(1/1482) Efficient synthesis of nucleic acids heavily modified with non-canonical ribose 2'-groups using a mutantT7 RNA polymerase (RNAP).
A T7 RNAP mutant (Y639F) which eliminates discrimination of the chemical character of the NTP ribose 2'-group, facilitates incorporation of non-canonicalsubstrates into nucleic acids. However, transcripts containing a high percentage of non-canonical NMPs are poorly extended due to effects of the 2'-substituents on the transcript:template hybrid conformation. We tested the addition of compounds that stabilize A-type helix geometry to the reaction. High concentrations of polyamines, together with other changes in reaction conditions, greatly increased the synthesis of transcripts heavily substituted with non-canonical ribose 2'-groups. Template structures that facilitate promoter opening increased the efficiency of reactions where non-canonical substrates were incorporated during transcription of +1 to +6. (+info)
(2/1482) An improved separation procedure for nucleoside monophosphates on polyethyleneimine-(PEI-)cellulose thin layers.
A procedure is described for the two-dimensional separation of the 4 major and 16 modified nucleoside-(5') monophosphates on anion-exchange thin layers of polyethyleneimine- (PEI-)cellulose. The method, which is simple and less time-consuming than existing partition chromatographic methods, may be used for the identification of 5'-termini of RNA and RNA fragments. (+info)
(3/1482) Allosteric control of three B12-dependent (class II) ribonucleotide reductases. Implications for the evolution of ribonucleotide reduction.
Three separate classes of ribonucleotide reductases are known, each with a distinct protein structure. One common feature of all enzymes is that a single protein generates each of the four deoxyribonucleotides. Class I and III enzymes contain an allosteric substrate specificity site capable of binding effectors (ATP or various deoxyribonucleoside triphosphates) that direct enzyme specificity. Some (but not all) enzymes contain a second allosteric site that binds only ATP or dATP. Binding of dATP to this site inhibits the activity of these enzymes. X-ray crystallography has localized the two sites within the structure of the Escherichia coli class I enzyme and identified effector-binding amino acids. Here, we have studied the regulation of three class II enzymes, one from the archaebacterium Thermoplasma acidophilum and two from eubacteria (Lactobacillus leichmannii and Thermotoga maritima). Each enzyme has an allosteric site that binds ATP or various deoxyribonucleoside triphosphates and that regulates its substrate specificity according to the same rules as for class I and III enzymes. dATP does not inhibit enzyme activity, suggesting the absence of a second active allosteric site. For the L. leichmannii and T. maritima enzymes, binding experiments also indicate the presence of only one allosteric site. Their primary sequences suggest that these enzymes lack the structural requirements for a second site. In contrast, the T. acidophilum enzyme binds dATP at two separate sites, and its sequence contains putative effector-binding amino acids for a second site. The presence of a second site without apparent physiological function leads to the hypothesis that a functional site was present early during the evolution of ribonucleotide reductases, but that its function was lost from the T. acidophilum enzyme. The other two B12 enzymes lost not only the function, but also the structural basis for the site. Also a large subgroup (Ib) of class I enzymes, but none of the investigated class III enzymes, has lost this site. This is further indirect evidence that class II and I enzymes may have arisen by divergent evolution from class III enzymes. (+info)
(4/1482) Impact of 9-(2-phosphonylmethoxyethyl)adenine on (deoxy)ribonucleotide metabolism and nucleic acid synthesis in tumor cells.
Following exposure to 9-(2-phosphonylmethoxyethyl)adenine (an inhibitor of the cellular DNA polymerases alpha, delta and epsilon), human erythroleukemia K562, human T-lymphoid CEM and murine leukemia L1210 cells markedly accumulated in the S phase of the cell cycle. In contrast to DNA replication, RNA synthesis (transcription) and protein synthesis (mRNA translation) were not affected by 9-(2-phosphonylmethoxyethyl)-adenine. The ribonucleoside triphosphate pools were slightly elevated, while the intracellular levels of all four deoxyribonucleoside triphosphates were 1.5-4-fold increased in 9-(2-phosphonylmethoxyethyl)adenine-treated K562, CEM and L1210 cells. The effect of 9-(2-phosphonylmethoxyethyl)adenine on de novo (thymidylate synthase-mediated) and salvage (thymidine kinase-mediated) dTTP synthesis was investigated using radio-labelled nucleoside precursors. The amount of thymidylate synthase-derived dTTP in the acid soluble pool was 2-4-fold higher in PMEA-treated than in untreated K562 cells, which is in accord with the 3-4-fold expansion of the global dTTP level in the presence of 9-(2-phosphonylmethoxyethyl)adenine. Strikingly, 2-derived dTTP accumulated to a much higher extent (i.e. 16-40-fold) in the soluble dTTP pool upon 9-(2-phosphonylmethoxyethyl)adenine treatment. In keeping with this finding, a markedly increased thymidine kinase activity could be demonstrated in extracts of 9-(2-phosphonylmethoxyethyl)adenine-treated K562 cell cultures. Also, in the presence of 200 microM 9-(2-phosphonylmethoxyethyl)adenine, 14-fold less thymidylate synthase-derived but only 3-fold less thymidine kinase-derived dTTP was incorporated into the DNA of the K562 cells. These data show that thymidine incorporation may be inappropriate as a cell proliferation marker in the presence of DNA synthesis inhibitors such as 9-(2-phosphonylmethoxyethyl)adenine. Our findings indicate that 9-(2-phosphonylmethoxyethyl)adenine causes a peculiar pattern of (deoxy)ribonucleotide metabolism deregulation in drug-treated tumor cells, as a result of the metabolic block imposed by the drug on the S phase of the cell cycle. (+info)
(5/1482) New thioredoxins and glutaredoxins as electron donors of 3'-phosphoadenylylsulfate reductase.
Reduction of inorganic sulfate to sulfite in prototrophic bacteria occurs with 3'-phosphoadenylylsulfate (PAPS) as substrate for PAPS reductase and is the first step leading to reduced sulfur for cellular biosynthetic reactions. The relative efficiency as reductants of homogeneous highly active PAPS reductase of the newly identified second thioredoxin (Trx2) and glutaredoxins (Grx1, Grx2, Grx3, and a mutant Grx1C14S) was compared with the well known thioredoxin (Trx1) from Escherichia coli. Trx1, Trx2, and Grx1 supported virtually identical rates of sulfite formation with a Vmax ranging from 6.6 units mg-1 (Trx1) to 5.1 units mg-1 (Grx1), whereas Grx1C14S was only marginally active, and Grx2 and Grx3 had no activity. The structural difference between active reductants had no effect upon Km PAPS (22.5 microM). Grx1 effectively replaced Trx1 with essentially identical Km-values: Km trx1 (13.7 microM), Km grx1 (14.9 microM), whereas the Km trx2 was considerably higher (34.2 microM). The results agree with previous in vivo data suggesting that Trx1 or Grx1 is essential for sulfate reduction but not for ribonucleotide reduction in E. coli. (+info)
(6/1482) 5'-Capping structures of Artemia salina mRNA and the translational inhibition by cap analogs.
The mRNA of the brain shrimp Artemia salina has two types of blocked methylated 5'-terminal structures (caps). About 75% of the mRNA molecules have the 5'-end structure of m7G5'ppp5'-AmpGp and about 25% have the structure of m7G5'ppp5'GmpGp. The only other type of methylated residue found in Artemia mRNA is N6-methyladenosine and which is located at internal positions along the mRNA chain. Translation of Artemia cyst or nauplius poly(A)-rich mRNA in wheat-germ extracts was found to be inhibited by 7-methylguanosine 5'-monophosphate, a chemical analog of the cap, as well as by snythetic caps such as m7G5'ppp5'Gm. On the other hand, the elongation activity on endonegous mRNA in an Artemia cell-free system was not sensitive to 7-methylguanosine 5'-monophosphate. (+info)
(7/1482) Apoptosis induced by growth factor withdrawal in fibroblasts overproducing fructose 2,6-bisphosphate.
Fructose 2,6-bisphosphate is a potent endogenous stimulator of glycolysis. A high aerobic glycolytic rate often correlates with increased cell proliferation. To investigate this relationship, we have produced clonal cell lines of Rat-1 fibroblasts that stably express transgenes coding for 6-phosphofructo-2-kinase, which catalyzes the synthesis of fructose 2,6-bisphosphate, or for fructose 2,6-bisphosphatase, which catalyzes its degradation. While serum deprivation in culture reduced the growth rate of control cells, it caused apoptosis in cells overproducing fructose 2,6-bisphosphate. Apoptosis was inhibited by 5-amino-4-imidazolecarboxamide riboside, suggesting that 5'-AMP-activated protein kinase interferes with this phenomenon. (+info)
(8/1482) Incorporation of 32Pi into nucleotides, polyphosphates, and other acid-soluble compounds by Myxococcus xanthus during myxospore formation.
When glycerol was used to induce myxospore formation in Myxococcu xanthus in the presence of 32Pi, the label was incorporated into a variety of acid-soluble compounds. Incorporation into ribonucleotides was approximately fivefold greater than in vegetative cells or noninducible mutants grown in glycerol. The label was also incorporated into some unknown compounds and material tentatively identified as guanosine tetraphosphate. Marked accumulation into polyphosphates, which were present mainly in culture supernatants, occurred relatively late during myxospore formation. The kinetics of accumulation of some of these compounds and their distribution into acid-soluble cell extracts and culture supernatants are described and compared with those in vegetative cells and noninducible mutants. (+info)