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1.
Plant J ; 112(6): 1337-1349, 2022 12.
Article in English | MEDLINE | ID: mdl-36288411

ABSTRACT

Structure-based high-throughput screening of chemical compounds that target protein-protein interactions (PPIs) is a promising technology for gaining insight into how plant development is regulated, leading to many potential agricultural applications. At present, there are no examples of using high-throughput screening to identify chemicals that target plant transcriptional complexes, some of which are responsible for regulating multiple physiological functions. Florigen, a protein encoded by FLOWERING LOCUS T (FT), was initially identified as a molecule that promotes flowering and has since been shown to regulate flowering and other developmental phenomena such as tuber formation in potato (Solanum tuberosum). FT functions as a component of the florigen activation complex (FAC) with a 14-3-3 scaffold protein and FD, a bZIP transcription factor that activates downstream gene expression. Although 14-3-3 is an important component of FAC, little is known about the function of the 14-3-3 protein itself. Here, we report the results of a high-throughput in vitro fluorescence resonance energy transfer (FRET) screening of chemical libraries that enabled us to identify small molecules capable of inhibiting FAC formation. These molecules abrogate the in vitro interaction between the 14-3-3 protein and the OsFD1 peptide, a rice (Oryza sativa) FD, by directly binding to the 14-3-3 protein. Treatment with S4, a specific hit molecule, strongly inhibited FAC activity and flowering in duckweed, tuber formation in potato, and branching in rice in a dose-dependent manner. Our results demonstrate that the high-throughput screening approach based on the three-dimensional structure of PPIs is suitable in plants. In this study, we have proposed good candidate compounds for future modification to obtain inhibitors of florigen-dependent processes through inhibition of FAC formation.


Subject(s)
Florigen , Oryza , Florigen/metabolism , Plant Proteins/metabolism , 14-3-3 Proteins/genetics , 14-3-3 Proteins/metabolism , High-Throughput Screening Assays , Oryza/metabolism , Gene Expression Regulation, Plant , Flowers/genetics
2.
Structure ; 26(4): 590-598.e5, 2018 04 03.
Article in English | MEDLINE | ID: mdl-29576320

ABSTRACT

The stator of the bacterial flagellar motor couples ion flow with torque generation. The ion-conducting stator channel opens only when incorporated into and anchored around the rotor via the peptidoglycan (PG) binding domain of the B subunit (MotBC). However, no direct evidence of PG binding coupled with channel activation has been presented. Here, we report the structural rearrangements of MotBC responsible for this coupling process. A MotBC fragment with the L119P replacement, which is known to cause channel activation, was able to bind PG. Nuclear magnetic resonance analysis of MotBC and the crystal structure of the MotBC-L119P dimer revealed major structural changes in helix α1. In vivo crosslinking results confirm that a major rearrangement occurs. Our results suggest that, upon stator incorporation into the motor, helix α1 of MotBC changes into an extended non-helical structure. We propose that this change allows the stator both to bind PG and to open its proton channel.


Subject(s)
Bacterial Proteins/chemistry , Flagella/chemistry , Peptidoglycan/chemistry , Periplasm/chemistry , Protons , Salmonella enterica/chemistry , Amino Acid Motifs , Amino Acid Substitution , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , Cloning, Molecular , Crystallography, X-Ray , Escherichia coli/genetics , Escherichia coli/metabolism , Flagella/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Ion Transport , Models, Molecular , Mutation , Peptidoglycan/metabolism , Periplasm/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Multimerization , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Salmonella enterica/metabolism
3.
Nucleic Acids Res ; 40(1): e7, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22080547

ABSTRACT

A site-specific isotope labeling technique of long RNA molecules was established. This technique is comprised of two simple enzymatic reactions, namely a guanosine transfer reaction of group I self-splicing introns and a ligation with T4 DNA ligase. The trans-acting group I self-splicing intron with its external cofactor, 'isotopically labeled guanosine 5'-monophosphate' (5'-GMP), steadily gave a 5'-residue-labeled RNA fragment. This key reaction, in combination with a ligation of 5'-remainder non-labeled sequence, allowed us to prepare a site-specifically labeled RNA molecule in a high yield, and its production was confirmed with (15)N NMR spectroscopy. Such a site-specifically labeled RNA molecule can be used to detect a molecular interaction and to probe chemical features of catalytically/structurally important residues with NMR spectroscopy and possibly Raman spectroscopy and mass spectrometry.


Subject(s)
Isotope Labeling/methods , RNA/chemistry , DNA Ligases , Introns , Nuclear Magnetic Resonance, Biomolecular , RNA, Catalytic/chemistry
4.
Nucleic Acids Symp Ser (Oxf) ; (53): 269-70, 2009.
Article in English | MEDLINE | ID: mdl-19749364

ABSTRACT

HAC1 is a transcription factor related to Unfolded Protein Response (UPR) signaling in yeast. Processing of HAC1 mRNA on Endoplasmic reticulum (ER) plays a key role in UPR signaling pathway, but the recognition mechanism of HAC1 mRNA by processing enzyme Ire1p is still unclear. Here, the solution structure of HAC1 mRNA was investigated by Nuclear Magnetic Resonance (NMR) spectroscopy, focusing on the structure of the recognition site of Ire1p in HAC1 mRNA. From the NOESY spectrum, imino proton signals of 5' processing regions of HAC1 mRNA were assigned and it was found that this region forms the stem-loop structure.


Subject(s)
Basic-Leucine Zipper Transcription Factors/genetics , Endoribonucleases/metabolism , Membrane Glycoproteins/metabolism , Protein Serine-Threonine Kinases/metabolism , RNA, Messenger/chemistry , Repressor Proteins/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Base Sequence , Basic-Leucine Zipper Transcription Factors/metabolism , Consensus Sequence , Nuclear Magnetic Resonance, Biomolecular , RNA Processing, Post-Transcriptional , RNA, Messenger/metabolism , Repressor Proteins/metabolism
5.
Nucleic Acids Symp Ser (Oxf) ; (53): 277-8, 2009.
Article in English | MEDLINE | ID: mdl-19749368

ABSTRACT

Recently, in hammerhead ribozymes, newly identified loop-loop interaction was found to be important for their activation. Therefore, we chemically synthesized a hammerhead ribozyme with this extra loop sequences and its mutant ribozymes, as well as their substrate RNA strands in order to clarify their cleavable sequences. After purification with an anion exchange column chromatography, we were able to obtain 44mer and 20mer RNA.


Subject(s)
RNA, Catalytic/chemistry , Base Sequence , Molecular Sequence Data , Mutation , RNA/chemistry , RNA/metabolism , RNA, Catalytic/chemical synthesis , RNA, Catalytic/metabolism
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