Genomic SELEX Screening of Regulatory Targets of Transcription Factors.

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.

Expanded roles of lactate-sensing LldR in transcription regulation of the K-12 genome: lactate utilisation and acid resistance.

LldR is a lactate-responsive transcription factor (TF) that transcriptionally regulates the operon consisting of lactate permease and lactate dehydrogenase. The operon facilitates the utilisation of lactic acid in bacteria. However, the role of LldR in whole genomic transcriptional regulation, and the mechanism involved in adaptation to lactate remains unclear.

ALS-linked TDP-43 mutations interfere with the recruitment of RNA recognition motifs to G-quadruplex RNA.

TDP-43 is a major pathological protein in sporadic and familial amyotrophic lateral sclerosis (ALS) and mediates mRNA fate. TDP-43 dysfunction leads to causes progressive degeneration of motor neurons, the details of which remain elusive. Elucidation of the molecular mechanisms of RNA binding could enhance our understanding of this devastating disease.

Metal-Responsive Transcription Factors Co-Regulate Anti-Sigma Factor (Rsd) and Ribosome Dimerization Factor Expression.

Bacteria exposed to stress survive by regulating the expression of several genes at the transcriptional and translational levels. For instance, in , when growth is arrested in response to stress, such as nutrient starvation, the anti-sigma factor Rsd is expressed to inactivate the global regulator RpoD and activate the sigma factor RpoS. However, ribosome modulation factor (RMF) expressed in response to growth arrest binds to 70S ribosomes to form inactive 100S ribosomes and inhibit translational activity.

Genomic SELEX Reveals Pervasive Role of the Flagella Master Regulator FlhDC in Carbon Metabolism.

Flagella are vital bacterial organs that allow microorganisms to move to favorable environments. However, their construction and operation consume a large amount of energy. The master regulator FlhDC mediates all flagellum-forming genes in through a transcriptional regulatory cascade, the details of which remain elusive.

Essential Roles and Risks of G-Quadruplex Regulation: Recognition Targets of ALS-Linked TDP-43 and FUS.

A non-canonical DNA/RNA structure, G-quadruplex (G4), is a unique structure formed by two or more guanine quartets, which associate through Hoogsteen hydrogen bonding leading to form a square planar arrangement. A set of RNA-binding proteins specifically recognize G4 structures and play certain unique physiological roles. These G4-binding proteins form ribonucleoprotein (RNP) through a physicochemical phenomenon called liquid-liquid phase separation (LLPS).

Transcription Factor SrsR (YgfI) Is a Novel Regulator for the Stress-Response Genes in Stationary Phase in K-12.

Understanding the functional information of all genes and the biological mechanism based on the comprehensive genome regulation mechanism is an important task in life science. YgfI is an uncharacterized LysR family transcription factor in . To identify the function of YgfI, the genomic SELEX (gSELEX) screening was performed for YgfI regulation targets on the genome.

Regulatory role of CsuR (YiaU) in determination of cell surface properties of K-12.

Genomic SELEX screening was performed to identify the binding sites of YiaU, an uncharacterized LysR family transcription factor, on the K-12 genome. Five high-affinity binding targets of YiaU were identified, all of which were involved in the structures of the bacterial cell surface such as outer and inner membrane proteins, and lipopolysaccharides. Detailed and analyses suggest that YiaU activates these target genes.

Whole set of constitutive promoters for RpoN sigma factor and the regulatory role of its enhancer protein NtrC in K-12.

The promoter selectivity of RNA polymerase (RNAP) is determined by its promoter-recognition sigma subunit. The model prokaryote K-12 contains seven species of the sigma subunit, each recognizing a specific set of promoters. Using genomic SELEX (gSELEX) screening , we identified the whole set of 'constitutive' promoters recognized by the reconstituted RNAP holoenzyme alone, containing RpoD (σ), RpoS (σ), RpoH (σ), RpoF (σ) or RpoE (σ), in the absence of other supporting regulatory factors.

ALS-linked FUS mutations dysregulate G-quadruplex-dependent liquid-liquid phase separation and liquid-to-solid transition.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the accumulation of protein aggregates in motor neurons. Recent discoveries of genetic mutations in ALS patients promoted research into the complex molecular mechanisms underlying ALS. FUS (fused in sarcoma) is a representative ALS-linked RNA-binding protein (RBP) that specifically recognizes G-quadruplex (G4)-DNA/RNAs.

Single-Target Regulators Constitute the Minority Group of Transcription Factors in K-12.

The identification of regulatory targets of all transcription factors (TFs) is critical for understanding the entire network of genome regulation. A total of approximately 300 TFs exist in the model prokaryote K-12, but the identification of whole sets of their direct targets is impossible with use of approaches. For this end, the most direct and quick approach is to identify the TF-binding sites on the genome.

Hierarchy of transcription factor network in Escherichia coli K-12: H-NS-mediated silencing and Anti-silencing by global regulators.

Transcriptional regulation for genome expression determines growth and adaptation of single-cell bacteria that are directly exposed to environment. The transcriptional apparatus in Escherichia coli K-12 is composed of RNA polymerase core enzyme and two groups of its regulatory proteins, seven species of promoter-recognition subunit sigma and about 300 species of transcription factors. The identification of regulatory targets for all these regulatory proteins is critical toward understanding the genome regulation as a whole.

Molecular Basis of Sulfosugar Selectivity in Sulfoglycolysis.

The sulfosugar sulfoquinovose (SQ) is produced by essentially all photosynthetic organisms on Earth and is metabolized by bacteria through the process of sulfoglycolysis. The sulfoglycolytic Embden-Meyerhof-Parnas pathway metabolizes SQ to produce dihydroxyacetone phosphate and sulfolactaldehyde and is analogous to the classical Embden-Meyerhof-Parnas glycolysis pathway for the metabolism of glucose-6-phosphate, though the former only provides one C3 fragment to central metabolism, with excretion of the other C3 fragment as dihydroxypropanesulfonate. Here, we report a comprehensive structural and biochemical analysis of the three core steps of sulfoglycolysis catalyzed by SQ isomerase, sulfofructose (SF) kinase, and sulfofructose-1-phosphate (SFP) aldolase.

Different recognition modes of G-quadruplex RNA between two ALS/FTLD-linked proteins TDP-43 and FUS.

Amyotrophic lateral sclerosis/frontotemporal lobar degeneration-linked proteins, TDP-43 and fused in sarcoma (FUS), bind to G-quadruplex-containing mRNAs and transport them to distal neurites for local translation. The specificity and mechanism of G4-RNA binding, however, remain largely unsolved. Using purified full-length TDP-43 and FUS and a set of seven G4-DNA/RNA, we compared their recognition properties of G4-RNAs.

Expanded roles of pyruvate-sensing PdhR in transcription regulation of the K-12 genome: fatty acid catabolism and cell motility.

The transcription factor PdhR has been recognized as the master regulator of the pyruvate catabolism pathway in , including both NAD-linked oxidative decarboxylation of pyruvate to acetyl-CoA by PDHc (pyruvate dehydrogenase complex) and respiratory electron transport of NADH to oxygen by Ndh-CyoABCD enzymes. To identify the whole set of regulatory targets under the control of pyruvate-sensing PdhR, we performed genomic SELEX (gSELEX) screening . A total of 35 PdhR-binding sites were identified along the K-12 genome, including previously identified targets.

Novel regulators of the gene encoding the master regulator of biofilm formation in K-12.

Under stressful conditions, forms biofilm for survival by sensing a variety of environmental conditions. CsgD, the master regulator of biofilm formation, controls cell aggregation by directly regulating the synthesis of Curli fimbriae. In agreement of its regulatory role, as many as 14 transcription factors (TFs) have so far been identified to participate in regulation of the promoter, each monitoring a specific environmental condition or factor.

Molecular dissection of ALS-linked TDP-43 - involvement of the Gly-rich domain in interaction with G-quadruplex mRNA.

TDP-43 is the major pathogenic protein of amyotrophic lateral sclerosis (ALS). Previously, we identified that TDP-43 interacts with G-quadruplex (G4)-containing RNA and is involved in their long-distance transport in neurons. For the molecular dissection of the TDP-43 and G4-RNA interaction, we analyzed it here in vitro and in cultured cells using a set of 10 mutant TDP-43 proteins from familial and sporadic ALS patients as well as using the TDP-43 C-terminal Gly-rich domain alone.

Cytotoxic Mechanism of Excess Polyamines Functions through Translational Repression of Specific Proteins Encoded by Polyamine Modulon.

Excessive accumulation of polyamines causes cytotoxicity, including inhibition of cell growth and a decrease in viability. We investigated the mechanism of cytotoxicity caused by spermidine accumulation under various conditions using an strain deficient in spermidine acetyltransferase (SAT), a key catabolic enzyme in controlling polyamine levels. Due to the excessive accumulation of polyamines by the addition of exogenous spermidine to the growth medium, cell growth and viability were markedly decreased through translational repression of specific proteins [RMF (ribosome modulation factor) and Fis (rRNA transcription factor) etc.

Author Correction: Regulatory Role of PlaR (YiaJ) for Plant Utilization in Escherichia coli K-12.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Regulatory Role of PlaR (YiaJ) for Plant Utilization in Escherichia coli K-12.

Outside a warm-blooded animal host, the enterobacterium Escherichia coli K-12 is also able to grow and survive in stressful nature. The major organic substance in nature is plant, but the genetic system of E. coli how to utilize plant-derived materials as nutrients is poorly understood.

Coordinated Regulation of Rsd and RMF for Simultaneous Hibernation of Transcription Apparatus and Translation Machinery in Stationary-Phase .

Transcription and translation in growing phase of , the best-studied model prokaryote, are coupled and regulated in coordinate fashion. Accordingly, the growth rate-dependent control of the synthesis of RNA polymerase (RNAP) core enzyme (the core component of transcription apparatus) and ribosomes (the core component of translation machinery) is tightly coordinated to keep the relative level of transcription apparatus and translation machinery constant for effective and efficient utilization of resources and energy. Upon entry into the stationary phase, transcription apparatus is modulated by replacing RNAP core-associated sigma (promoter recognition subunit) from growth-related RpoD to stationary-phase-specific RpoS.

Regulatory role of pyruvate-sensing BtsSR in biofilm formation by Escherichia coli K-12.

Pyruvate, the key regulator in connection of a variety of metabolic pathways, influences transcription of the Escherichia coli genome through controlling the activity of two pyruvate-sensing two-component systems (TCSs), BtsSR and PyrSR. Previously, we identified the whole set of regulatory targets of PyrSR with low-affinity to pyruvate. Using gSELEX screening system, we found here that BtsSR with high-affinity to pyruvate regulates more than 100 genes including as many as 13 transcription factors genes including the csgD gene encoding the master regulator of biofilm formation.

Regulatory roles of pyruvate-sensing two-component system PyrSR (YpdAB) in Escherichia coli K-12.

When the rate of production of metabolites in bacteria exceeds the amounts needed for cell growth, excess metabolites are secreted into the extracellular environment. Upon entry into poor nutrient conditions, overflowed exometabolites are reused to continue cell growth and survival. At present, however, the genetic system for utilization of exometabolites is poorly understood even for the best-characterized model prokaryote Escherichia coli.

Regulatory role of CsqR (YihW) in transcription of the genes for catabolism of the anionic sugar sulfoquinovose (SQ) in Escherichia coli K-12.

The binding sites of YihW, an uncharacterized DeoR-family transcription factor (TF) of Escherichia coli K-12, were identified using Genomic SELEX screening at two closely located sites, one inside the spacer between the bidirectional transcription units comprising the yihUTS operon and the yihV gene, and another one upstream of the yihW gene itself. Recently the YihUTS and YihV proteins were identified as catalysing the catabolism of sulfoquinovose (SQ), a hydrolysis product of sulfoquinovosyl diacylglycerol (SQDG) derived from plants and other photosynthetic organisms. Gel shift assay in vitro and reporter assay in vivo indicated that YihW functions as a repressor for all three transcription units.

Coordinated Hibernation of Transcriptional and Translational Apparatus during Growth Transition of Escherichia coli to Stationary Phase.

In the process of Escherichia coli K-12 growth from exponential phase to stationary, marked alteration takes place in the pattern of overall genome expression through modulation of both parts of the transcriptional and translational apparatus. In transcription, the sigma subunit with promoter recognition properties is replaced from the growth-related factor RpoD by the stationary-phase-specific factor RpoS. The unused RpoD is stored by binding with the anti-sigma factor Rsd.