Presence Of CTP Research Articles

Biochemical and structural bioinformatics studies of fungal CutA nucleotidyltransferases explain their unusual specificity toward CTP and increased tendency for cytidine incorporation at the 3'-terminal positions of synthesized tails.

Noncanonical RNA nucleotidyltransferases (NTases), including poly(A), poly(U) polymerases (PAPs/PUPs), and C/U-adding enzymes, modify 3′-ends of different transcripts affecting their functionality and stability. They contain PAP/OAS1 substrate-binding domain (SBD) with inserted NTase domain. Aspergillus nidulans CutA (AnCutA), synthesizes C/U-rich 3′-terminal extensions in vivo. Here, using high-throughput sequencing of the 3′-RACE products for tails generated by CutA proteins in vitro in the presence of all four NTPs, we show that even upon physiological ATP excess synthesized tails indeed contain an unprecedented number of cytidines interrupted by uridines and stretches of adenosines, and that the majority end with two cytidines. Strikingly, processivity assays documented that in the presence of CTP as a sole nucleotide, the enzyme terminates after adding two cytidines only. Comparison of our CutA 3D model to selected noncanonical NTases of known structures revealed substantial differences in the nucleotide recognition motif (NRM) within PAP/OAS1 SBD. We demonstrate that CutA specificity toward CTP can be partially changed to PAP or PUP by rational mutagenesis within NRM and, analogously, Cid1 PUP can be converted into a C/U-adding enzyme. Collectively, we suggest that a short cluster of amino acids within NRM is a determinant of NTases’ substrate preference, which may allow us to predict their specificity.

P.35 - Translational biochemistry for dystroglycanopathies

ISPD (isoprenoid synthase domain containing) mutations cause a wide clinical spectrum of muscular dystrophies (dystroglycanopathies). Human ISPD is a cytidyltransferase, catalyses the reaction from CTP and Ribitol-5-phosphate to CDP-Ribitol, a novel nucleotide sugar for functional alpha-dystroglycan. hISPD has one conserved cytidyltransferase domain and a second functional unknown non-conserved domain. We aim to correlate clinical phenotypes to structural impacts of ISPD mutations to decipher the underlying molecular mechanisms. First we established a high throughput in vitro protein thermostablity screening assay (Thermofluor assay, TF), by which we screened for the ligands of recombinant hISPD (purified from E.coli) and found out that Ribitol-5-Phosphate can only alter the protein thermostability in the presence of CTP. The thermostablities of missense mutants are also assessed by TF assay, together with protein half-life cycloheximide assay in HEK cells transfected with mutants revealing that some mutants have significant lower stabilities than wild type (p.Thr238Ile). Surprisingly, few mutants have a higher stability than wild type. Interestingly, we found that C-terminal truncating mutations of the second domain showed very low protein levels in HEK cells (p.Arg395, p.Arg396*), consistent with the fact that secondary domain is essential for protein stability and dimerization and with the associated severe form of CMD. Enzyme activates were measured by Pyrophosphate Fluormetric assay, revealed that most missense mutants have 30%-50% reduced activity. However, few mutations have a high enzymatic activity, those are currently being investigated for their ligand binding affinity (Km). In summary, we assessed the effects of the single amino acid changes on ISPD protein stability, protein structure, enzyme activities, and correlated this with the patient phenotype. Ultimately, using the high throughput Thermofluor assay, we screened a small chemical chaperones library as a proof-of-principle for personalized treatment of dystroglycanopathy patients.

Distinct features of protein folding by the GroEL system from a psychrophilic bacterium, Colwellia psychrerythraea 34H

We investigated the protein folding mechanism of the GroEL system of a psychrophilic bacterium, Colwellia psychrerythraea 34H. The amount of mRNA of the groESL operon of C. psychrerythraea was increased about 6-fold after a temperature upshift from 8 to 18 °C for 30 min, suggesting that this temperature causes heat stress in this bacterium. A σ(32)-type promoter was found upstream of the groESL, suggesting that the C. psychrerythraea groESL is regulated by the σ(32) system, like the groESL in E. coli. The maximum ATPase and CTPase activities of CpGroEL were observed at 45 and 35 °C, respectively, which are much higher than the growth temperatures of C. psychrerythraea. We found that the refolding activity of the CpGroEL system in the presence of ATP is lower than that in the presence of CTP. This suggests that ATP is not the optimum energy source of the CpGroEL system. Analyses for the interaction of CpGroEL-CpGroES revealed that CTP could weaken this interaction, resulting in effective refolding function of the CpGroEL system. From these findings, we consider that the CpGroEL system possesses an energy-saving mechanism for avoiding excess consumption of ATP to ensure growth in a low-temperature environment.

Metal ion involvement in the allosteric mechanism of Escherichia coli aspartate transcarbamoylase.

Escherichia coli aspartate transcarbamoylase (ATCase) allosterically regulates pyrimidine nucleotide biosynthesis. The enzyme is inhibited by CTP and can be further inhibited by UTP, although UTP alone has little or no influence on activity; however, the mechanism for the synergistic inhibition is still unknown. To determine how UTP is able to synergistically inhibit ATCase in the presence of CTP, we determined a series of X-ray structures of ATCase·nucleotide complexes. Analysis of the X-ray structures revealed that (1) CTP and dCTP bind in a very similar fashion, (2) UTP, in the presence of dCTP or CTP, binds at a site that does not overlap the CTP/dCTP site, and (3) the triphosphates of the two nucleotides are parallel to each other with a metal ion, in this case Mg(2+), coordinated between the β- and γ-phosphates of the two nucleotides. Kinetic experiments showed that the presence of a metal ion such as Mg(2+) is required for synergistic inhibition. Together, these results explain how the binding of UTP can enhance the binding of CTP and why UTP binds more tightly in the presence of CTP. A mechanism for the synergistic inhibition of ATCase is proposed in which the presence of UTP stabilizes the T state even more than CTP alone. These results also call into question many of the past kinetic and binding experiments with ATCase with nucleotides as the presence of metal contamination was not considered important.

A Second Allosteric Site in Escherichia coli Aspartate Transcarbamoylase

Escherichia coli aspartate transcarbamoylase is feedback inhibited by CTP and UTP in the presence of CTP. Here, we show by X-ray crystallography that UTP binds to a unique site on each regulatory chain of the enzyme that is near but not overlapping with the known CTP site. These results bring into question all of the previously proposed mechanisms of allosteric regulation in aspartate transcarbamoylase.

Structure-based Mechanism of CMP-2-keto-3-deoxymanno-octulonic Acid Synthetase

The enzyme CMP-Kdo synthetase (KdsB) catalyzes the addition of 2-keto-3-deoxymanno-octulonic acid (Kdo) to CTP to form CMP-Kdo, a key reaction in the biosynthesis of lipopolysaccharide. The reaction catalyzed by KdsB and the related CMP-acylneuraminate synthase is unique among the sugar-activating enzymes in that the respective sugars are directly coupled to a cytosine monophosphate. Using inhibition studies, in combination with isothermal calorimetry, we show the substrate analogue 2beta-deoxy-Kdo to be a potent competitive inhibitor. The ligand-free Escherichia coli KdsB and ternary complex KdsB-CTP-2beta-deoxy-Kdo crystal structures reveal that Kdo binding leads to active site closure and repositioning of the CTP phosphates and associated Mg(2+) ion (Mg-B). Both ligands occupy conformations compatible with an S(n)2-type attack on the alpha-phosphate by the Kdo 2-hydroxyl group. Based on strong similarity with DNA/RNA polymerases, both in terms of overall chemistry catalyzed as well as active site configuration, we postulate a second Mg(2+) ion (Mg-A) is bound by the catalytically competent KdsB-CTP-Kdo ternary complex. Modeling of this complex reveals the Mg-A coordinated to the conserved Asp(100) and Asp(235) in addition to the CTP alpha-phosphate and both the Kdo carboxylic and 2-hydroxyl groups. EPR measurements on the Mn(2+)-substituted ternary complex support this model. We propose the KdsB/CNS sugar-activating enzymes catalyze the formation of activated sugars, such as the abundant CMP-5-N-acetylneuraminic acid, by recruitment of two Mg(2+) to the active site. Although each metal ion assists in correct positioning of the substrates and activation of the alpha-phosphate, Mg-A is responsible for activation of the sugar-hydroxyl group.

Molecular Basis of Transcriptional Mutagenesis at 8-Oxoguanine

Structure-function analysis has revealed the mechanism of yeast RNA polymerase II transcription at 8-oxoguanine (8-oxoG), the major DNA lesion resulting from oxidative stress. When polymerase II encounters 8-oxoG in the DNA template strand, it can misincorporate adenine, which forms a Hoogsteen bp with 8-oxoG at the active center. This requires rotation of the 8-oxoG base from the standard anti- to an uncommon syn-conformation, which likely occurs during 8-oxoG loading into the active site. The misincorporated adenine escapes intrinsic proofreading, resulting in transcriptional mutagenesis that is observed directly by mass spectrometric RNA analysis.

Fuel Specificity of the Hepatitis C Virus NS3 Helicase

The hepatitis C virus (HCV) NS3 protein is a helicase capable of unwinding duplex RNA or DNA. This study uses a newly developed molecular-beacon-based helicase assay (MBHA) to investigate how nucleoside triphosphates (NTPs) fuel HCV helicase-catalyzed DNA unwinding. The MBHA monitors the irreversible helicase-catalyzed displacement of an oligonucleotide-bound molecular beacon so that rates of helicase translocation can be directly measured in real time. The MBHA reveals that HCV helicase unwinds DNA at different rates depending on the nature and concentration of NTPs in solution, such that the fastest reactions are observed in the presence of CTP followed by ATP, UTP, and GTP. 3′-Deoxy-NTPs generally support faster DNA unwinding, with dTTP supporting faster rates than any other canonical (d)NTP. The presence of an intact NS3 protease domain makes HCV helicase somewhat less specific than truncated NS3 bearing only its helicase region (NS3h). Various NTPs bind NS3h with similar affinities, but each NTP supports a different unwinding rate and processivity. Studies with NTP analogs reveal that specificity is determined by the nature of the Watson–Crick base-pairing region of the NTP base and the nature of the functional groups attached to the 2′ and 3′ carbons of the NTP sugar. The divalent metal bridging the NTP to NS3h also influences observed unwinding rates, with Mn 2+ supporting about 10 times faster unwinding than Mg 2+. Unlike Mg 2+, Mn 2+ does not support HCV helicase-catalyzed ATP hydrolysis in the absence of stimulating nucleic acids. Results are discussed in relation to models for how ATP might fuel the unwinding reaction.

Biosynthesis of Isoprenoids: Characterization of a Functionally Active Recombinant 2-C-methyl-D-erythritol 4-phosphate Cytidyltransferase (IspD) from Mycobacterium tuberculosis H37Rv

Tuberculosis, caused by Mycobacterium tuberculosis, continues to be one of the leading infectious diseases to humans. It is urgent to discover novel drug targets for the development of antitubercular agents. The 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway for isoprenoid biosynthesis has been considered as an attractive target for the discovery of novel antibiotics for its essentiality in bacteria and absence in mammals. MEP cytidyltransferase (IspD), the third-step enzyme of the pathway, catalyzes MEP and CTP to form 4-diphosphocytidyl-2-C-methylerythritol (CDP-ME) and PPi. In the work, ispD gene from M. tuberculosis H37Rv (MtIspD) was cloned and expressed. With N-terminal fusion of a histidine-tagged sequence, MtIspD could be purified to homogeneity by one-step nickel affinity chromatography. MtIspD exists as a homodimer with an apparent molecular mass of 52 kDa. Enzyme property analysis revealed that MtIspD has high specificity for pyrimidine bases and narrow divalent cation requirements, with maximal activity found in the presence of CTP and Mg(2+). The turnover number of MtIspD is 3.4 s(-1). The Km for MEP and CTP are 43 and 92 muM, respectively. Furthermore, MtIspD shows thermal instable above 50 degrees C. Circular dichroism spectra revealed that the alteration of tertiary conformation is closely related with sharp loss of enzyme activity at higher temperature. This study is expected to help better understand the features of IspD and provide useful information for the development of novel antibiotics to treat M. tuberculosis.

Role of the linker between the zinc binding domain and the polymerization domain of the bacteriophage T7 DNA primase

T7 DNA primase is composed of a catalytic RNA polymerase domain (RPD) and a zinc binding domain (ZBD) that are connected by an unstructured linker of 16 residues. The two domains are required for initiating the formation of a functional tetraribonucleotide, 5′-pppACCC-3′, from ATP and CTP (de novo synthesis) and for extending 5′-AC-3′ diribonucleotides or 5′-ACC-3′ triribonucleotides into a functional 5′-ACCC-3′ in the presence of CTP (diribonucleotide/triribonucleotide extension) in a template-directed manner. We have changed the distance between these domains by deleting or adding residues from the linker connecting the two domains. We find that the wild type primase is superior in de novo synthesis compared to primases having longer (21 residues) or shorter (10 residues) linkers. The activity of the altered primases is reduced by 2-fold and 10-fold, respectively. Interestingly, the primase with the shorter linker exhibits a 2-fold enhancement in its ability to extend di- and triribonucleotides, whereas the primase with the longer linker extends ribonucleotides 2-fold less efficiently compared to the wild type primase. The wild type and the altered T7 DNA primases do not extend diribonucleotides by the ZBD of one subunit acting on the RPD of an adjacent subunit (trans mode), whereas de novo synthesis is feasible in this mode. We propose that the formation of the diribonucleotide and the extension of the dimer or trimer into a functional tetramer is a stepwise process in which distinct interactions of the ZBD are required. This work was supported by United States Public Health Services Grant GM 54397

Behavior of Osteoblasts Cultured on Collagen Coated Bioactive Glass

In the current work, we investigated cellular viability, proliferation, and metabolic activity of rat primary culture osteoblasts in contact with a sample of collagen type I (C) and of this same collagen chemically treated (CTP). The chemical process used here consisted in recover the collagen surface with silica glass obtained from a sol-gel process. The cell viability, the cell death, the alkaline phosphate production and collagen secretion, after 72 hours of incubating the samples with osteoblasts, were measured by MTT assay, propidium iodide, NBT-BCIP assay and SIRCOL method, respectively. The viability and proliferation of osteoblasts had a significant decrease in the presence of samples when compared to control. The alkaline phosphatase production by the cells had a significant increase in the presence of CTP and collagen secretion by the cells was practically the same when compared to control.

A Fluorescent Probe-labeled Escherichia coli Aspartate Transcarbamoylase That Monitors the Allosteric Conformational State

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.

Nucleotide specificity of an archaeal group II chaperonin from Thermococcus strain KS-1 with reference to the ATP-dependent protein folding cycle

The archaeal chaperonin-mediated folding of green fluorescent protein (GFP) was examined in the presence of various nucleotides. The recombinant α- and β-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.

Solvent perturbation of the allosteric regulation of aspartate transcarbamylase

Escherichia coli aspartate transcarbamylase (ATCase) catalyzes the first committed step in pyrimidine biosynthesis, the condensation of aspartate and carbamyl phosphate. ATCase is positively allosterically regulated by ATP and negatively regulated by CTP. We have used mild solvent perturbation to gain global molecular information about the mechanism of heterotropic allostery. The [NaCl], temperature, and osmotic pressure dependence of the enzymatic activity of ATCase has been examined in the presence and absence of allosteric effectors. The results indicate that: 1) Regulation of aspartate binding by CTP appears to involve a unique set of electrostatic interactions not involved in enzyme function in the presence of ATP or in the absence of effectors. 2) Aspartate binding is enthalpically driven in the presence and absence of allosteric effectors. 3) The apparent enthalpy and entropy of aspartate binding (Δ H, Δ S), and activation energy of catalysis ( E a) are substantially altered in the presence of CTP but not ATP. 4) The change in hydration of ATCase upon substrate binding is the same in the presence and absence of allosteric effectors. 5) The linkage between heterotropic and homotropic allostery is different for ATP and CTP.

Roles of the helicase and primase domain of the gene 4 protein of bacteriophage T7 in accessing the primase recognition site.

The 63 kDa gene 4 protein of bacteriophage T7 provides both helicase and primase activities. The C-terminal helicase domain of the gene 4 protein is responsible for DNA-dependent NTP hydrolysis and for hexamer formation, whereas the N-terminal primase domain contains the zinc motif that is, in part, responsible for template-directed oligoribonucleotide synthesis. In the presence of beta, gamma-methylene dTTP, the protein forms a hexamer that surrounds and binds tightly to single-stranded DNA and consequently is unable to translocate to primase recognition sites, 5'-GTC-3', or to dissociate from the molecule to which it is bound. Nonetheless, in the presence of beta,gamma-methylene dTTP, it catalyzes the synthesis of pppAC dimers at primase sites on M13 DNA. When bound to single-stranded DNA in the presence of beta,gamma-methylene dTTP, the primase can function at recognition sites on the same molecule to which it is bound provided that a sufficient distance exists between the recognition site and the site to which it is bound. Furthermore, the primase bound to one DNA strand can function at a primase site located on a second DNA strand. The results indicate that the primase domain resides on the outside of the hexameric ring, a location that enables it to access sites distal to its site of binding.

Gene 4 DNA Primase of Bacteriophage T7 Mediates the Annealing and Extension of Ribo-oligonucleotides at Primase Recognition Sites

The 63-kDa gene 4 primase of bacteriophage T7 recognizes a core trinucleotide sequence, 5'-GTC-3', on single-stranded DNA at which it catalyzes the synthesis of the ribodinucleotide pppAC. The dinucleotide is extended to a tetranucleotide primer at the sites 5'-(G/T)GGTC-3' and 5'-GTGTC-3'. In the presence of T7 primase, T7 DNA polymerase extends the synthetic ribotetranucleotide pACCA (1 microM), but not pCACA, on M13 DNA templates. The reaction is specific for T7 DNA polymerase and depends on dTTP and translocation of the gene 4 protein. T7 primase extends the dinucleotide AC and trinucleotide ACC to ACCC in the presence of CTP and an appropriate template, whereas other dinucleotides are extended less efficiently; the deoxyribodinucleotide dAC is not extended. The Cys4 zinc motif of the primase is essential for extension of the dinucleotides. The 5'-cryptic cytidine of the recognition sequence is essential for extension of the dinucleotide AC to tri- and tetranucleotides. At a preformed replication fork, the dinucleotide AC provides for primer synthesis on the lagging strand. The synthesis of all Okazaki fragments is initiated by primers arising from the recognition sequence 5'-GGGTC-3'; none arise at an adjacent 5'-GGGTT-3' sequence. If ADP or AMP replaces ATP in the primase reaction, primers terminating in di- or monophosphate, respectively, are synthesized.

Nucleotide-Dependent Contractile Properties of Ca 2+-Activated Fast and Slow Skeletal Muscle Fibers

The relation between single skinned skeletal fiber contractile mechanics and the myosin mechanoenzyme was examined by perturbing the actomyosin interaction with the ATP analog CTP in fibers from both rabbit psoas and rat soleus. Tension, instantaneous stiffness, and the rate of tension redevelopment (ktr), under software-based sarcomere length control, were examined at 15 degrees C for a range of Ca2+ concentrations in both fiber types. CTP produced 94% of the maximum ATP-generated tension in psoas fibers and 77% in soleus fibers. In psoas, CTP also increased stiffness to 106% of the ATP stiffness, whereas in soleus stiffness decreased to 92%. Thus, part of the greater difference between maximum ATP- and CTP-generated tension in soleus fibers appears to be due to a decrease in strongly bound cross-bridge number. Interestingly, although the nucleotide exchange produced substantial increases in the steepness (nH) of the tension- and stiffness-pCa relationships in soleus fibers, only minor changes were seen in psoas fibers. At maximum Ca2+ and nominal P(i) levels, ktr in psoas fibers increased from 11.7 s-1 with ATP to 16.6 s-1 with CTP and in soleus fibers from 4.9 to 8.4 s-1. Increased P(i) levels decreased the maximum Ca(2+)-activated tension in both fiber types and increased the ktr of psoas fibers, but the ktr of soleus fibers was not significantly altered. Thus, although the nucleotide exchange generally produced similar changes in the mechanics, there were significant muscle lineage differences in the tension- and stiffness-pCa relations and in the effects of P(i) on ktr, such that differences in contractile mechanics were lessened in the presence of CTP.

Effects of nucleotides on assembly of the 26S proteasome and degradation of ubiquitin conjugates.

We have investigated three aspects of nucleotide usage by the 26S proteasome and its regulatory complex (RC). Both particles hydrolyze the four major ribonucleotides, but ATP and CTP have substantially lower Kms for hydrolysis than do GTP and UTP. The Km for ATP hydrolysis is 15 microm for the 26S proteasome and 30 microm for the regulatory complex. Formation of the 26S proteasome from the RC and the 20S proteasome requires about 5 microm ATP. Although measurable degradation of Ubiquitin(Ub)-lysozyme conjugates occurs in the presence of CTP, GTP, and UTP, the best nucleotide for Ub-conjugate degradation by the 26S proteasome is ATP, with an estimated Km of 12 microm. In summary, our studies show that micromolar concentrations of ATP are sufficient for several 26S proteasome activities.

A Mutant RNA Polymerase Reveals a Kinetic Mechanism for the Switch between Nonproductive Stuttering Synthesis and Productive Initiation during Promoter Clearance

A Mutant RNA Polymerase Reveals a Kinetic Mechanism for the Switch between Nonproductive Stuttering Synthesis and Productive Initiation during Promoter Clearance

Involvement of the gamma-phosphate of UTP in the synergistic inhibition of Escherichia coli aspartate transcarbamylase by CTP and UTP.

The allosteric control of Escherichia coli aspartate transcarbamylase (ATCase) involves synergistic feedback inhibition by CTP and UTP. Previously reported results [England, P., & Hervé, G. (1992) Biochemistry 31, 9725-9732] suggest that this phenomenon relies entirely on interactions between the two neighboring allosteric sites, which belong to the same regulatory dimer. Furthermore, it has been demonstrated that UTP alone binds to the enzyme, but that it is only in the presence of CTP that this binding inhibits the catalytic activity. The properties of mutants in which the synergistic inhibition is totally abolished suggested that the terminal gamma-phosphate of the pyrimidine triphosphate nucleotides may play a crucial role in promoting site-site interactions within the regulatory dimer. In the present work, kinetic studies and binding experiments by continuous-flow dialysis were performed, using combinations of diphosphate and triphosphate nucleotides. The results obtained show that the gamma-phosphate lf UTP is indeed essential for synergistic inhibition to occur, as UDP is unable to inhibit ATCase activity, whether alone or in combination with CTP. On the contrary, the gamma-phosphate of CTP can be suppressed without modifying the inhibitory properties of this nucleotide and its synergy of action with UTP. These results indicate that the mutual effects of CTP and UTP on their respective binding are not symmetrical and that the signals emitted upon binding of the two triphosphate pyrimidine nucleotides to the regulatory sites do not follow the same pathway and involve different mechanisms.