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The riboflavin kinase in Methanocaldococcus jannaschii has been identified as the product of the MJ0056 gene. Recombinant expression of the MJ0056 gene in Escherichia coli led to a large increase in the amount of flavin mononucleotide (FMN) in the E. coli cell extract. The unexpected features of the purified recombinant enzyme were its use of CTP as the phosphoryl donor and the absence of a requirement for added metal ion to catalyze the formation of FMN. Identification of this riboflavin kinase fills another gap in the archaeal flavin biosynthetic pathway. Some divalent metals were found to be potent inhibitors of the reaction. The enzyme represents a unique CTP-dependent family of kinases.

A novel separation method was developed to isolate directly cytidine triphosphate (CTP) from fermentation broth of yeast using anion-exchange supermacroporous cryogel. The anion-exchange cryogel with tertiary amine groups was prepared by graft polymerization. The breakthrough characteristics and elution performance of pure CTP in the cryogel bed were investigated experimentally and the CTP binding capacity was determined. Then the separation experiments of CTP from crude fermentation broth of yeast using the cryogel column were carried out using deionized water and 0.01 M HCl as washing buffer, respectively. The chromatographic behavior was monitored and analyzed. The purity and concentration of the obtained CTP in these processes were determined quantitatively by HPLC. The maximal purity of CTP obtained at the condition of 0.01 M HCl as washing buffer and 0.5 M NaCl in 0.01 M HCl as elution buffer reached 93%.

Purpose: The aim of the study is to synthesize and characterize nanogel carriers composed of amphiphilic polymers and cationic polyethylenimine for encapsulation and delivery of cytotoxic nucleoside analogs 5'-triphosphates (NTPs) into cancer cells., Methods: Nanogels were synthesized by a novel micellar approach and compared with carriers prepared by the emulsification/evaporation method. Complexes of nanogels with NTP were prepared; particle size and in vitro drug release were characterized. Resistance of the nanogel-encapsulated NTP to enzymatic hydrolysis was analyzed by ion-pair high-performance liquid chromatography. Binding to isolated cellular membranes, cellular accumulation and cytotoxicity were compared using breast carcinoma cell lines CL-66, MCF-7, and MDA-MB-231. In vivo biodistribution of the 3H-labeled NTP encapsulated in different types of nanogels was evaluated in comparison to the injected NTP alone., Results: Nanogels with a particle size of 100-300 nm in the unloaded form and less than 140 nm in the NTP-loaded form were prepared. An in vitro release of NTP was >50% during the first 24 h. Nanogel formulations ensured increased NTP drug stability against enzymatic hydrolysis as compared to the drug alone. Pluronic-based nanogels NG(F68), NG(F127), NG(P85), and NGM(P123) demonstrated 2-2.5 times enhanced interaction with cellular membranes and association with various cancer cells compared to NG(PEG). Among them, NG(F68) and NG(F127) exhibited the lowest cytotoxicity. Injection of nanogel-formulated NTP significantly modulated the drug accumulation in different mouse organs., Conclusions: Nanogels composed of Pluronic F68 and P123 were shown to display certain advanced properties compared to NG(PEG) as a drug delivery system for NTP analogs. Formulations of nucleoside analogs in active NTP form with these nanogels will improve the delivery of these cytotoxic drugs to cancer cells and the therapeutic potential of this anticancer chemotherapy.

Efforts to correlate genetic variations with phenotypic differences are intensifying due to the availability of high-density maps of single nucleotide polymorphisms (SNPs) and the development of high throughput scoring methods. These recent advances have led to an increased interest for improved multiplex preparations of genetic material to facilitate such whole genome analyses. Here we propose a strategy for the parallel amplification of polymorphic loci based on a reduced set of nucleotides. The technique denoted Tri-nucleotide Threading (TnT), allows SNPs to be amplified via controlled linear amplification followed by complete removal of the target material and subsequent amplification with a pair of universal primers. A dedicated software tool was developed for this purpose and variable positions in genes associated with different forms of cancer were analyzed using sub-nanogram amounts of starting material. The amplified fragments were then successfully scored using a microarray-based PrASE technique. The results of this study, in which 75 SNPs were analyzed, show that the TnT technique circumvents potential problems associated with multiplex amplification of SNPs from minute amounts of material. The technique is specific, sensitive and can be readily adapted to equipment and genotyping techniques used in other research laboratories without requiring changes to the preferred typing method.

Human DNA apurinic/apyrimidinic (AP-) endonuclease 1 (APE1) is involved in the base excision repair (BER) pathway. The enzyme hydrolyzes DNA from the 5 side of the AP site. In addition to endonuclease activity, APE1 also possesses other slight activities including 3 -5 exonuclease activity. The latter is preferentially exhibited towards mispaired (non-canonical) nucleotides, this being the reason why APE1 is considered as a proofreading enzyme correcting the misincorporations introduced by DNA polymerase beta. We have studied 3 -5 exonuclease activity of APE1 towards dCMP and dTMP residues and modified dCMP analogs with photoreactive groups at the 3 end of the nicked DNA. Photoreactive dNMP residues were incorporated at the 3 end of the lesion using DNA polymerase beta and photoreactive dNTPs. The dependence of exonuclease activity on the "canonicity" of the base pair formed by dNMP flanking the nick at the 3 end, on the nature of the group flanking the nick at the 5 end, and on the reaction conditions has been determined. Optimal reaction conditions for the 3 -5 exonuclease hydrolysis reaction catalyzed by APE1 in vitro have been established, and conditions when photoreactive residues are not removed by APE1 have been chosen. These reaction conditions are suitable for using photoreactive nicked DNAs bearing 3 -photoreactive dNMP residues for photoaffinity labeling of proteins in cellular/nuclear extracts and model APE1-containing systems. We recommend using FAPdCTP for photoaffinity modification in APE1-containing systems because the FAPdCMP residue is less prone to exonuclease degradation, in contrast to FABOdCTP, which is not recommended.

We engineer colloidal quantum dot nanocrystals through the choice of biomolecular ligands responsible for nanoparticle nucleation, growth, stabilization, and passivation. We systematically vary the presence of, and thereby elucidate the role of, phosphate groups and a multiplicity of functionalities on the mononucleotides used as ligands. The results provide the basis for synthesis of nanoparticles using precisely controlled synthetic oligonucleotide sequences.

The interactions between pyrimidine nucleotides: cytidine-5'-diphosphate (CDP) and cytidine-5'-triphosphate (CTP) and Cu(II) ions, spermine (Spm) and 1,11-diamino-4,8-diazaundecane (3,3,3-tet) have been studied. The composition and stability constants of the complexes formed have been determined by means of the potentiometric method, while the centres of interactions in the ligands have been identified by the spectral methods (UV-Vis, Ultraviolet and Visible spectroscopy; EPR, electron spin resonance; NMR). In the systems without metal, formation of the molecular complexes nucleotide-polyamine with the interaction centres at the endocyclic nitrogen atom of purine ring N3, the oxygen atoms of the phosphate group from the nucleotide and protonated nitrogen atoms of the polyamine have been detected. Significant differences have been found in the metallation between the systems with Spm and with 3,3,3-tet. In the systems with spermine, mainly protonated species are formed with the phosphate group of the nucleotide and deprotonated nitrogen atoms of the polyamine making the coordination centres, while the donor nitrogen atom of the nucleotide N3 is involved in the intramolecular interligand interactions, additionally stabilising the complex. In the systems with 3,3,3-tet, the MLL' type species are formed in which the oxygen atoms of the phosphate group and nitrogen atoms of the polyamine are involved in metallation, whereas the N3 atom from the pyrimidine ring of the nucleotide is located outside the inner coordination sphere of copper ion. The main centre of Cu(II) interaction in the nucleotide, both in the system with Spm and 3,3,3-tet is the phosphate group of the nucleotide.

The DEAH protein Prp22 is important for the second transesterification step of pre-mRNA splicing, and it is essential for releasing mature mRNA from the spliceosome. Recombinant Prp22 has RNA-stimulated ATPase and ATP-dependent unwinding activities, which are crucial for the mRNA release step. In this study, we characterize the RNA-binding, NTP hydrolysis, and RNA unwinding functions of Prp22. Using nitrocellulose filter binding assays, we determined that the apparent affinity of Prp22 is approximately 20-fold greater for single-stranded RNA than for single-stranded DNA or duplex nucleic acids. Inclusion of hydrolyzable ATP in binding reactions increased the apparent K(D) for RNA by 3-4-fold. The Prp22-RNA interaction is influenced by the length of the RNA chain, and the apparent K(D) values for poly(A)(40) and poly(A)(10) are 17 and 140 nM, respectively. RNA-stimulated ATP hydrolysis is similarly affected by chain length, and optimal activity requires RNA oligomers of >or=20 nt. We show that Prp22 can hydrolyze all common NTPs and dNTPs with comparable efficiencies and that Prp22 unwinds RNA duplexes with 3' to 5' directionality.

Polyadenylate polymerase (PAP) catalyzes the synthesis of 3'-polyadenylate tails onto mRNA. A comprehensive steady-state kinetic analysis of PAP was conducted which included initial velocity studies of the forward and reverse reactions, inhibition studies, and the use of alternative substrates. The reaction (A(n) + ATP <--> A(n+1) + PP(i)) is adequately described by a rapid equilibrium random mechanism. Several thermodynamic parameters for the reaction were determined or calculated, including the overall equilibrium constant (K(eq) = 84) and the apparent equilibrium constant of the internal step (K(int) = 4) which involves the rate-determining interconversion of central complexes. A large (100-fold) difference in Vmax accounts for nucleotide specificity (ATP vs CTP), despite an only 3-fold difference in Km. Comparison of the sulfur elemental effect on Vmax for ATP and CTP suggests that the chemical step is rate-determining for both reactions. Comparison of the sulfur elemental effect on Vmax/Km revealed differences in the mechanism by which either nucleotide is incorporated. Consistent with these data, an induced fit mechanism for nucleotide specificity is proposed whereby PAP couples a uniform binding mechanism, which selects for ATP, with a ground-state destabilization mechanism, which serves to accelerate the velocity for the correct substrate.

The synthesis of the triphosphates of 4'-thiouridine and 4'-thiocytidine, 4'-thioUTP (7; thioUTP) and 4'-thioCTP (8; thioCTP), and their utility for SELEX (systematic evolution of ligands by exponential enrichment) is described. The new nucleoside triphosphate (NTP) analogs 7 and 8 were prepared from appropriately protected 4'-thiouridine and -cytidine derivatives using the one-pot method reported by J. Ludwig and F. Eckstein [(1989) J. Org. Chem., 54, 631-635]. Because SELEX requires both in vitro transcription and reverse transcription, we examined the ability of 7 and 8 for SELEX by focusing on the two steps. Incorporation of 7 and 8 by T7 RNA polymerase to give 4'-thioRNA (thioRNA) proceeded well and was superior to those of the two sets of frequently used modified NTP analogs for SELEX (2'-NH2dUTP and 2'-NH2dCTP; 2'-FdUTP and 2'-FdCTP), when an adequate leader sequence of DNA template was selected. We revealed that a leader sequence of about +15 of DNA template is important for the effective incorporation of modified NTP analogs by T7 RNA polymerase. In addition, reverse transcription of the resulting thioRNA into the complementary DNA in the presence of 2'-deoxynucleoside triphosphates (dNTPs) also proceeded smoothly and precisely. The stability of the thioRNA toward RNase A was 50 times greater than that of the corresponding natural RNA. With these successful results in hand, we attempted the selection of thioRNA aptamers to human alpha-thrombin using thioUTP and thioCTP, and found a thioRNA aptamer with high binding affinity (K(d) = 4.7 nM).

Polymerases form a class of enzymes that act as molecular motors as they move along their nucleic acid substrate during catalysis, incorporating nucleotide triphosphates at the end of the growing chain and consuming chemical energy. A debated issue is how the enzyme converts chemical energy into motion [J. Gelles and R. Landick, Cell 93, 13 (1998)]. In a single molecule assay, we studied how an opposing mechanical force affects the translocation rate of T7 RNA polymerase. Our measurements show that force acts as a competitive inhibitor of nucleotide binding. This result is interpreted in the context of possible models, and with respect to published crystal structures of T7 RNA polymerase. The transcribing complex appears to utilize only a small fraction of the energy of hydrolysis to perform mechanical work, with the remainder being converted to heat.

Tyvelose is a 3,6-dideoxyhexose found in the O-antigen of the surface lipopolysaccharides of some pathogenic bacteria. It is synthesized via a complex biochemical pathway that is initiated by the formation of CDP-D-glucose. The production of this ligand is catalyzed by the enzyme glucose-1-phosphate cytidylyltransferase, which utilizes alpha-D-glucose 1-phosphate and MgCTP as substrates. Previous x-ray crystallographic investigations have demonstrated that the Salmonella typhi enzyme complexed with the product CDP-glucose is a fully integrated hexamer displaying 32 point group symmetry. The binding pocket for CDP-glucose is shared between two subunits. Here we describe both a detailed kinetic analysis of the cytidylyltransferase and a structural investigation of the enzyme complexed with MgCTP. These data demonstrate that the reaction catalyzed by the cytidylyltransferase proceeds via a sequential rather than a Bi Bi ping-pong mechanism as was previously reported. Additionally, the enzyme utilizes both CTP and UTP equally well as substrates. The structure of the enzyme with bound MgCTP reveals that the binding pocket for the nucleotide is contained within one subunit rather than shared between two. Key side chains involved in nucleotide binding include Thr(14), Arg(15), Lys(25), and Arg(111). In the previous structure of the enzyme complexed with CDP-glucose, those residues defined by Thr(14) to Ile(21) were disordered. The kinetic and x-ray crystallographic data presented here support a mechanism for this enzyme that is similar to that reported for the glucose-1-phosphate thymidylyltransferases.

Catalytic DNA cleavage reactions by an ATP-dependent deoxyribonuclease (DNase) from Micrococcus luteus were monitored directly with a DNA-immobilized 27-MHz quartz-crystal microbalance (QCM). The 27-MHz QCM is a very sensitive mass-measuring device in aqueous solution, as the frequency decreases linearly with increasing mass on the electrode at a nanogram level. Three steps in ATP-dependent DNA hydrolysis reactions, including (1) binding of DNase to the end of double-stranded DNA (dsDNA) on the QCM electrode (mass increase), (2) degradation of one strand of dsDNA in the 3' --> 5' direction depending on ATP (mass decrease), and (3) release of the enzyme from the nonhydrolyzed 5'-free-ssDNA (mass decrease), could be monitored stepwise from the time dependencies of QCM frequency changes. Kinetic parameters for each step were obtained as follows. The binding constant (K(a)) of DNase to the dsDNA was determined as (28 +/- 2) x 10(6) M(-)(1) (k(on) = (8.0 +/- 0.3) x 10(3) M (-)(1) s(-)(1) and k(off) = (0.29 +/-0.01) x 10(-)(3) s(-)(1)), and it decreased to (0.79 +/- 0.16) x 10(6) M(-)(1) (k'(on) = (2.3 +/- 0.2) x 10(3) M (-)(1) s(-)(1) and k'(off) = (2.9 +/- 0.1) x 10(-)(3) s(-)(1)) for the completely nonhydrolyzed 5'-free ssDNA. This is the reason the DNase bound to the dsDNA substrate can easily release from the nonhydrolyzed 5'-free-ssDNA after the complete hydrolysis of the 3' --> 5' direction of the complementary ssDNA. K(a) values depended on the DNA structures on the QCM, and the order of these values was as follows: the dsDNA having a 4-base-mismatched base-pair end (3) > the dsDNA having a 5' 15-base overhanging end (2) > the dsDNA having a blunt end (1) > the ssDNA having a 3'-free end (4) >> the ssDNA having a 5'-free end (5). Thus, DNase hardly recognized the free 5' end of ssDNA. Michaelis-Menten parameters (K(m) for ATP and k(cat)) of the hydrolysis process also could be obtained, and the order of k(cat)/K(m) was as follows: the dsDNA having a blunt end (1) approximately the dsDNA having a 4-base-mismatched base-pair end (3) > the ssDNA having a free 3' end (4) >> the ssDNA having a free 5' end (5). Thus, DNase could not recognize and not hydrolyze the free 5' end of ssDNA. The DNA hydrolysis reaction could be driven by dATP and GTP (purine base) as well as ATP, whereas the cleavage efficiency was very low driven with UTP, CTP (pyrimidine base), ADP, and AMP.

CMP-Kdn synthetase catalyses the reaction of sialic acids (Sia) and cytidine-5'-triphosphate (CTP) to the corresponding activated sugar nucleotide CMP-Sia and pyrophosphate PP(i). STD NMR experiments of a recombinant nucleotide cytidine-5'-monophosphate-3-deoxy-d-glycero-d-galacto-nonulosonic acid synthetase (CMP-Kdn synthetase) were performed to map the binding epitope of the substrate CTP and the product CMP-Neu5Ac. The STD NMR analysis clearly shows that the anomeric proton of the ribose moiety of both investigated compounds is in close proximity to the protein surface and is likely to play a key role in the binding process. The relative rates of the enzyme reaction, derived from (1)H NMR signal integrals, show that Kdn is activated at a rate 2.5 and 3.1 faster than Neu5Ac and Neu5Gc, respectively. Furthermore, proton-decoupled (31)P NMR spectroscopy was successfully used to follow the enzyme reaction and clearly confirmed the appearance of CMP-Sia and the inorganic pyrophosphate by-product.

N4-Acetyl-1-(2, 3-di-O-acetyl-4-thio-beta-D-arabinofuranosyl) cytosine (2) was synthesized in three steps from 1-(4-thio-beta-D-arabinofuranosyl) cytosine (1). The reaction of this partially blocked 4'-thio-ara-C derivative 2 with 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one gave the 5-phosphitylate derivative 3, which on reaction with pyrophosphate gave the 5'-nucleosidylcyclotriphosphite 4. Product 4 was then oxidized with iodine/pyridine/water and deblocked with concentrated ammonium hydroxide to provide the desired 4'-thio-ara-C-5'-triphosphate 5. This triphosphate 5 was converted to 4'-thio-ara-C -5'-monophosphate 6 by treatment with snake venom phosphodiesterase I. The details of the synthesis, purification, and characterization of both nucleotides are described.

Expression of the Escherichia coli nucleoid-associated protein Fis (factor for inversion stimulation) is controlled at the transcriptional level in accordance with the nutritional availability. It is highly expressed during early logarithmic growth phase in cells growing in rich medium but poorly expressed in late logarithmic and stationary phase. However, fis mRNA expression is prolonged at high levels throughout the logarithmic and early stationary phase when the preferred transcription initiation site (+1C) is replaced with A or G, indicating that initiation with CTP is a required component of the regulation pattern. We show that RNA polymerase-fis promoter complexes are short lived and that transcription is stimulated over 20-fold from linear or supercoiled DNA if CTP is present during formation of initiation complexes, which serves to stabilize these complexes. Use of fis promoter fusions to lacZ indicated that fis promoter transcription is sensitive to the intracellular pool of the predominant initiating NTP. Growth conditions resulting in increases in CTP pools also result in corresponding increases in fis mRNA levels. Measurements of NTP pools performed throughout the growth of the bacterial culture in rich medium revealed a dramatic increase in all four NTP levels during the transition from stationary to logarithmic growth phase, followed by reproducible oscillations in their levels during logarithmic growth, which later decrease during the transition from logarithmic to stationary phase. In particular, CTP pools fluctuate in a manner consistent with a role in regulating fis expression. These observations support a model whereby fis expression is subject to regulation by the availability of its initiating NTP.

Cytidine 5'-triphosphate (CTP) synthase catalyses the ATP-dependent formation of CTP from uridine 5'-triphosphate using either NH(3) or l-glutamine as the nitrogen source. The hydrolysis of glutamine is catalysed in the C-terminal glutamine amide transfer domain and the nascent NH(3) that is generated is transferred via an NH(3) tunnel [Endrizzi, J.A., Kim, H., Anderson, P.M. & Baldwin, E.P. (2004) Biochemistry43, 6447-6463] to the active site of the N-terminal synthase domain where the amination reaction occurs. Replacement of Leu109 by alanine in Escherichia coli CTP synthase causes an uncoupling of glutamine hydrolysis and glutamine-dependent CTP formation [Iyengar, A. & Bearne, S.L. (2003) Biochem. J.369, 497-507]. To test our hypothesis that L109A CTP synthase has a constricted or a leaky NH(3) tunnel, we examined the ability of wild-type and L109A CTP synthases to utilize NH(3), NH(2)OH, and NH(2)NH(2) as exogenous substrates, and as nascent substrates generated via the hydrolysis of glutamine, gamma-glutamyl hydroxamate, and gamma-glutamyl hydrazide, respectively. We show that the uncoupling of the hydrolysis of gamma-glutamyl hydroxamate and nascent NH(2)OH production from N(4)-hydroxy-CTP formation is more pronounced with the L109A enzyme, relative to the wild-type CTP synthase. These results suggest that the NH(3) tunnel of L109A, in the presence of bound allosteric effector guanosine 5'-triphosphate, is not leaky but contains a constriction that discriminates between NH(3) and NH(2)OH on the basis of size.

CTP synthetase (CTPs) catalyzes the last step in CTP biosynthesis, in which ammonia generated at the glutaminase domain reacts with the ATP-phosphorylated UTP at the synthetase domain to give CTP. Glutamine hydrolysis is active in the presence of ATP and UTP and is stimulated by the addition of GTP. We report the crystal structures of Thermus thermophilus HB8 CTPs alone, CTPs with 3SO4(2-), and CTPs with glutamine. The enzyme is folded into a homotetramer with a cross-shaped structure. Based on the binding mode of sulfate anions to the synthetase site, ATP and UTP are computer modeled into CTPs with a geometry favorable for the reaction. Glutamine bound to the glutaminase domain is situated next to the triad of Glu-His-Cys as a catalyst and a water molecule. Structural information provides an insight into the conformational changes associated with the binding of ATP and UTP and the formation of the GTP binding site.

A new system has been developed capable of monitoring conformational changes of the 240s loop of aspartate transcarbamoylase, which are tightly correlated with the quaternary structural transition, with high sensitivity in solution. Pyrene, a fluorescent probe, was conjugated to residue 241 in the 240s loop of aspartate transcarbamoylase to monitor changes in conformation by fluorescence spectroscopy. Pyrene maleimide was conjugated to a cysteine residue on the 240s loop of a previously constructed double catalytic chain mutant version of the enzyme, C47A/A241C. The pyrene-labeled enzyme undergoes the normal T to R structural transition, as demonstrated by small-angle x-ray scattering. Like the wild-type enzyme, the pyrene-labeled enzyme exhibits cooperativity toward aspartate, and is activated by ATP and inhibited by CTP at subsaturating concentrations of aspartate. The binding of the bisubstrate analogue N-(phosphonoacetyl)-l-aspartate (PALA), or the aspartate analogue succinate, in the presence of saturating carbamoyl phosphate, to the pyrenelabeled enzyme caused a sigmoidal change in the fluorescence emission. Saturation with ATP and CTP (in the presence of either subsaturating amounts of PALA or succinate and carbamoyl phosphate) caused a hyperbolic increase and decrease, respectively, in the fluorescence emission. The half-saturation values from the fluorescence saturation curves and kinetic saturation curves were, within error, identical. Fluorescence and small-angle x-ray scattering stopped-flow experiments, using aspartate and carbamoyl phosphate, confirm that the change in excimer fluorescence and the quaternary structure change correlate. These results in conjunction with previous studies suggest that the allosteric transition involves both global and local conformational changes and that the heterotropic effect of the nucleotides may be exerted through local conformational changes in the active site by directly influencing the conformation of the 240s loop.

We synthesized 4'-thioUTP (1) and 4'-thioCTP (2) with the aim of developing new NTP analogs for in vitro selection. Since in vitro selection requires both in vitro transcription and reverse transcription, we examined the ability of 1 and 2 for in vitro selection by focusing on both steps. Incorporation of 1 and 2 by T7 RNA polymerase to give 4'-thioRNA proceeded well and was superior to those of the two sets of frequently used modified NTP analogs (2'-NH2dUTP and 2'-NH2dCTP, and 2'-FdUTP and 2'-FdCTP) for in vitro selection. In addition, reverse transcription of the resulting 4'-thioRNA into the complementary DNA in the presence of dNTPs also proceeded smoothly and precisely. With these successful results in hand, in vitro selection of human a-thrombin RNA aptamer using 1 and 2 is in progress.

Charged droplet processing methodology, that utilizes electrodynamic levitation technology to control the trajectories of picoliter volume charged droplets and deliver them to a target plate at atmospheric pressure, has been developed. Termed wall-less sample preparation (WaSP), this methodology offers several features that could prove beneficial to the preparation of sample spots from separation column effluents for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis. These features include solute pre-concentration factors of 10(1) to 10(3) due to volatile solvent evaporation prior to droplet deposition onto the target plate, high spatial accuracy of the deposition position of each processed droplet (+/-5 microm), and the ability to prepare sample spots as small as 20 microm in diameter from a single droplet. Here a new mode of operation of this methodology is described and used as an offline post-column pre-concentrating interface between capillary liquid chromatography (capLC) and a target plate for offline MALDI-MS. Using a fraction from the capLC separation of peptides produced by the proteolytic digestion of the protein cytidine 5'-triphosphate:phosphocholine cytidylyltransferase, MALDI sample spots were prepared using the dried-droplet method, direct piezoelectric droplet dispensing, and the processing of piezo-dispensed droplets by WaSP. The sample spot morphology was investigated using light microscopy, and peptide ion abundances produced by MALDI were measured using time-of-flight (TOF) MS. The advantages of developing an online capLC/WaSP interface with MALDI-MS in the future are discussed along with some of the challenges that may be encountered in such an endeavor., (Copyright (c) 2004 John Wiley & Sons, Ltd.)

The extreme T and R quaternary structures of the allosteric enzyme aspartate transcarbamoylase have been trapped by encapsulation in a silica sol-gel matrix. Detection of the specific quaternary structure present in the sol-gel was accomplished using a pyrene-labeled version of the enzyme that exhibited monomer fluorescence in the T quaternary structure and excimer fluorescence in the R quaternary structure. Using thin films of the encapsulated enzyme, kinetics of the T and R states could be determined without interconversion of the states. Using a monolith form of the encapsulated enzyme, the transition from the T or the R structure was monitored. Within the sol-gel matrix, the rate of the transition was slowed approximately 105 over that observed in solution.

Cytidine 5'-triphosphate synthase catalyses the ATP-dependent formation of CTP from UTP using either ammonia or l-glutamine as the source of nitrogen. When glutamine is the substrate, GTP is required as an allosteric effector to promote catalysis. Limited trypsin-catalysed proteolysis, Edman degradation, and site-directed mutagenesis were used to identify peptide bonds C-terminal to three basic residues (Lys187, Arg429, and Lys432) of Escherichia coli CTP synthase that were highly susceptible to proteolysis. Lys187 is located at the CTP/UTP-binding site within the synthase domain, and cleavage at this site destroyed all synthase activity. Nucleotides protected the enzyme against proteolysis at Lys187 (CTP > ATP > UTP > GTP). The K187A mutant was resistant to proteolysis at this site, could not catalyse CTP formation, and exhibited low glutaminase activity that was enhanced slightly by GTP. K187A was able to form tetramers in the presence of UTP and ATP. Arg429 and Lys432 appear to reside in an exposed loop in the glutamine amide transfer (GAT) domain. Trypsin-catalyzed proteolysis occurred at Arg429 and Lys432 with a ratio of 2.6 : 1, and nucleotides did not protect these sites from cleavage. The R429A and R429A/K432A mutants exhibited reduced rates of trypsin-catalyzed proteolysis in the GAT domain and wild-type ability to catalyse NH3-dependent CTP formation. For these mutants, the values of kcat/Km and kcat for glutamine-dependent CTP formation were reduced approximately 20-fold and approximately 10-fold, respectively, relative to wild-type enzyme; however, the value of Km for glutamine was not significantly altered. Activation of the glutaminase activity of R429A by GTP was reduced 6-fold at saturating concentrations of GTP and the GTP binding affinity was reduced 10-fold. This suggests that Arg429 plays a role in both GTP-dependent activation and GTP binding.

By following the kinetics of abortive and productive synthesis in single-round transcription assays, we confirm the existence of two general classes of initial transcribing complexes (ITCs), which we term "productive ITC" and "unproductive ITC". The productive ITCs are able to escape from the promoter rapidly to produce full-length transcripts, but only after carrying out an obligate series of abortive initiation steps. The unproductive ITCs were found to synthesize mostly abortive transcripts of 2-3 nucleotides and escape from the promoter extremely slowly, if at all. Formation of the unproductive ITC is not due to the inactive RNA polymerase. Instead, RNA polymerase molecules recovered from both the productive and unproductive ITC fractions were shown to carry out abortive and productive synthesis with both the partitioning tendency and transcription kinetics similar to those of the original enzyme. Our results suggest that early transcription complexes are partitioned into the productive and unproductive ITCs most likely during the formation of open promoter complexes. The extent of partitioning varies with individual promoter sequences and is dependent on the nature and concentration of the initiating nucleotide. Thus, multiple classes of ITCs can be formed during promoter binding and transcript initiation.

RNA chain initiation and promoter escape is the latter stage of transcription initiation. This stage is characterized by several well-defined biochemical events: synthesis and release of short RNA products ranging 2 to 15 nucleotides in length, release of the sigma subunit from the enzyme-promoter complex, and initial translocation of the polymerase away from the promoter. In this paper, we report the use of a steady-state transcription assay with [gamma-(32)P]ATP labeling to subject the RNA chain initiation-promoter escape reaction to quantitative analysis. The specific parameters we follow to describe the chain initiation-promoter escape process include the abortive and productive rates, the abortive probability, the abortive:productive ratio, and the maximal size of the abortive product. In this study, we measure these parameters for three bacteriophage promoters transcribed by Escherichia coli RNA polymerase: T7 A1, T5 N25, and T5 N25(antiDSR). Our studies show that all three promoters form substantial amounts of abortive products under all conditions we tested. However, each of the promoters shows distinct differences from the others when the various parameters are compared. At 100 microM NTP, in a 10 min reaction, the abortive and productive yields are 87 and 13%, respectively, for T7 A1; 97 and 3%, respectively, for T5 N25; and 99.4 and 0.6%, respectively, for T5 N25(antiDSR). These values correspond to approximately 7, 32, and 165 abortive transcripts per productive transcript for the three promoters, respectively. The yield of most of the abortive products is not affected by the elevated concentration of the NTP substrate corresponding to the next template-specified nucleotide; hence, abortive products are not normally formed through a simple process of "kinetic competition". Instead, formation of abortive products appears to be determined by intrinsic DNA signals embedded in the promoter recognition region and the initial transcribed sequence region of each promoter.

Abortive initiation and promoter escape are two principal biochemical reactions occurring in the latter stage of transcript initiation. We have analyzed the influences of individual DNA elements within the promoter recognition region (PRR) on these reactions by measuring the quantitative initiation parameters that describe abortive initiation and promoter escape; these parameters are the abortive rate, the productive rate, the abortive:productive ratio, the abortive probability, and the maximum size of abortive transcripts. Changes in the individual DNA elements within the PRR can have a substantial effect on each of these parameters. The discriminator region and the -10 element primarily influence the abortive probability at positions 2-5 and 6-10, respectively, while the -10 and -35 conserved hexamers and the spacer region affect the abortive probability at positions 11-15. Surprisingly, transcription of a consensus promoter invariably gives a higher abortive yield, a higher abortive probability, a longer abortive ladder, and a lower productive rate than promoter variants carrying even a single deviation in the consensus hexamers. These results suggest that strong RNA polymerase-PRR interactions stall the polymerase at the promoter, thereby reducing the rate of promoter escape and consequently enhancing the extent of abortive initiation.

The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is essential for the replication of viral RNA and thus constitutes a valid target for the chemotherapeutic intervention of HCV infection. In this report, we describe the identification of 2'-substituted nucleosides as inhibitors of HCV replication. The 5'-triphosphates of 2'-C-methyladenosine and 2'-O-methylcytidine are found to inhibit NS5B-catalyzed RNA synthesis in vitro, in a manner that is competitive with substrate nucleoside triphosphate. NS5B is able to incorporate either nucleotide analog into RNA as determined with gel-based incorporation assays but is impaired in its ability to extend the incorporated analog by addition of the next nucleotide. In a subgenomic replicon cell line, 2-C-methyladenosine and 2'-O-methylcytidine inhibit HCV RNA replication. The 5'-triphosphates of both nucleosides are detected intracellularly following addition of the nucleosides to the media. However, significantly higher concentrations of 2'-C-methyladenosine triphosphate than 2'-O-methylcytidine triphosphate are detected, consistent with the greater potency of 2'-C-methyladenosine in the replicon assay, despite similar inhibition of NS5B by the triphosphates in the in vitro enzyme assays. Thus, the 2'-modifications of natural substrate nucleosides transform these molecules into potent inhibitors of HCV replication.

CTP:glycerol 3-phosphate cytidylyltransferase catalyzes the formation of CDP-glycerol, an activated form of glycerol 3-phosphate and key precursor to wall teichoic acid biogenesis in Gram-positive bacteria. There is high sequence identity (69%) between the CTP:glycerol 3-phosphate cytidylyltransferases from Bacillus subtilis 168 (TagD) and Staphylococcus aureus (TarD). The B. subtilis TagD protein was shown to catalyze cytidylyltransferase via a random mechanism with millimolar K(m) values for both CTP and glycerol 3-phosphate [J. Biol. Chem. 268, (1993) 16648] and exhibited negative cooperativity in the binding of substrates but not in catalysis [J. Biol. Chem. 276, (2001) 37922]. In the work described here on the S. aureus TarD protein, we have elucidated a steady state kinetic mechanism that is markedly different from that determined for B. subtilis TagD. Steady state kinetic experiments with recombinant, purified TarD employed a high-performance liquid chromatography assay developed in this work. The data were consistent with a ternary complex model. The K(m) values for CTP and glycerol 3-phosphate were 36 and 21 microM, respectively, and the k(cat) was 2.6 s(-1). Steady state kinetic analysis of the reverse (pyrophosphorylase) reaction was also consistent with a ternary complex model. Product inhibition studies indicated an ordered Bi-Bi reaction mechanism where glycerol 3-phosphate was the leading substrate and the release of CDP-glycerol preceded that of pyrophosphate. Finally, we investigated the capacity of S. aureus tarD to substitute for tagD in B. subtilis. The tarD gene was placed under control of the xylose promoter in a B. subtilis 168 mutant defective in tagD (temperature-sensitive, tag-12). Growth of the resulting strain at the restrictive temperature (47 degrees C) was shown to be xylose-dependent.

Assuming that the efficiency of the incorporation of 5-methyl-2'-deoxyisocytosine-5' triphosphate (dMiCTP) and dTTP opposite isoguanine (iG) is a measure of the proportion of the keto and enol tautomers of iG in the Thermus aquaticus DNA polymerase active centre, we studied the influence of temperature and iG-neighbouring bases in the template on base-pairing of iG. On the basis of experiments with four sequences (3'-TXT-5', 3'-GXG-5', 3'-CXC-5', 3'-CXT-5', where X=iG) at 37, 50, 65 and 80 degrees C, we found that 3'-neighbours decrease the fraction of the keto tautomer in the order C>G>or=T, whereas temperature apparently does not influence the tautomeric equilibrium of iG. The variability of the ratio of incorporation of dMiCTP versus dTTP (5-20) primarily reflects the variability of K (m) values, since V (max) values are roughly similar, which indicates that the iG.MiC and iG.T pairs fit the polymerase active centre equally well. The altering of the base-pairing of iG by sequence context is discussed in relation to tautomerism and miscoding of this oxidized adenine derivative. A key derivative for preparation oligodeoxynucleotides, O (2)-diphenylcarbamoyl- N (6)-[(dimethylamino)ethylidene]-2'-deoxyisoguanosine, is extremely labile (t (1/2)=3.5 min) in 3% trichloroacetic acid/dichloromethane, i.e. under the conditions of automated DNA synthesis, which results in low yield and length heterogeneity of templates.

CCA-adding enzymes polymerize CCA onto the 3' terminus of immature tRNAs without using a nucleic acid template. The 3.0 A resolution crystal structures of the CCA-adding enzyme from Bacillus stearothermophilus and its complexes with ATP or CTP reveal a seahorse-shaped subunit consisting of four domains: head, neck, body, and tail. The head is structurally homologous to the palm domain of DNA polymerase beta but has additional structural features and functions. The neck, body, and tail represent new protein folding motifs. The neck provides a specific template for the incoming ATP or CTP, whereas the body and tail may bind tRNA. Each subunit has one active site capable of switching its base specificity between ATP and CTP, an important component of the CCA-adding mechanism.

Early studies showed that in addition to GTP, the pyrimidine nucleotides UTP and CTP support activation of the adenylyl cyclase (AC)-stimulating G(s) protein. The aim of this study was to elucidate the mechanism by which UTP and CTP support G(s) activation. As models, we used S49 wild-type lymphoma cells, representing a physiologically relevant system in which the beta(2)-adrenoreceptor (beta(2)AR) couples to G(s), and Sf9 insect cell membranes expressing beta(2)AR-Galpha(s) fusion proteins. Fusion proteins provide a higher sensitivity for the analysis of beta(2)AR-G(s) coupling than native systems. Nucleoside 5'-triphosphates (NTPs) supported agonist-stimulated AC activity in the two systems and basal AC activity in membranes from cholera toxin-treated S49 cells in the order of efficacy GTP > or = UTP > CTP > ATP (ineffective). NTPs disrupted high affinity agonist binding in beta(2)AR-Galpha(s) in the order of efficacy GTP > UTP > CTP > ATP (ineffective). In contrast, the order of efficacy of NTPs as substrates for nucleoside diphosphokinase, catalyzing the formation of GTP from GDP and NTP was ATP > or = UTP > or = CTP > or = GTP. NTPs inhibited beta(2)AR-Galpha(s)-catalyzed [gamma-(32)P]GTP hydrolysis in the order of potency GTP > UTP > CTP. Molecular dynamics simulations revealed that UTP is accommodated more easily within the binding pocket of Galpha(s) than CTP. Collectively, our data indicate that GTP, UTP, and CTP interact differentially with G(s) proteins and that transphosphorylation of GDP to GTP is not involved in this G protein activation. In certain cell systems, intracellular UTP and CTP concentrations reach approximately 10 nmol/mg of protein and are higher than intracellular GTP concentrations, indicating that G protein activation by UTP and CTP can occur physiologically. G protein activation by UTP and CTP could be of particular importance in pathological conditions such as cholera and Lesch-Nyhan syndrome.

To design combination strategies for chronic hepatitis B therapy, we evaluated in vitro the inhibitory activity of 4 nucleoside analogs, (-)FTC, L-FMAU, DXG, and DAPD, in comparison with lamivudine (3TC) and PMEA. In a cell-free assay for the expression of wild-type duck hepatitis B virus (DHBV) reverse transcriptase, DAPD-TP was found to be the most active on viral minus strand DNA synthesis, including the priming reaction, followed by 3TC-TP, (-)FTC-TP, and DXG-TP, whereas L-FMAU-TP was a weak inhibitor. In cell culture experiments, important differences in drug concentration allowing a 50% inhibition of viral replication or polymerase activity (IC50s) were observed depending on the cell type used, showing that antiviral effect of nucleoside analogs may depend on their intracellular metabolism. IC50s obtained for wild-type DHBV replication in primary duck hepatocytes were much lower than with DHBV transfected LMH cells. IC50s were also significantly lower in the 2.2.1.5 and HepG2 cells compared with HBV transfected HuH7 cells. Moreover, L-FMAU inhibited preferentially HBV plus strand DNA synthesis in these cell lines. The antiviral effect of these inhibitors was also evaluated against 3TC-resistant mutants of the DHBV and HBV polymerases. These mutants were found to be cross resistant to (-)FTC. By contrast, the double DHBV polymerase mutant was sensitive to DXG-TP and DAPD-TP. Moreover, both purine analogs remained active against DHBV and HBV 3TC-resistant mutants in transfected LMH and HepG2 cells, respectively. In conclusion, the unique mechanism of action of these new inhibitors warrants further evaluation in experimental models to determine their capacity to delay or prevent the selection of drug resistant mutants.

Among the acquired immunodeficiency syndrome (AIDS) drugs approved by the FDA for clinical use, two are modified cytosine analogs, Zalcitabine (ddC) and Lamivudine [(-)3TC]. (-)3TC is the only analog containing an unnatural L (-) nucleoside configuration. Similar to other dideoxy nucleosides, these analogs are metabolically activated to the triphosphate that is incorporated into DNA by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) resulting in DNA chain termination and ultimately cessation of viral replication. The natural D (+) 3TC isomer also acts in a similar manner to inhibit HIV-1 RT. In cell culture, (-)3TC is less toxic than its D (+) isomer, (+)3TC, containing the natural nucleoside configuration, and both are considerably less toxic than 2',3'-dideoxycytidine (ddC). The mechanistic basis for the stereochemical selectivity and differential toxicity of the isomeric 2',3'-dideoxy-3'-thiacytidine (3TC) and ddC compounds is not completely understood although a number of factors may clearly come into play. We have previously investigated the mechanistic basis for the differential stereoselective inhibition and toxicity of these three cytosine analogs by comparing the effects of 2',3'-deoxycytidine-5'-triphosphate (ddCTP), beta-D-(+)-2'3'-dideoxy-3'-thiacytidine-5'-triphosphate [(+)3TC-TP] and beta-L-(-)-2'3'-dideoxy-3'-thiacytidine-5'-triphosphate [(-)3TC-TP] on the HIV-1 RT as well as a recombinant form of the human mitochondrial DNA polymerase gamma (Pol gamma), the holoenzyme polymerase responsible for mitochondrial DNA replication. In this review, we discuss studies which may provide insight into the molecular mechanism for the stereochemical selectivity and differential toxicity.

The archaeal chaperonin-mediated folding of green fluorescent protein (GFP) was examined in the presence of various nucleotides. The recombinant alpha- and beta-subunit homo-oligomers and natural chaperonin oligomer from Thermococcus strain KS-1 exhibited folding activity with not only ATP but also with CTP, GTP, or UTP. The ADP-bound form of both recombinant and natural chaperonin had the ability to capture non-native GFP, but could not refold it in the presence of CTP, GTP or UTP until ATP was supplied. The archaeal chaperonin thus utilized ATP, but could not use other nucleoside triphosphates in the cytoplasm where ADP was present.

The enzyme CMP-Kdo synthetase (CKS) catalyzes the activation of the sugar Kdo (2-keto-3-deoxy-manno-octonic acid) by forming a monophosphate diester. CKS is a pharmaceutical target because CMP-Kdo is used in the biosynthesis of lipopolysaccharides that are vital for Gram-negative bacteria. We have refined the structure of the unligated capsule-specific CKS from Escherichia coli at 1.8 A resolution (1 A=0.1 nm) and we have established the structures of its complexes with the substrate CTP, with CDP and CMP as well as with the product analog CMP-NeuAc (CMP-sialate) by X-ray diffraction analyses at resolutions between 2.1 A and 2.5 A. The N-terminal domains of the dimeric enzyme bind CTP in a peculiar nucleotide-binding fold, whereas the C-terminal domains form the dimer interface. The observed binding geometries together with the amino acid variabilities during evolution and the locations of a putative Mg(2+) and of a very strongly bound water molecule suggest a pathway for the catalysis. The N-terminal domain shows sequence homology with the CMP-NeuAc synthetases. Moreover, the chain fold and the substrate-binding position of CKS resemble those of other enzymes processing nucleotide-sugars., (Copyright 2001 Academic Press.)

The YgbP protein of Escherichia coli encodes the enzyme 4-diphosphocytidyl-2-C-methylerythritol (CDP-ME) synthetase, a member of the cytidyltransferase family of enzymes. CDP-ME is an intermediate in the mevalonate-independent pathway for isoprenoid biosynthesis in a number of prokaryotic organisms, algae, the plant plastids and the malaria parasite. Because vertebrates synthesize isoprenoid precursors using a mevalonate pathway, CDP-ME synthetase and other enzymes of the mevalonate-independent pathway for isoprenoid production represent attractive targets for the structure-based design of selective antibacterial, herbicidal and antimalarial drugs. The high-resolution structures of E. coli CDP-ME synthetase in the apo form and complexed with both CTP-Mg2+ and CDP-ME-Mg2+ reveal the stereochemical principles underlying both substrate and product recognition as well as catalysis in CDP-ME synthetase. Moreover, these complexes represent the first experimental structures for any cytidyltransferase with both substrates and products bound.

Success in treating hepatitis B virus (HBV) infection with nucleoside analog drugs like lamivudine is limited by the emergence of drug-resistant viral strains upon prolonged therapy. The predominant lamivudine resistance mutations in HBV-infected patients are Met552IIe and Met552Val (Met552Ile/Val), frequently in association with a second mutation, Leu528Met. The effects of Leu528Met, Met552Ile, and Met552Val mutations on the binding of HBV polymerase inhibitors and the natural substrate dCTP were evaluated using an in vitro HBV polymerase assay. Susceptibility to lamivudine triphosphate (3TCTP), emtricitabine triphosphate (FTCTP), adefovir diphosphate, penciclovir triphosphate, and lobucavir triphosphate was assessed by determination of inhibition constants (K(i)). Recognition of the natural substrate, dCTP, was assessed by determination of Km values. The results from the in vitro studies were as follows: (i) dCTP substrate binding was largely unaffected by the mutations, with Km changing moderately, only in a range of 0.6 to 2.6-fold; (ii) K(i)s for 3TCTP and FTCTP against Met552Ile/Val mutant HBV polymerases were increased 8- to 30-fold; and (iii) the Leu528Met mutation had a modest effect on direct binding of these beta-L-oxathiolane ring-containing nucleotide analogs. A three-dimensional homology model of the catalytic core of HBV polymerase was constructed via extrapolation from retroviral reverse transcriptase structures. Molecular modeling studies using the HBV polymerase homology model suggested that steric hindrance between the mutant amino acid side chain and lamivudine or emtricitabine could account for the resistance phenotype. Specifically, steric conflict between the Cgamma2-methyl group of Ile or Val at position 552 in HBV polymerase and the sulfur atom in the oxathiolane ring (common to both beta-L-nucleoside analogs lamivudine and emtricitabine) is proposed to account for the resistance observed upon Met552Ile/Val mutation. The effects of the Leu528Met mutation, which also occurs near the HBV polymerase active site, appeared to be less direct, potentially involving rearrangement of the deoxynucleoside triphosphate-binding pocket residues. These modeling results suggest that nucleotide analogs that are beta-D-enantiomers, that have the sulfur replaced by a smaller atom, or that have modified or acyclic ring systems may retain activity against lamivudine-resistant mutants, consistent with the observed susceptibility of these mutants to adefovir, lobucavir, and penciclovir in vitro and adefovir in vivo.

We have characterized the misincorporation properties of wild-type (wt) T7 RNAP and of 45 T7RNAP point mutants. The wt enzyme selects strongly against incorporation of an incorrect nucleotide. From the measured rates of misincorporation, an average error frequency of 1 in 2 x 10(4) is estimated. RNAs bearing 3'-mismatches are extended more slowly than correctly paired 3'-termini, and mismatches one or two bases away from the RNA 3'-end can also slow extension severely even when the 3'-base is correctly paired. Though it has been reported that T7RNAP has a 3' --> 5' nuclease activity, we were unable to detect any endogenous T7RNAP RNase activity in elongation complexes. Pyrophosphorolysis was detected but does not appear to contribute to proofreading. Therefore, unlike other RNAPs, T7RNAP fidelity appears to depend entirely on discrimination against incorporation of the incorrect nucleotide and not on post-misincorporation proofreading. Alanine substitution of the H784 side chain, which interacts with the 3' RNA.template base pair, increases both misincorporation and mismatch extension, while substitutions at G640, F644, and G645 increase misincorporation, but not mismatch extension. The latter three amino acids are in a part of the RNAP which interacts with the templating base and with the base immediately 5' to the templating base. Mutation of these amino acids not only increases misincorporation, but also eliminates pausing during promoter clearance. The effects of these mutations and the interactions observed in a crystal structure of a transcribing complex indicate that these mutations disrupt interactions which limit misincorporation rates by stabilizing the catalytically incompetent open conformation of the RNAP.

Previous studies have shown that the S5' beta-strand (r93-r97) of the regulatory polypeptides of the aspartate transcarbamoylases (ATCases) from Serratia marcescens and Escherichia coli are responsible for their diverged allosteric regulatory patterns, including conversion of CTP from an inhibitor in E. coli to an activator in S. marcescens. Similarly, mutation of residues located in the interface between the allosteric and the zinc domains resulted in conversion of the ATP responses of the E. coli enzyme from activation to inhibition, suggesting that this interface not only mediates but also discriminates the allosteric responses of ATP and CTP. To further decipher the roles and the interrelationships of these regions in allosteric communication, allosteric-zinc interface mutations (Y77F and V106A) have been introduced into both the native and the S5' beta-strand chimeric backgrounds. While the significance of this interface in the allosteric regulation has been confirmed, there is no direct evidence supporting the presence of distinct pathways for the ATP and CTP signals through this interface. The analysis of the mutational effects reported here suggested that the S5' beta-strand transmits the allosteric signal by modulating the hydrophobic allosteric-zinc interface rather than disturbing the allosteric ligand binding. Intragenic suppression by substitutions in the hydrophobic interface between the allosteric and the zinc domains of the regulatory chains resulted in the partial recovery of allosteric responses in the EC:rS5'sm chimera and reduced the activation by ATP in the Sm:rS5'ec chimera. Thus, it seems that there is a synergy between these two structural units., (Copyright 2000 Academic Press.)

CTP synthetase [EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)] from the yeast Saccharomyces cerevisiae catalyzes the ATP-dependent transfer of the amide nitrogen from glutamine to the C-4 position of UTP to form CTP. In this work, we demonstrated that CTP synthetase utilized dUTP as a substrate to synthesize dCTP. The dUTP-dependent activity was linear with time and with enzyme concentration. Maximum dUTP-dependent activity was dependent on MgCl(2) (4 mM) and GTP (K(a) = 14 microM) at a pH optimum of 8.0. The apparent K(m) values for dUTP, ATP, and glutamine were 0.18, 0.25, and 0.41 mM, respectively. dUTP promoted the tetramerization of CTP synthetase, and the extent of enzyme tetramerization correlated with dUTP-dependent activity. dCTP was a poor inhibitor of dUTP-dependent activity, whereas CTP was a potent inhibitor of this activity. The enzyme catalyzed the synthesis of dCTP and CTP when dUTP and UTP were used as substrates together. CTP was the major product synthesized when dUTP and UTP were present at saturating concentrations. When dUTP and UTP were present at concentrations near their K(m) values, the synthesis of dCTP increased relative to that of CTP. The synthesis of dCTP was favored over the synthesis of CTP when UTP was present at a concentration near its K(m) value and dUTP was varied from subsaturating to saturating concentrations. These data suggested that the dUTP-dependent synthesis of dCTP by CTP synthetase activity may be physiologically relevant.

In vitro selection of RNA aptamer containing biotin-carrying nucleotide was carried out used for development of a new type of molecular sensor. Cytidine 5'-triphosphate (CTP) carrying biotinyl group at the N6-position was used in this technique. A pool of biotin-containing RNAs, which binds specifically to adenosine 5'-triphosphate (ATP), was obtained and used for competitive binding assay of ATP. The selected nonnatural RNA possesses many biotinyl groups to render it a high sensitivity toward ATP.

The noncovalent fluorescent probe 6-propionyl-2-(dimethylamino)naphthalene (prodan) binds stoichiometrically to myosin subfragment-1 (S-1) without affecting the ATPase and actin-binding properties of S-1. Neither ATP nor actin interferes with the prodan binding. Free prodan exhibits a green emission peak at 520 nm. However, the prodan bound to S-1 and the S-1.ADP complex shows blue emission peaks at 460 and 450 nm, respectively, which allow easy separation of the fluorescence contributions from the free and bound probes. In the S-1.ADP.Pi state, the blue emission peak is further shifted to 445 nm with a large (4.5-fold) fluorescence enhancement. Thus, prodan in the presence of S-1 exhibits predominantly blue fluorescence only during ATP hydrolysis, and so visualizes the ATPase reaction continuously. The initial velocities of the steady state of the Mg2+-, Ca2+-, and actin-activated ATPases can be conveniently calculated from the blue fluorescence changes. The ability of different nucleoside triphosphates (NTP) to enhance the blue fluorescence of prodan follows the order ATP > CTP > UTP > ITP > GTP. This order agrees with those of the extent of hydrophobicity near the ribose of the corresponding nucleoside diphosphates (NDP) trapped to S-1 with orthovanadate (Vi) [Hiratsuka, T. (1984) J. Biochem. (Tokyo) 96, 155-162] and the ability of different NTPs to support force production in muscle fibers [Regnier, M., et al. (1993) Biophys. J. 64, A250]. The rate of formation of the corresponding S-1.NDP.Vi complex also follows this order, whereas the NTPase rate follows the reverse order. These results indicate that nucleotide-induced changes in prodan fluorescence correspond to the nucleotide-induced conformational states of S-1. Thus, the use of prodan in studies of the myosin ATPase offers a new and promising approach not only to monitoring the ATPase reaction but also to investigating the structural changes during ATP hydrolysis.

4-N-exo-base-substituted photoreactive analogs of CTP were designed and synthesized. Two flavivirus proteins NS5 and NS3 are shown to be labelled after RNA synthesis in the presence of the analogs, irradiation by UV-light (313 nm) and subsequent [alpha-32P]NTP incorporation.

During general genetic recombination and recombinational DNA repair, DNA damages and heterologies are often encountered which must be efficiently processed by the cellular recombination machinery. In RecA-mediated three-strand exchange reactions between single-stranded circular and linear duplex DNA, or four-strand exchange reactions between gapped circular and linear duplex DNA, heterologies can only be bypassed in vitro when they are short in length and are followed by homologous DNA downstream. Larger DNA inserts block RecA-mediated strand exchange, indicating that effective bypass requires other components of the recombination machinery. The RuvA and RuvB proteins of Escherichia coli form an important part of this machinery. In this work, we have analysed the ability of RuvA and RuvB to bypass large tracts of DNA heterology in both three- and four-strand exchange reactions, using recombination intermediates made by the E. coli RecA protein. Under optimal reaction conditions for RuvAB, up to 1000 bp of DNA heterology can by bypassed in three-strand reactions and 300 bp of DNA heterology can be bypassed in four-strand reactions. Whereas high concentrations of RuvB (in the absence of RuvA) can promote homologous branch migration, we find that RuvB alone is unable to catalyse heterologous bypass, indicating an essential role for both proteins in homologous recombination and recombinational DNA repair processes. Under certain conditions, the bypass of heterology is stimulated by the single-strand binding protein SSB.

Aspartate transcarbamylase (ATCase) is an important control enzyme in the pyrimidine biosynthetic pathway in Escherichia coli. It is a classic example of an allosteric protein and has been extensively studied biochemically, kinetically and structurally. As yet, however, a detailed model for the cooperative transition between the tensed (T) and relaxed (R) forms of the protein does not exist. In this work we have calculated the low frequency normal modes of the CTP-ligated T-state of ATCase with the aim of identifying some of the motions that could be important in initiating the transition. The calculated modes, of frequencies lower than 5 per cm, produce root-mean-square coordinate deviations for the atoms which are a substantial fraction of those derived from the crystallographic B-factors. Some of the modes result in displacements which change the quaternary structure of the protein (in particular the elongation of the protein and the relative rotation of the subunits) in such a way that the R-state structure is approached. The implication of these mode motions for the overall T-->R transition process is discussed.

A new photocrosslinking CTP analog that functioned as a substrate during transcription was synthesized and used to photoaffinity label E. coli and bacteriophage T7 RNA polymerases. This analog, 5-((4-azidophenacyl)thio) cytidine-5'-triphosphate (5-APAS-CTP) contains an aryl azide group approximately 10 A from the nucleotide base and specifically replaced CTP during synthesis of RNA by both polymerases. Analog was placed at the 3' end or internally within RNA. Both polymerases inefficiently incorporated two 5-APAS-CMP molecules sequentially, as was found for the related 5-APAS-UMP. Analog was placed at the 3' end of RNA in transcription complexes paused at the site of Q-modification of E. coli RNA polymerase, downstream of the lambda PR' promoter (+16), a pause that requires specific DNA sequences but no apparent RNA hairpin. Crosslinking was examined in the presence and absence of the NusA protein, which enhances the transcriptional pause at this site and is required for Q modification of the polymerase. Crosslinking of the 3' end of the RNA to NusA was not observed, consistent with our earlier results involving a NusA-enhanced pause site downstream from an RNA hairpin.

The contractile protein actin contains one mole of firmly bound nucleotide and a number of divalent cations bound with different affinities. During recent years evidence for a second nucleotide interacting site on actin has been reported. Therefore, a specific search for the presence of a second nucleotide-interacting site on actin was undertaken. For this purpose G- and F-actin or actin in complex with deoxyribonuclease I (DNase I) was passed over ADP-agarose which was found to retain all three forms of actin. Nucleotide bound to the high affinity site of actin did not exchange during passage and retention to agarose-immobilized ADP, thus indicating the presence of a second nucleotide interacting site. This site was found to be equally accessible in G- and F-actin and in the actin-DNase I complex, whereas DNase I alone passed unretained through this column. A number of nucleotides and phosphorylated compounds were tested for their ability to compete with immobilized ADP for actin interaction. It was found that all forms of actin are liberated only by high concentrations (5mM) of ADP, ATP and NADH, by 1mM CTP and ITP, and by high salt concentrations (150mM NaCl). Since it was found that EDTA- and heat-treated actin were also retained on ADP-agarose, we conclude that this second nucleotide interacting site is of limited specificity, low affinity, and not dependent on the native configuration of actin. It exhibits characteristics of an unspecific, polyanionic site, but may represent the low affinity phosphate binding site.