Allele-Specific Reprogramming of Cancer Metabolism by the Long Non-coding RNA CCAT2.

Altered energy metabolism is a cancer hallmark as malignant cells tailor their metabolic pathways to meet their energy requirements. Glucose and glutamine are the major nutrients that fuel cellular metabolism, and the pathways utilizing these nutrients are often altered in cancer. Here, we show that the long ncRNA CCAT2, located at the 8q24 amplicon on cancer risk-associated rs6983267 SNP, regulates cancer metabolism in vitro and in vivo in an allele-specific manner by binding the Cleavage Factor I (CFIm) complex with distinct affinities for the two subunits (CFIm25 and CFIm68). The CCAT2 interaction with the CFIm complex fine-tunes the alternative splicing of Glutaminase (GLS) by selecting the poly(A) site in intron 14 of the precursor mRNA. These findings uncover a complex, allele-specific regulatory mechanism of cancer metabolism orchestrated by the two alleles of a long ncRNA.

A Universal Stress Protein That Controls Bacterial Stress Survival in Micrococcus luteus.

Bacteria have remarkable mechanisms to survive severe external stresses, and one of the most enigmatic is the nonreplicative persistent (NRP) state. Practically, NRP bacteria are difficult to treat, and so inhibiting the proteins underlying this survival state may render such bacteria more susceptible to external stresses, including antibiotics. Unfortunately, we know little about the proteins and mechanisms conferring survival through the NRP state. Here, we report that a universal stress protein (Usp) is a primary regulator of bacterial survival through the NRP state in Micrococcus luteus NCTC 2665, a biosafety level 1 (BSL1) mycobacterial relative. Usps are widely conserved, and bacteria, including Mycobacterium tuberculosis, Mycobacterium smegmatis, and Escherichia coli, have multiple paralogs with overlapping functions that have obscured their functional roles. A kanamycin resistance cassette inserted into the M. luteus universal stress protein A 616 gene (ΔuspA616::kanM. luteus) ablates the UspA616 protein and drastically impairs M. luteus survival under even short-term starvation (survival, 83% wild type versus 32% ΔuspA616::kanM. luteus) and hypoxia (survival, 96% wild type versus 48% ΔuspA616::kanM. luteus). We observed no detrimental UspA616 knockout phenotype in logarithmic growth. Proteomics demonstrated statistically significant log-phase upregulation of glyoxylate pathway enzymes isocitrate lyase and malate synthase in ΔuspA616::kanM. luteus We note that these enzymes and the M. tuberculosis UspA616 homolog (Rv2623) are important in M. tuberculosis virulence and chronic infection, suggesting that Usps are important stress proteins across diverse bacterial species. We propose that UspA616 is a metabolic switch that controls survival by regulating the glyoxylate shunt.IMPORTANCE Bacteria tolerate severe external stresses, including antibiotics, through a nonreplicative persistent (NRP) survival state, yet the proteins regulating this survival state are largely unknown. We show a specific universal stress protein (UspA616) controls the NRP state in Micrococcus luteus Usps are widely conserved across bacteria, but their biological function(s) has remained elusive. UspA616 inactivation renders M. luteus susceptible to stress: bacteria die instead of adapting through the NRP state. UspA616 regulates malate synthase and isocitrate lyase, glyoxylate pathway enzymes important for chronic Mycobacterium tuberculosis infection. These data show that UspA616 regulates NRP stress survival in M. luteus and suggest a function for homologous proteins in other bacteria. Importantly, inhibitors of UspA616 and homologs may render NRP bacteria more susceptible to stresses, including current antibiotics.

A Proteomic Signature of Dormancy in the Actinobacterium Micrococcus luteus.

Dormancy is a protective state in which diverse bacteria, including Mycobacterium tuberculosis, Staphylococcus aureus, Treponema pallidum (syphilis), and Borrelia burgdorferi (Lyme disease), curtail metabolic activity to survive external stresses, including antibiotics. Evidence suggests dormancy consists of a continuum of interrelated states, including viable but nonculturable (VBNC) and persistence states. VBNC and persistence contribute to antibiotic tolerance, reemergence from latent infections, and even quorum sensing and biofilm formation. Previous studies indicate that the protein mechanisms regulating persistence and VBNC states are not well understood. We have queried the VBNC state of Micrococcus luteus NCTC 2665 (MI-2665) by quantitative proteomics combining gel electrophoresis, high-performance liquid chromatography, and tandem mass spectrometry to elucidate some of these mechanisms. MI-2665 is a nonpathogenic actinobacterium containing a small (2.5-Mb), high-GC-content genome which exhibits a well-defined VBNC state induced by nutrient deprivation. The MI-2665 VBNC state demonstrated a loss of protein diversity accompanied by increased levels of 18 proteins that are conserved across actinobacteria, 14 of which have not been previously identified in VNBC. These proteins implicate an anaplerotic strategy in the transition to VBNC, including changes in the glyoxylate shunt, redox and amino acid metabolism, and ribosomal regulatory processes. Our data suggest that MI-2665 is a viable model for dissecting the protein mechanisms underlying the VBNC stress response and provide the first protein-level signature of this state. We expect that this protein signature will enable future studies deciphering the protein mechanisms of dormancy and identify novel therapeutic strategies effective against antibiotic-tolerant bacterial infections.IMPORTANCE Dormancy is a protective state enabling bacteria to survive antibiotics, starvation, and the immune system. Dormancy is comprised of different states, including persistent and viable but nonculturable (VBNC) states that contribute to the spread of bacterial infections. Therefore, it is imperative to identify how bacteria utilize these different dormancy states to survive antibiotic treatment. The objective of our research is to eliminate dormancy as a route to antibiotic tolerance by understanding the proteins that control dormancy in Micrococcus luteus NCTC 2665. This bacterium has unique advantages for studying dormancy, including a small genome and a well-defined and reproducible VBNC state. Our experiments implicate four previously identified and 14 novel proteins upregulated in VBNC that may regulate this critical survival mechanism.

The ability of human nuclear DNA to cause false positive low-abundance heteroplasmy calls varies across the mitochondrial genome.

BACKGROUND: Low-abundance mutations in mitochondrial populations (mutations with minor allele frequency ≤ 1%), are associated with cancer, aging, and neurodegenerative disorders. While recent progress in high-throughput sequencing technology has significantly improved the heteroplasmy identification process, the ability of this technology to detect low-abundance mutations can be affected by the presence of similar sequences originating from nuclear DNA (nDNA). To determine to what extent nDNA can cause false positive low-abundance heteroplasmy calls, we have identified mitochondrial locations of all subsequences that are common or similar (one mismatch allowed) between nDNA and mitochondrial DNA (mtDNA). RESULTS: Performed analysis revealed up to a 25-fold variation in the lengths of longest common and longest similar (one mismatch allowed) subsequences across the mitochondrial genome. The size of the longest subsequences shared between nDNA and mtDNA in several regions of the mitochondrial genome were found to be as low as 11 bases, which not only allows using these regions to design new, very specific PCR primers, but also supports the hypothesis of the non-random introduction of mtDNA into the human nuclear DNA. CONCLUSION: Analysis of the mitochondrial locations of the subsequences shared between nDNA and mtDNA suggested that even very short (36 bases) single-end sequencing reads can be used to identify low-abundance variation in 20.4% of the mitochondrial genome. For longer (76 and 150 bases) reads, the proportion of the mitochondrial genome where nDNA presence will not interfere found to be 44.5 and 67.9%, when low-abundance mutations at 100% of locations can be identified using 417 bases long single reads. This observation suggests that the analysis of low-abundance variations in mitochondria population can be extended to a variety of large data collections such as NCBI Sequence Read Archive, European Nucleotide Archive, The Cancer Genome Atlas, and International Cancer Genome Consortium.

Epigenetic alteration by DNA-demethylating treatment restores apoptotic response to glucocorticoids in dexamethasone-resistant human malignant lymphoid cells.

BACKGROUND: Glucocorticoids (GCs) are often included in the therapy of lymphoid malignancies because they kill several types of malignant lymphoid cells. GCs activate the glucocorticoid receptor (GR), to regulate a complex genetic network, culminating in apoptosis. Normal lymphoblasts and many lymphoid malignancies are sensitive to GC-driven apoptosis. Resistance to GCs can be a significant clinical problem, however, and correlates with resistance to several other major chemotherapeutic agents. METHODS: We analyzed the effect of treatment with the cytosine analogue 5 aza-2' deoxycytidine (AZA) on GC resistance in two acute lymphoblastic leukemia (T or pre-T ALL) cell lines- CEM and Molt-4- and a (B-cell) myeloma cell line, RPMI 8226. Methods employed included tissue culture, flow cytometry, and assays for clonogenicity, cytosine extension, immunochemical identification of proteins, and gene transactivation. High throughput DNA sequencing was used to confirm DNA methylation status. CONCLUSIONS: Treatment of these cells with AZA resulted in altered DNA methylation and restored GC-evoked apoptosis in all 3 cell lines. In CEM cells the altered epigenetic state resulted in site-specific phosphorylation of the GR, increased GR potency, and GC-driven induction of the GR from promoters that lie in CpG islands. In RPMI 8226 cells, expression of relevant coregulators of GR function was altered. Activation of p38 mitogen-activated protein kinase (MAPK), which is central to a feed-forward mechanism of site-specific GR phosphorylation and ultimately, apoptosis, occurred in all 3 cell lines. These data show that in certain malignant hematologic B- and T-cell types, epigenetically controlled GC resistance can be reversed by cell exposure to a compound that causes DNA demethylation. The results encourage studies of application to in vivo systems, looking towards eventual clinical applications.

Functional and structural characterization of Hyp730, a highly conserved and dormancy-specific hypothetical membrane protein.

Membrane proteins represent major drug targets, and the ability to determine their functions, structures, and conformational changes will significantly advance mechanistic approaches to both biotechnology and bioremediation, as well as the fight against pathogenic bacteria. A pertinent example is Mycobacterium tuberculosis (H37Rv), which contains ~4000 protein-coding genes, with almost a thousand having been categorized as 'membrane protein', and a few of which (~1%) have been functionally characterized and structurally modeled. However, the functions and structures of most membrane proteins that are sparsely, or only transiently, expressed, but essential in small phenotypic subpopulations or under stress conditions such as persistence or dormancy, remain unknown. Our deep quantitative proteomics profiles revealed that the hypothetical membrane protein 730 (Hyp730) WP_010079730 (protein ID Mlut_RS11895) from M. luteus is upregulated in dormancy despite a ~5-fold reduction in overall protein diversity. Its H37Rv paralog, Rv1234, showed a similar proteomic signature, but the function of Hyp730-like proteins has never been characterized. Here, we present an extensive proteomic and transcriptomic analysis of Hyp730 and have also characterized its in vitro recombinant expression, purification, refolding, and essentiality as well as its tertiary fold. Our biophysical studies, circular dichroism, and tryptophan fluorescence are in immediate agreement with in-depth in silico 3D-structure prediction, suggesting that Hyp730 is a double-pass membrane-spanning protein. Ablation of Hyp730-expression did not alter M. luteus growth, indicating that Hyp730 is not essential. Structural homology comparisons showed that Hyp730 is highly conserved and non-redundant in G+C rich Actinobacteria and might be involved, under stress conditions, in an energy-saving role in respiration during dormancy.

A color-based competition assay for studying bacterial stress responses in Micrococcus luteus.

Competition assays measure differences between populations of bacteria after stress adaptation, populations of different bacteria and mutations in antibiotic resistance genes. We have developed a competition-based assay to evaluate if genes upregulated under starvation are important for bacterial survival. Stress responses are critical for survival in non-pathogenic and pathogenic bacteria alike including Mycobacterium tuberculosis, Enterococcus fecaelis, Escherichia coli and Staphylococcus aureus. Unfortunately, most stress-survival proteins are poorly understood because suitable model bacteria and techniques are limited. To address this problem, we have engineered Micrococcus luteus NCTC 2665 (M. luteus) for competition assays by inactivating the sarcinaxanthin biosynthesis gene crtE (ΔcrtE), changing M. luteus colonies from yellow to white. This change allows easy identification in mixed cultures. The crtE knockout is relatively neutral for growth in complex and minimal acetate media and shows a measured fitness of one in competition with yellow wild-type bacteria. The ΔcrtE M. luteus competition assay identified a competition defect in a M. luteus strain when a specific universal stress protein was inactivated, suggesting a negative survival phenotype for this protein. We anticipate this competition assay can identify defects in other gene knockouts and mutational studies in M. luteus and will enhance our understanding of bacterial survival mechanisms.

Thermodynamic and EPR studies of slowly relaxing ubisemiquinone species in the isolated bovine heart complex I.

Previously, we investigated ubisemiquinone (SQ) EPR spectra associated with NADH-ubiquinone oxidoreductase (complex I) in the tightly coupled bovine heart submitochondrial particles (SMP). Based upon their widely differing spin relaxation rate, we distinguished SQ spectra arising from three distinct SQ species, namely SQ(Nf) (fast), SQ(Ns) (slow), and SQ(Nx) (very slow). The SQ(Nf) signal was observed only in the presence of the proton electrochemical gradient (deltamu(H)(+)), while SQ(Ns) and SQ(Nx) species did not require the presence of deltamu(H+). We have now succeeded in characterizing the redox and EPR properties of SQ species in the isolated bovine heart complex I. The potentiometric redox titration of the g(z,y,x)=2.00 semiquinone signal gave the redox midpoint potential (E(m)) at pH 7.8 for the first electron transfer step [E(m1)(Q/SQ)] of -45 mV and the second step [E(m2)(SQ/QH(2))] of -63 mV. It can also be expressed as [E(m)(Q/QH(2))] of -54 mV for the overall two electron transfer with a stability constant (K(stab)) of the SQ form as 2.0. These characteristics revealed the existence of a thermodynamically stable intermediate redox state, which allows this protein-associated quinone to function as a converter between n=1 and n=2 electron transfer steps. The EPR spectrum of the SQ species in complex I exhibits a Gaussian-type spectrum with the peak-to-peak line width of approximately 6.1 G at the sample temperature of 173 K. This indicates that the SQ species is in an anionic Q(-) state in the physiological pH range. The spin relaxation rate of the SQ species in isolated complex I is much slower than the SQ counterparts in the complex I in situ in SMP. We tentatively assigned slow relaxing anionic SQ species as SQ(Ns), based on the monophasic power saturation profile and several fold increase of its spin relaxation rate in the presence of reduced cluster N2. The current study also suggests that the very slowly relaxing SQ(Nx) species may not be an intrinsic complex I component. The functional role of SQ(Ns) is further discussed in connection with the SQ(Nf) species defined in SMP in situ.

Bismuth-dithiol inhibition of the Escherichia coli rho transcription termination factor.

Bismuth-dithiol mixtures are proven antimicrobial agents with unknown mechanism(s) of action. We show that select bismuth-dithiol solutions inhibit the Escherichia coli rho transcription termination factor. Rho is an essential enzyme in most Gram-negative prokaryotes and without rho function the cells are not viable. Bismuth complexes with 2,3-dimercapto-1-propanol (BiBAL) (3:1 solutions) functioned as a noncompetitive inhibitor with respect to ATP in the rho poly(C)-dependent ATPase assay (I50=60 microM) and as a competitive inhibitor with respect to ribo(C)10 in the poly(dC)-ribo(C)10-dependent ATPase assay. The minimum inhibitory concentration (MIC) of bacterial growth for BiBAL (3:1) in the liquid culture assay using E. coli W3350 was 16 microM. Using the tnaA/lacZ fusion reporter assay we showed that sublethal amounts (3 microM) of BiBAL (3:1 solution) led to a small increase (37%) in in vivo beta-galactosidase activity in E. coli SVS1144, which corresponds to antitermination of the tna operon as a result of rho inhibition. We concluded that BiBAL was a potent in vitro rho inhibitor but its effect on in vivo rho processes was modest indicating that other mechanisms contributed to the antibacterial activity of BiBAL. Our study suggests that structural changes in the dithiol unit that provide greater bismuth binding may improve rho specificity, a macromolecular target not previously recognized for bismuth therapy.

Cyanobacterial signature genes.

A comparison of 8 cyanobacterial genomes reveals that there are 181 shared genes that do not have obvious orthologs in other bacteria. These signature genes define aspects of the genotype that are uniquely cyanobacterial. Approximately 25% of these genes have been associated with some function. These signature genes may or may not be involved in photosynthesis but likely they will be in many cases. In addition, several examples of widely conserved gene order involving two or more signature genes were observed. This suggests there may be regulatory processes that have been preserved throughout the long history of the cyanobacterial phenotype. The results presented here will be especially useful because they identify which of the many genes of unassigned function are likely to be of the greatest interest.

NADH-Ubiquinone oxidoreductase: substrate-dependent oxygen turnover to superoxide anion as a function of flavin mononucleotide.

Bovine heart mitochondrial NADH-ubiquinone oxidoreductase (complex I) catalyzed NADH- and ubiquinone-1-dependent oxygen (O2) turnover to hydrogen peroxide that was stimulated by piericidin A and superoxide dismutase (SOD), but was insensitive to antimycin A, myxothiazol, and potassium cyanide. The extent of O2 consumption as a function of ubiquinone-1 did not correlate with piericidin A-sensitive rates of ubiquinone reduction. Decylubiquinone did not stimulate O2 consumption, but did initiate an SOD-sensitive cytochrome c reduction when complex I was isolated away from ubiquinol-cytochrome c oxidoreductase. Rates and extent of O2 turnover (ROS production) and ubiquinone reduction were higher than previously reported for submitochondrial particles (SMP) and isolated complex I. This ROS production was shown to co-isolate with complex I flavin.

Role of the C(1) Triol Group in Bicyclomycin: Synthesis and Biochemical and Biological Properties.

Bicyclomycin (1) is a commercial antibiotic whose primary site of action in Escherichia coli is the essential cellular protein transcription termination factor rho. The bicyclomycin binding domain in rho is unknown; however, enzyme irreversible inactivators that modify rho upon activation may identify the site. In this study, we investigated the importance for rho binding of the C(1) triol group in 1. Twelve bicyclomycin derivatives were prepared, and the C(1) triol group was modified at the C(1'), the C(2'), and the C(3') sites. The compounds were evaluated by rho-dependent ATPase and transcription termination assays and their antimicrobial activities assessed using a filter disc assay. Bicyclomycin inhibited both rho-dependent ATPase (I(50) = 60 &mgr;M) and rho-dependent transcription termination (I(50) approximately 5 &mgr;M) processes and had a minimum inhibitory concentration value of 0.25 mg/mL against E. coli W3350 cells. None of the 12 C(1) triol bicyclomycin derivatives significantly inhibited rho-dependent ATPase (I(50) > 400 &mgr;M) and transcription termination (I(50) > 100 &mgr;M) activities or exhibited antibiotic activity at a 32 mg/mL concentration. These results indicated that there was a strong molecular complement between the C(1) triol group and its rho binding site. We concluded that the C(1) triol group in 1 is a critical structural element necessary for drug binding to rho and that an enzyme irreversible inactivating unit placed at this site would prohibit the bicyclomycin derivative from efficiently binding to rho.

Role of the [4.2.2] Bicyclic Unit in Bicyclomycin: Synthesis, Structure, Chemical, Biochemical, and Biological Properties.

Twelve bicyclomycin derivatives were synthesized to determine the effect of modification of the [4.2.2] bicyclic unit in bicyclomycin (1) on drug function. Few bicyclomycin derivatives have been described in which the [4.2.2] ring system has been modified. The compounds evaluated were divided into two categories: the two N-methyl-modified bicyclomycins (2, 3) and the ten C(6)-substituted bicyclomycins (4-13). Substituents introduced at the C(6) site included alkoxy, thioalkoxy, thiophenoxy, anilino, and hydrogen. A procedure was developed to synthesize select C(6)-substituted bicyclomycins. Bicyclomycin was first converted to bicyclomycin C(2'),C(3')-acetonide (16) and then treated with methanesulfonyl chloride to give in situ the corresponding C(6) mesylate 17. Treatment of 17 with the appropriate nucleophile followed by removal of the C(2'),C(3')-acetonide group gave the desired C(6)-substituted bicyclomycin. The chemical properties of C(6) O-methylbicyclomycin (4) were examined. Treatment of THF-H(2)O mixtures of 4 with excess EtSH maintained at "pH" 8.0-9.0 led to no detectable reaction, while at more basic "pH" values 4 underwent stereospecific conversion to the bis-spiro derivative 33 and no appreciable EtSH addition to the C(5)-C(5a) exomethylene unit. These results were compared to the reactivity of 1 with EtSH. The stability (pH 7.4, 37 degrees C) of C(6)-substituted bicyclomycins 4, 6, and 10-13 in aqueous solutions were examined. We observed that most of these compounds (4, 6, 10-12) underwent near complete change (>75%) within 200 h. The [4.2.2] bicyclic-modified bicyclomycins were evaluated in the rho-dependent ATPase assay and their antimicrobial activities determined using a filter disc assay. Most of the compounds were also tested in the transcription termination assay. We observed that all structural modifications conducted within the [4.2.2] bicyclic unit led to a loss of rho-dependent ATPase (I(50) > 400 &mgr;M) and to transcription termination (I(50) > 100 &mgr;M) inhibitory activities, as well as a loss of antimicrobial activity (MIC > 32 mg/mL). Only N(10)-methylbicyclomycin (2) displayed moderate inhibitory activities in these assays. These findings indicated that the [4.2.2] bicyclic unit played an important role in the antibiotic-rho recognition process. Potential factors that govern this interaction are briefly discussed. We concluded that placement of an irreversible inactivating unit at the N- and O-sites within the [4.2.2] bicyclic unit in 1 would likely prohibit the bicyclomycin derivative from efficiently binding to rho.

Role of the C(5)-C(5a) Exomethylene Group in Bicyclomycin: Synthesis, Structure, and Biochemical and Biological Properties.

Thirty-two C(5)-C(5a) exomethylene-modified bicyclomycin derivatives were prepared to determine the effect of structural modification of this unit on bicyclomycin (1) function. The compounds were grouped into three categories: the C(5)-unsaturated bicyclomycins, the C(5a)-substituted C(5)-C(5a)-dihydrobicyclomycin derivatives, and the C(5)-modified norbicyclomycins. An efficient three-step procedure was developed to synthesize C(5a)-substituted C(5),C(5a)-dihydrobicyclomycins. Bicyclomycin was converted to bicyclomycin C(2'),C(3')-acetonide (36) and then treated with a nucleophile in 50% aqueous methanol ("pH" 10.5) to give the C(5a)-substituted C(5),C(5a)-dihydrobicyclomycin C(2'),C(3')-acetonide. Removal of the acetonide group (trifluoroacetic acid in 50% aqueous methanol) in the final step provided the desired bicyclomycin derivative. All the compounds were evaluated using the rho-dependent ATPase assay and their antimicrobial activities determined using a filter disc assay. Most of the compounds were also tested in the transcription termination assay. We observed that many of the C(5)-unsaturated bicyclomycins effectively inhibited ATP hydrolysis at 400 &mgr;M and inhibited the production of rho-dependent transcripts at 100 &mgr;M. The biochemical activities of C(5a)-bicyclomycincarboxylic acid (5), methyl C(5a)-bicyclomycincarboxylate (6), ethyl C(5a)-bicyclomycincarboxylate (7), and bicyclomycin C(5)-norketone O-methyloxime (11) were all similar to 1. Compounds 6, 7, and 11 exhibited diminished antibiotic activity compared to 1, and 5 displayed no detectable activity. Several C(5a)-substituted C(5),C(5a)-dihydrobicyclomycins showed significant inhibition of rho-dependent ATPase and transcription termination activities. The inhibitory properties of C(5),C(5a)-dihydrobicyclomycin C(5a)-methyl sulfide (18), C(5),C(5a)-dihydrobicyclomycin C(5a)-phenyl sulfide (23), and C(5)-C(5a)-dihydrobicyclomycin-5,5a-diol (31) approached those of 1. Compounds 18, 23, and 31 did not exhibit antibiotic activity. Two of the four C(5)-modified norbicyclomycin adducts showed moderate inhibitory activities in the ATPase assay, and none showed significant antibiotic activity. Our findings showed that the C(5)-C(5a) exomethylene unit retention in 1 was not essential for inhibition of in vitro rho activity. The structure-activity relationship data indicated that bicyclomycins that contained a small unsaturated C(5) unit or C(5),C(5a)-dihydrobicyclomycins that possessed a small, nonpolar C(5a) substituent effectively inhibited rho function in in vitro biochemical assays. We concluded that the C(5)-C(5a) unit in 1 was not a critical structural element necessary for drug binding to rho and that irreversible, inactivating units placed at this site would permit the bicyclomycin derivative to bind efficiently to rho.

Nonlinear dielectric spectroscopy as an indirect probe of metabolic activity in thylakoid membrane.

Nonlinear dielectric spectroscopy (NDS) is a non-invasive probe of cellular metabolic activity with potential application in the development of whole-cell biosensors. However, the mechanism of NDS interaction with metabolic membrane proteins is poorly understood, partly due to the inherent complexity of single cell organisms. Here we use the light-activated electron transport chain of spinach thylakoid membrane as a model system to study how NDS interacts with metabolic activity. We find protein modification, as opposed to membrane pump activity, to be the dominant source of NDS signal change in this system. Potential mechanisms for such protein modifications include reactive oxygen species generation and light-activated phosphorylation.

Development of a technique to determine bicyclomycin-rho binding and stoichiometry by isothermal titration calorimetry and mass spectrometry.

Bicyclomycin (1) is the only natural product inhibitor of the transcription termination factor rho. Rho is a hexameric helicase that terminates nascent RNA transcripts utilizing ATP hydrolysis and is an essential protein for many bacteria. The paucity of information concerning the 1-rho interaction stems from the weak binding affinity of 1. We report a novel technique using imine formation with rho to enhance the affinity of a bicyclomycin analogue and determine the binding stoichiometry by isothermal titration calorimetry (ITC) and mass spectrometry (MS). Our designed bicyclomycin ligand, 5a-(3-formyl-phenylsulfanyl)-dihydrobicyclomycin (2) (apparent I(50) = 4 muM), inhibits rho an order of magnitude more efficiently than 1 (I(50) = 60 muM). MS shows that 2 selectively forms an imine with K181 in rho. We found that despite the heterogeneity of ATP binding (three tight and three weak) imposed on the rho hexamer, the nearby bicyclomycin binding pocket is not affected, and both 1 and 2 bind with equal affinity to all six subunits.

The Mg2+ requirements for rho transcription termination factor: catalysis and bicyclomycin inhibition.

Kinetic studies document that the essential Escherichia coli transcription termination factor rho utilizes Mg(2+) and ATP as a substrate and requires a second Mg(2+) ion for maximum poly(C)-dependent ATP hydrolysis activity. The velocity curves show a classic nonessential Mg(2+) activation pattern in which Mg(2+) augments hydrolysis by 39% and gives a K(1)' for MgATP of 9.5 microM in the presence of excess Mg(2+) and a K(1) for MgATP of 21.2 microM under limiting Mg(2+) concentrations. Bicyclomycin (1), a commercial antibiotic that inhibits rho, weakened Mg(2+) binding at the nonessential site and disrupted the nonessential Mg(2+) activation pathway for poly(C)-dependent ATP hydrolysis. The K(i) values for 1 were 23 microM and 35 microM under excess and limiting Mg(2+) conditions, respectively, while the K(Mg(app)) for nonessential Mg(2+) increased with increasing 1 concentrations. These findings, when combined with reported mechanistic studies, provide an emerging picture of key catalytic and substrate binding sites that are necessary for rho function and that are proximal to the 1 binding site.

ATP binding to Rho transcription termination factor. Mutant F355W ATP-induced fluorescence quenching reveals dynamic ATP binding.

Rho transcription termination factor mutant, F355W, showed tryptophan fluorescence intensity approximately twice that of wild-type Rho at equivalent protein concentrations and underwent a decrease in relative fluorescence intensity at 350 nm when 100 microm ATP was added in the presence or absence of RNA. Titration of this fluorescence quenching with varying concentrations of ATP (0-600 microm), where Rho is shown to exist as a hexamer (400 nm Rho), revealed tight and loose ATP-binding sites. Bicyclomycin, a specific inhibitor of Rho, increased the tight ATP binding and was used to calibrate ATP-induced fluorescence quenching by using [gamma-(32)P]ATP filter binding. For the Rho mutant F355W, three tight (K(d)(1) = 3 +/- 0.3 microm) and three loose (K(d)(2) = 58 +/- 3 microm) ATP-binding sites per hexamer were seen on Scatchard analysis in the absence of bicyclomycin and poly(C). In the presence of bicyclomycin, the K(d)(1) changed from 3.0 to 1.4 microm, but K(d)(2) underwent a lesser change. The non-hydrolyzable ATP analogue, gamma-S-ATP, gave a similar profile with three tight (K(d)(1) = 0.2 microm) and three loose (K(d)(2) = 70 microm) ATP-binding sites per hexamer. Adding poly(C) to F355W did not alter the K(d)(1) or K(d)(2) for ATP or for gamma-S-ATP. ADP-induced quenching produced 5.5 loose (K(d) = 92 microm) binding sites in the absence of poly(C), and the binding became weaker (K(d) = 175 microm) in the presence of poly(C). The data suggest that in the presence of ADP Rho has six equivalent nucleotide-binding sites. When ATP was added these sites converted to three tight and three loose binding loci. We propose an alternating ATP site mechanism where ATP binding creates heterogeneity in the ATP binding in adjacent subunits, and we suggest that ATP binding to a neighboring loose site stimulates hydrolysis at a neighboring tight binding site such that all six subunits can be potential "active" sites for ATP hydrolysis. The dynamic nature of the ATP binding to Rho is discussed in the terms of the mechanism of RNA tracking driven by ATP hydrolysis.

Mutations in the rho transcription termination factor that affect RNA tracking.

Model studies have identified 16 conserved positively charged amino acids that form a positive strip pointing toward the center hole of Rho. Fourteen residues were individually changed to either an alanine or a glycine and one to a glutamate. Residues Arg(269), Arg(272), Lys(283), Arg(296), Lys(298), and Arg(299) form a subdomain (locus) located N-terminal to (above) the ATP hydrolysis domain (P-loop) and mutations in these residues led to either inactive Rho or to proteins displaying decreased k(cat) for poly(C)-dependent ATP hydrolysis, increased K(m) for ribo(C)(10) activation, and decreased transcription termination efficiencies (57-77%) compared with wild-type Rho. Residues Arg(347), Lys(348), Lys(352), and Arg(353) form a subdomain (locus) C-terminal to (below) the ATP hydrolysis domain, and mutations in these residues also show a decreased k(cat) for poly(C)-dependent ATP hydrolysis, an increased K(m) for ribo(C)(10) activation, and a 50-70% decrease in transcription termination, compared with wild-type Rho. Residues Arg(212) and Lys(336) surround the ATP hydrolysis domain, and mutations in these residues also altered the kinetic properties of Rho. We conclude that the secondary RNA-tracking site consists of amino acids whose putative orientation faces the central hole in Rho and in part reside in two clusters of positively charged residues located above and below the ATP hydrolysis domain.