Phosphoethanolamine cytidylyltransferase ameliorates mitochondrial function and apoptosis in hepatocytes in T2DM in vitro.

Liver function indicators are often impaired in patients with type 2 diabetes mellitus (T2DM), who present higher concentrations of aspartate aminotransferase, alanine aminotransferase, and gamma-glutamyl transferase than individuals without diabetes. However, the mechanism of liver injury in patients with T2DM has not been clearly elucidated. In this study, we performed a lipidomics analysis on the liver of T2DM mice, and we found that phosphatidylethanolamine (PE) levels were low in T2DM, along with an increase in diglyceride, which may be due to a decrease in the levels of phosphoethanolamine cytidylyltransferase (Pcyt2), thus likely affecting the de novo synthesis of PE. The phosphatidylserine decarboxylase pathway did not change significantly in the T2DM model, although both pathways are critical sources of PE. Supplementation with CDP-ethanolamine (CDP-etn) to increase the production of PE from the CDP-etn pathway reversed high glucose and FFA (HG&FFA)-induced mitochondrial damage including increased apoptosis, decreased ATP synthesis, decreased mitochondrial membrane potential, and increased reactive oxygen species, whereas supplementation with lysophosphatidylethanolamine, which can increase PE production in the phosphatidylserine decarboxylase pathway, did not. Additionally, we found that overexpression of PCYT2 significantly ameliorated ATP synthesis and abnormal mitochondrial morphology induced by HG&FFA. Finally, the BAX/Bcl-2/caspase3 apoptosis pathway was activated in hepatocytes of the T2DM model, which could also be reversed by CDP-etn supplements and PCYT2 overexpression. In summary, in the liver of T2DM mice, Pcyt2 reduction may lead to a decrease in the levels of PE, whereas CDP-etn supplementation and PCYT2 overexpression ameliorate partial mitochondrial function and apoptosis in HG&FFA-stimulated L02 cells.

Structural basis for template-independent RNA polymerization.

The 3'-terminal CCA nucleotide sequence (positions 74-76) of transfer RNA is essential for amino acid attachment and interaction with the ribosome during protein synthesis. The CCA sequence is synthesized de novo and/or repaired by a template-independent RNA polymerase, 'CCA-adding enzyme', using CTP and ATP as substrates. Despite structural and biochemical studies, the mechanism by which the CCA-adding enzyme synthesizes the defined sequence without a nucleic acid template remains elusive. Here we present the crystal structure of Aquifex aeolicus CCA-adding enzyme, bound to a primer tRNA lacking the terminal adenosine and an incoming ATP analogue, at 2.8 A resolution. The enzyme enfolds the acceptor T helix of the tRNA molecule. In the catalytic pocket, C75 is adjacent to ATP, and their base moieties are stacked. The complementary pocket for recognizing C74-C75 of tRNA forms a 'protein template' for the penultimate two nucleotides, mimicking the nucleotide template used by template-dependent polymerases. These results are supported by systematic analyses of mutants. Our structure represents the 'pre-insertion' stage of selecting the incoming nucleotide and provides the structural basis for the mechanism underlying template-independent RNA polymerization.

Recombinant RNase Z does not recognize CCA as part of the tRNA and its cleavage efficieny is influenced by acceptor stem length.

One of the essential maturation steps to yield functional tRNA molecules is the removal of 3'-trailer sequences by RNase Z. After RNase Z cleavage the tRNA nucleotidyl transferase adds the CCA sequence to the tRNA 3'-terminus, thereby generating the mature tRNA. Here we investigated whether a terminal CCA triplet as 3'-trailer or embedded in a longer 3'-trailer influences cleavage site selection by RNase Z using three activities: a recombinant plant RNase Z, a recombinant archaeal RNase Z and an RNase Z active wheat extract. A trailer of only the CCA trinucleotide is left intact by the wheat extract RNase Z but is removed by the recombinant plant and archaeal enzymes. Thus the CCA triplet is not recognized by the RNase Z enzyme itself, but rather requires cofactors still present in the extract. In addition, we investigated the influence of acceptor stem length on cleavage by RNase Z using variants of wild-type tRNATyr. While the wild type and the variant with 8 base pairs in the acceptor stem were processed efficiently by all three activities, variants with shorter and longer acceptor stems were poor substrates or were not cleaved at all.

U2 small nuclear RNA is a substrate for the CCA-adding enzyme (tRNA nucleotidyltransferase).

The CCA-adding enzyme builds and repairs the 3' terminus of tRNA. Approximately 65% of mature human U2 small nuclear RNA (snRNA) ends in 3'-terminal CCA, as do all mature tRNAs; the other 35% ends in 3' CC or possibly 3' C. The 3'-terminal A of U2 snRNA cannot be encoded because the 3' end of the U2 snRNA coding region is CC/CC, where the slash indicates the last encoded nucleotide. The first detectable U2 snRNA precursor contains 10-16 extra 3' nucleotides that are removed by one or more 3' exonucleases. Thus, if 3' exonuclease activity removes the encoded 3' CC during U2 snRNA maturation, as appears to be the case in vitro, the cell may need to build or rebuild the 3'-terminal A, CA, or CCA of U2 snRNA. We asked whether homologous and heterologous class I and class II CCA-adding enzymes could add 3'-terminal A, CA, or CCA to human U2 snRNA lacking 3'-terminal A, CA, or CCA. The naked U2 snRNAs were good substrates for the human CCA-adding enzyme but were inactive with the Escherichia coli enzyme; activity was also observed on native U2 snRNPs. We suggest that the 3' stem/loop of U2 snRNA resembles a tRNA minihelix, the smallest efficient substrate for class I and II CCA-adding enzymes, and that CCA addition to U2 snRNA may take place in vivo after snRNP assembly has begun.

A single rep protein initiates replication of multiple genome components of faba bean necrotic yellows virus, a single-stranded DNA virus of plants.

Faba bean necrotic yellows virus (FBNYV) belongs to the nanoviruses, plant viruses whose genome consists of multiple circular single-stranded DNA components. Eleven distinct DNAs, 5 of which encode different replication initiator (Rep) proteins, have been identified in two FBNYV isolates. Origin-specific DNA cleavage and nucleotidyl transfer activities were shown for Rep1 and Rep2 proteins in vitro, and their essential tyrosine residues that catalyze these reactions were identified by site-directed mutagenesis. In addition, we showed that Rep1 and Rep2 proteins hydrolyze ATP, and by changing the key lysine residue in the proteins' nucleoside triphosphate binding sites, demonstrated that this ATPase activity is essential for multiplication of virus DNA in vivo. Each of the five FBNYV Rep proteins initiated replication of the DNA molecule by which it was encoded, but only Rep2 was able to initiate replication of all the six other genome components. Furthermore, of the five rep components, only the Rep2-encoding DNA was always detected in 55 FBNYV samples from eight countries. These data provide experimental evidence for a master replication protein encoded by a multicomponent single-stranded DNA virus.

Product release is the major contributor to kcat for the hepatitis C virus helicase-catalyzed strand separation of short duplex DNA.

Hepatitis C virus (HCV) helicase catalyzes the ATP-dependent strand separation of duplex RNA and DNA containing a 3' single-stranded tail. Equilibrium and velocity sedimentation centrifugation experiments demonstrated that the enzyme was monomeric in the presence of DNA and ATP analogues. Steady-state and pre-steady-state kinetics for helicase activity were monitored by the fluorescence changes associated with strand separation of F21:HF31 that was formed from a 5'-hexachlorofluorescein-tagged 31-mer (HF31) and a complementary 3'-fluorescein-tagged 21-mer (F21). kcat for this reaction was 0.12 s-1. The fluorescence change associated with strand separation of F21:HF31 by excess enzyme and ATP was a biphasic process. The time course of the early phase (duplex unwinding) suggested only a few base pairs ( approximately 2) were disrupted concertedly. The maximal value of the rate constant (keff) describing the late phase of the reaction (strand separation) was 0. 5 s-1, which was 4-fold greater than kcat. Release of HF31 from E. HF31 in the presence of ATP (0.21 s-1) was the major contributor to kcat. At saturating ATP and competitor DNA concentrations, the enzyme unwound 44% of F21:HF31 that was initially bound to the enzyme (low processivity). These results are consistent with a passive mechanism for strand separation of F21:HF31 by HCV helicase.

RNA helicase A mediates association of CBP with RNA polymerase II.

The coactivator CBP has been proposed to stimulate the expression of certain signal-dependent genes via its association with RNA polymerase II complexes. Here we show that complex formation between CBP and RNA polymerase II requires RNA helicase A (RHA), a nuclear DNA/RNA helicase that is related to the Drosophila male dosage compensation factor mle. In transient transfection assays, RHA was found to cooperate with CBP in mediating target gene activation via the CAMP responsive factor CREB. As a mutation in RHA that compromised its helicase activity correspondingly reduced CREB-dependent transcription, we propose that RHA may induce local changes in chromatin structure that promote engagement of the transcriptional apparatus on signal responsive promoters.

Kinetics of the RNA-DNA helicase activity of Escherichia coli transcription termination factor rho. 1. Characterization and analysis of the reaction.

The kinetics of the ATP-dependent RNA-DNA helicase activity of Escherichia colitranscription termination factor rho have been analyzed. Helicase substrates were assembled using 255 nt and 391 nt RNA sequences from the trp t' RNA transcript of E. coli. These RNA sequences each carry a rho "loading site" at a position near the 5'-end, and a rho-dependent terminator sequence at the 3'-end to which complementary approximately 20 nt DNA oligonucleotides have been annealed. A rapid ( approximately 30 s) pre-steady-state burst of helicase activity (DNA oligomer release), followed by a slow linear phase, is observed in reactions carried out at low salt concentrations (50 mM KCl). Using poly(rC) or poly(dC) as traps for the rho that is released after one round of activity, we have shown that the first (burst) phase of the reaction represents the processive translocation of prebound rho hexamers from the rho loading site to the 3'-end of the RNA molecule. The slow phase of the reaction is complex and represents a combination of many different processes, including the slow release of RNA from rho, the reannealing of complementary DNA oligonucleotides to the RNA substrate, and the recycling of rho hexamers onto additional RNA molecules. Reactions carried out at higher salt concentrations (150 mM KCl) consist of only one phase, since under these conditions rho dissociates more rapidly from the RNA, with an amplitude corresponding to several DNA oligomers removed per rho hexamer. Thus, rho can recycle and function as a catalytic helicase under reaction conditions resembling those found in the cell.

Kinetics of the RNA-DNA helicase activity of Escherichia coli transcription termination factor rho. 2. Processivity, ATP consumption, and RNA binding.

The RNA-binding and RNA-DNA helicase activities of the Escherichia coli transcription termination factor rho have been investigated using natural RNA molecules that are 255 and 391 nucleotide residues in length and that contain the trp t' rho-dependent termination sequence of E. coli. Helicase substrates were prepared from these RNA molecules by annealing one or more DNA oligomers to complementary sequences located at or near the 3'-ends of the RNA molecules to form defined RNA-DNA hybrid sequences ranging in length from 20 to 100 bp. By comparing the fraction of the RNA molecules bound to rho with the fraction of bound DNA oligomers removed from the RNA during one round of the helicase reaction, we have shown that rho translocates processively at 37 degrees C in buffer containing 50 mM KCl. Helicase reactions and ATPase measurements were performed in parallel in the presence of RNA molecules containing RNA-DNA hybrids of various lengths, and we show that both the rate of translocation of the rho hexamer along the RNA chain and the rate of ATP consumption are similar, whether or not DNA is hybridized to the RNA transcript. By combining measurements of translocation and ATPase rates, we estimate that rho consumes approximately 1-2 ATP molecules in translocating over 1 nucleotide residue of the RNA chain at 37 degrees C in 50 mM KCl. The ATPase activity of rho remains the same after one round of the helicase reaction, indicating that rho appears to hydrolyze ATP at the same rate, whether it is translocating along the RNA, separating RNA-DNA hybrids, or bound at the 3'-end of the RNA substrate. We also show that rho binds cooperatively ( approximately 2-4 rho hexamers per RNA chain) to the RNA substrates under our standard helicase reaction conditions. However, cooperative binding is not essential for helicase activity, since this binding stoichiometry can be reduced to approximately 1.5 rho hexamers per 255-nucleotide residue RNA chain by blocking approximately 100 nt of either end of the rho binding site of the helicase substrate with complementary DNA oligonucleotides, with no change in helicase properties. The implications of these results for models of rho helicase function and for the role of rho in termination are discussed.

Interaction of Escherichia coli primase with a phage G4ori(c)-E. coli SSB complex.

We earlier reported that Escherichia coli single-stranded DNA-binding protein (SSB) bound in a fixed position to the stem-loop structure of the origin of complementary DNA strand synthesis in phage G4 (G4ori(c)), leaving stem-loop I and the adjacent 5' CTG 3', the primer RNA initiation site, as an SSB-free region (W. Sun and G. N. Godson, J. Biol. Chem. 268:8026-8039, 1993). Using a small 278-nucleotide (nt) G4ori(c) single-stranded DNA fragment that supported primer RNA synthesis, we now demonstrate by gel shift that E. coli primase can stably interact with the SSB-G4ori(c) complex. This stable interaction requires Mg2+ for specificity. At 8 mM Mg2+, primase binds to an SSB-coated 278-nt G4ori(c) fragment but not to an SSB-coated control 285-nt LacZ ss-DNA fragment. In the absence of Mg2+, primase binds to both SSB-coated fragments and gives a gel shift. T4 gene 32 protein cannot substitute for E. coli SSB in this reaction. Stable interaction of primase with naked G4ori(c). single-stranded DNA was not observed. DNase I and micrococcal nuclease footprinting, of both 5' and 3' 32P-labeled DNA, demonstrated that primase interacts with two regions of G4ori(c): one covering stem-loop I and the 3' sequence flanking stem-loop I which contains the pRNA initiation site and another located on the 5' sequence flanking stem-loop III.

Primer synthesis kinetics by Escherichia coli primase on single-stranded DNA templates.

The kinetics of primer RNA initiation and elongation by Escherichia coli primase were measured. The single-stranded DNA template that was used to develop the system, d(CAGA-(CA)5CTGCAAAGC), contained: (1) the preferred initiating trinucleotide d(CTG); (2) five residues 3' to the d(CTG), the minimum required for efficient primer synthesis; and (3) a single guanine placed near the 5'-end to facilitate study of cytidine triphosphate analog incorporation at a unique site. The assay monitored radiolabeled nucleotide incorporation into the RNA primers. The various primers were separated by length using denaturing polyacrylamide gel electrophoresis. Different types of primers were observed when synthesis was monitored using gamma- versus alpha-radiolabeled nucleotides as the probe. When [gamma-32P]-ATP incorporation was the probe, only primers initiated with ATP from the unique template thymine were observed. The sequences of these primers were complementary to that of the template. No primers shorter than a 12-mer accumulated, demonstrating that formation of the first phosphodiester bond was much slower than that of the next 10 phosphodiester bonds. The longest primer observed when monitoring [gamma-32P]ATP incorporation was 16 nucleotides long, the correct length for a primer completely template-directed and initiated at the unique thymine. Misinsertion of a noncognate nucleotide at the template's guanine indicated very poor nucleotide discrimination by this enzyme. When [alpha-32P]UTP was the probe for primer synthesis, all primers synthesized were observed whether or not they were initiated with ATP. Under these conditions, "overlong" primers and the above-described template length-dependent primers were observed. The template length-dependent primers accumulated faster than the overlong primers, but, at long incubation times, the overlong primers became the dominant species. The overlong primers were not fully related to the template length-dependent primers since they were not initiated complementary to the template d(CTG). Nevertheless, the overlong primers did appear to arise as a consequence of the template length-dependent species since their length was double and they arose in the time course after the length-dependent species.

Cell cycle-dependent phosphorylation and dephosphorylation of the yeast DNA polymerase alpha-primase B subunit.

The yeast DNA polymerase alpha-primase B subunit functions in initiation of DNA replication. This protein is present in two forms, of 86 and 91 kDa, and the p91 polypeptide results from cell cycle-regulated phosphorylation of p86. The B subunit present in G1 arises by dephosphorylation of p91 while cells are exiting from mitosis, becomes phosphorylated in early S phase, and is competent and sufficient to initiate DNA replication. The B subunit transiently synthesized as a consequence of periodic transcription of the POL12 gene is phosphorylated no earlier than G2. Phosphorylation of the B subunit does not require execution of the CDC7-dependent step and ongoing DNA synthesis. We suggest that posttranslational modifications of the B subunit might modulate the role of DNA polymerase alpha-primase in DNA replication.

Characterization and partial purification of tRNA processing activities from potato mitochondria.

In plant mitochondria, as in most other genetic systems, several enzymatic processing and modification steps are required to yield mature tRNAs from primary transcripts. Three of the enzymes involved, RNase P, 3'-processing activity, and tRNA nucleotidyl transferase, were identified in potato (Solanum tuberosum) mitochondria and have been separated by several purification steps. RNase P was partially purified, with only a few proteins detectable in active fractions after a final glycerol gradient step. A small RNA molecule present in fractions with RNase P activity contains the heptanucleotide conserved in the other known RNase P RNA sequences and may be a fragment of the RNA moiety of the plant mitochondrial RNase P.

Enriched sources of Escherichia coli replication proteins. The dnaG primase is a zinc metalloprotein.

Primase, the product of the Escherichia coli dnaG gene, is the enzyme responsible for RNA primer synthesis on both template strands at replication forks during chromosomal DNA synthesis. The dnaG gene was modified by replacement of the natural ribosome-binding site with one complementary to the 3' end of 16S rRNA, and then inserted downstream of tandem bacteriophage lambda PR and PL promoters in the pUC9-derived vector pCE30. Following thermal induction of transcription, the resulting plasmid pPL195 directed synthesis of primase activity to levels corresponding to approx. 120,000 molecules per cell. The overproduced protein was soluble and was readily purified in high yield (31 mg per 1 of culture). Purified primase was monomeric, was fully active in priming replication at the bacteriophage G4 complementary strand origin, and was shown to contain 0.92 +/- 0.08 g atom of tightly-bound zinc per mol of protein. Potential zinc-binding amino-acid residues near the N-terminus of the protein were identified. Although a mutant primase lacking 27 amino acid residues from the N-terminus was partly soluble, it was completely inactive.

The major RNA in prosomes of HeLa cells and duck erythroblasts is tRNA(Lys,3).

Two-dimensional gel electrophoresis of HeLa cell prosomal RNAs, 3'-end labeled by RNA ligase, revealed one prominent spot. Determination of a partial sequence at the 3'-end indicated full homology to the 18 nucleotides at the 3'-end of tRNA(Lys,3) from rabbit, the bovine and the human species. An oligonucleotide complementary to the 3'-end of tRNA(Lys,3) hybridized on Northern blots with prosomal RNA from both HeLa cells and duck erythroblasts. In two-dimensional PAGE, the major pRNA of HeLa cells co-migrated with bovine tRNA(Lys,3). Reconstitution of the CCA 3'-end of RNA from both human and duck prosomes, by tRNA-nucleotidyl-transferase, confirmed the tRNA character of this type of RNA. Furthermore, it revealed at least one additional tRNA band about 85 nt long among the prosomal RNA from both species. Finally, confirming an original property of prosomal RNA, we show that in vitro synthesized tRNA(Lys,3) hybridizes stably to duck globin mRNA, and to poly(A)(+)- and poly(A)(-)-RNA from HeLa cells.

Pea chloroplast DNA primase: characterization and role in initiation of replication.

A DNA primase activity was isolated from pea chloroplasts and examined for its role in replication. The DNA primase activity was separated from the majority of the chloroplast RNA polymerase activity by linear salt gradient elution from a DEAE-cellulose column, and the two enzyme activities were separately purified through heparin-Sepharose columns. The primase activity was not inhibited by tagetitoxin, a specific inhibitor of chloroplast RNA polymerase, or by polyclonal antibodies prepared against purified pea chloroplast RNA polymerase, while the RNA polymerase activity was inhibited completely by either tagetitoxin or the polyclonal antibodies. The DNA primase activity was capable of priming DNA replication on single-stranded templates including poly(dT), poly(dC), M13mp19, and M13mp19 + 2.1, which contains the AT-rich pea chloroplast origin of replication. The RNA polymerase fraction was incapable of supporting incorporation of 3H-TTP in in vitro replication reactions using any of these single-stranded DNA templates. Glycerol gradient analysis indicated that the pea chloroplast DNA primase (115-120 kDa) separated from the pea chloroplast DNA polymerase (90 kDa), but is much smaller than chloroplast RNA polymerase. Because of these differences in size, template specificity, sensitivity to inhibitors, and elution characteristics, it is clear that the pea chloroplast DNA primase is an distinct enzyme form RNA polymerase. In vitro replication activity using the DNA primase fraction required all four rNTPs for optimum activity. The chloroplast DNA primase was capable of priming DNA replication activity on any single-stranded M13 template, but shows a strong preference for M13mp19 + 2.1. Primers synthesized using M13mp19 + 2.1 are resistant to DNase I, and range in size from 4 to about 60 nucleotides.

DNA primase-DNA polymerase alpha from simian cells: sequence specificity of initiation sites on simian virus 40 DNA.

Unique single-stranded regions of simian virus 40 DNA, phage M13 virion DNA, and several homopolymers were used as templates for the synthesis of (p)ppRNA-DNA chains by CV-1 cell DNA primase-DNA polymerase alpha. Intact RNA primers, specifically labeled with an RNA capping enzyme, were typically 6 to 8 ribonucleotides long, although their lengths ranged from 1 to 9 bases. The fraction of intact RNA primers 1 to 4 ribonucleotides long was 14 to 73%, depending on the template used. RNA primer length varied among primers initiated at the same nucleotide, as well as with primers initiated at different sites. Thus, the size of an RNA primer depended on template sequence. Initiation sites were identified by mapping 5' ends of nascent RNA-DNA chains on the template sequence, identifying the 5'-terminal ribonucleotide, and partially sequencing one RNA primer. A total of 56 initiation events were identified on simian virus 40 DNA, an average of 1 every 16 bases. Some sites were preferred over others. A consensus sequence for initiation sites consisted of either 3'-dCTTT or 3'-dCCC centered within 7 to 25 pyrimidine-rich residues; the 5' ends of RNA primers were complementary to the dT or dC. High ATP/GTP ratios promoted initiation of RNA primer synthesis at 3'-dCTTT sites, whereas low ATP/GTP ratios promoted initiation at 3'-dCCC sites. Similarly, polydeoxythymidylic acid and polydeoxycytidylic acid were the only effective homopolymer templates. Thus, both template sequence and ribonucleoside triphosphate concentrations determine which initiation sites are used by DNA primase-DNA polymerase alpha. Remarkably, initiation sites selected in vitro were strikingly different from initiation sites selected during simian virus 40 DNA replication in vivo.

Reactions in vitro of the DNA polymerase-primase from Xenopus laevis eggs. A role for ATP in chain elongation.

A form of DNA polymerase alpha was purified several thousandfold from a protein extract of Xenopus laevis eggs. The enzyme effectively converts, in the presence of ribonucleoside triphosphates, a circular single-stranded phage fd DNA template into a double-stranded DNA form and, therefore, must be associated with a DNA primase. We first show by gel electrophoresis in the presence of sodium dodecyl sulfate that both enzymatic activities, DNA polymerase and primase, most probably reside on a greater than 100 000-Da subunit of the DNA polymerase holoenzyme. We then assayed the polymerase-primase at various template/enzyme ratios and found that the DNA complementary strand sections synthesized in vitro belong to defined size classes in the range of 600-2000 nucleotides, suggesting preferred start and/or stop sites on the fd DNA template strand. We show that the stop sites coincide with stable hairpin structures in fd DNA. We have used a fd DNA template, primed by a restriction fragment of known size, to show that the polymerase-primase stops at the first stable hairpin structure upstream from the 3'-OH primer site when the reaction was carried out at 0.1 mM ATP. However, at 2 mM ATP the enzyme was able to travers this and other stop sites on the fd DNA template strand leading to the synthesis of 2-4 times longer DNA strands. Our results suggest a role for ATP in the polymerase-primase-catalyzed chain-elongation reaction.