- and C-termini of lysozyme were also involved in the interactions. The nonapeptide RAWVAWRNR (aa 107-115 of lysozyme) reported previously to block HIV-1 viral entrance and replication was also able to bind DNA with its KD value comparable to that of histones. The possibilities of ligand-binding-induced conformational changes were investigated using the circular dichroism spectroscopy. The CD spectra (200-320 nm) reveal that the conformational changes indeed occur as the spectra of lysozyme-DNA interactions are much less at the major trough region than the sum of individual spectra. The interaction of lysozyme with DNA molecules may interfere with DNA replication, modulate gene expression, and block bacterial and viral infections. These all suggest that human lysozyme may represent part of the innate immune system with a very broad protective spectrum.

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Section: Discussionmentioning

confidence: 62%

Characterization of the Interactions of Lysozyme with DNA by Surface Plasmon Resonance and Circular Dichroism Spectroscopy

Lin¹,

Wey

Kan

et al. 2008

Appl Biochem Biotechnol

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“…P2 C has been shown to generate two retarded bands in the EMSA, compared to WΦ C that generated three retarded bands (36). The distance between the half-sites of P2 is two helical turns, and that of WΦ three helical turns.…”

Section: Resultsmentioning

confidence: 99%

“…The observed difference in spontaneous phage production from strains carrying either P2 or WΦ prophage might reflect differences in the components of the transcriptional switch, such as relative strengths of the Pe and Pc promoters and the kinetics of the interactions between the C and the Cox repressors and their respective operator. The kinetics of the interactions between the C proteins to their respective operator has been studied (36), but not the interactions of the Cox proteins with their operators. Other phage or cellular factors could also affect the burst size.…”

Section: Discussionmentioning

confidence: 99%

A comparison of the DNA binding and bending capacities and the oligomeric states of the immunity repressors of heteroimmune coliphages P2 and W

Ahlgren-Berg¹,

Henriksson-Peltola²,

Sehlén³

et al. 2007

Nucleic Acids Research

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Bacteriophages P2 and WΦ are heteroimmune members of the P2-like family of temperate Escherichia coli phages. Temperate phages can grow lytically or form lysogeny after infection. A transcriptional switch that contains two con-vergent promoters, Pe and Pc, and two repressors regulate what life mode to enter. The immunity repressor C is the first gene of the lysogenic operon, and it blocks the early Pe promoter. In this work, some characteristics of the C proteins of P2 and WΦ are compared. An in vivo genetic analysis shows that WΦ C, like P2 C, has a strong dimerization activity in the absence of its DNA target. Both C proteins recognize two directly repeated sequences, termed half-sites and a strong bending is induced in the respective DNA target upon binding. P2 C is unable to bind to one half-site as opposed to WΦ, but both half-sites are required for repression of WΦ Pe. A reduction from three to two helical turns between the centers of the half-sites in WΦ has no significant effect on the capacity to repress Pe. However, the protein–DNA complexes formed differ, as determined by electrophoretic mobility shift experiments. A difference in spontaneous phage production is observed in isogenic lysogens.

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“…All experiments were performed using a Biacore 3000 instrument and streptavadin‐coated biosensor chips (SA‐Chip; GE Healthcare) (Henriksson‐Peltola et al ., ; Wang et al ., ). All buffers were freshly prepared, filtered using 0.22‐μm syringe filters and de‐gassed.…”

Section: Methodsmentioning

confidence: 99%

OsMADS57 together with OsTB1 coordinates transcription of its target OsWRKY94 and D14 to switch its organogenesis to defense for cold adaptation in rice

et al. 2018

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Summary Plants modify their development to adapt to their environment, protecting themselves from detrimental conditions such as chilling stress by triggering a variety of signaling pathways; however, little is known about how plants coordinate developmental patterns and stress responses at the molecular level.Here, we demonstrate that interacting transcription factors OsMADS57 and OsTB1 directly target the defense gene OsWRKY94 and the organogenesis gene D14 to trade off the functions controlling/moderating rice tolerance to cold.Overexpression of OsMADS57 maintains rice tiller growth under chilling stress. OsMADS57 binds directly to the promoter of OsWRKY94, activating its transcription for the cold stress response, while suppressing its activity under normal temperatures. In addition, OsWRKY94 was directly targeted and suppressed by OsTB1 under both normal and chilling temperatures. However, D14 transcription was directly promoted by OsMADS57 for suppressing tillering under the chilling treatment, whereas D14 was repressed for enhancing tillering under normal condition.We demonstrated that OsMADS57 and OsTB1 conversely affect rice chilling tolerance via targeting OsWRKY94.Our findings highlight a molecular genetic mechanism coordinating organogenesis and chilling tolerance in rice, which supports and extends recent work suggesting that chilling stress environments influence organ differentiation.

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Determination of the DNA-binding kinetics of three related but heteroimmune bacteriophage repressors using EMSA and SPR analysis

Cited by 20 publications

References 23 publications

Characterization of the Interactions of Lysozyme with DNA by Surface Plasmon Resonance and Circular Dichroism Spectroscopy

Characterization of the Interactions of Lysozyme with DNA by Surface Plasmon Resonance and Circular Dichroism Spectroscopy

A comparison of the DNA binding and bending capacities and the oligomeric states of the immunity repressors of heteroimmune coliphages P2 and W

OsMADS57 together with OsTB1 coordinates transcription of its target OsWRKY94 and D14 to switch its organogenesis to defense for cold adaptation in rice

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