Localization of the replicase recognition site within brome mosaic virus RNA by hybrid-arrested RNA synthesis

Summary 3 terminal fragments of BMV RNA as short as 153 bases in length serve as efficient templates in vitro for BMV-specific RNA polymerase. Template activity of such fragments or of native BMV RNA is abolished when cDNA fragments as short as 39 bases are hybridized to their 3 termini. Hybridization of cDNa fragments to regions of BMV RNA 200 or more bases distal to the 3 end has no discernible effect on initiation and little effect on elongation. We conclude that BMV RNA polymerase initiates binding with an RNA template through a mechanism mediated by the tRNA-like 3 end of BMV RNA, requiring at least some of the last 39, but no more than the last 153 bases.     

The nature of initiation sites on DNA for the core RNA polymerase

Summary The biological significance of the low level of symmetric and non-specific RNA synthesis catalyzed by the core RNA polymerase devoid of the sigma factor has been analyzed. Shearing of DNA's including T4 DNA markedly increased the template activities with the core enzyme but not with the holoenzyme. This finding suggests that RNA synthesis by the core enzyme increases concomittantly with the production of termini in DNA. Double-stranded circular DNA's such as dv and fd-RFI were found to be inactive as templates for the core enzyme, but were made active by introduction of single-strand nicks with deoxyribonuclease. In contrast, single-stranded circular DNA (X 174) served as a good template for RNA synthesis by the core RNA polymerase. These findings suggest that the sigma factor may activate double-stranded DNA at the promotor sites by creating proper initiation points for RNA synthesis. Partial separation of duplex DNA into single-stranded forms at the promotor sites could be one of the processes in the reaction catalyzed by the holoenzyme containing the sigma factor.     

Mechanism of inhibition of Escherichia coli RNA polymerase by captan.

Captan (N-trichloromethylthiocyclohex-4-ene-1,2-dicarboximide) was shown to inhibit RNA synthesis in vitro catalysed by Escherichia coli RNA polymerase. Incorporation of [gamma-32P]ATP and [gamma-32P]GTP was inhibited by captan to the same extent as overall RNA synthesis. The ratio of [3H]UTP incorporation to that of [gamma-32P]ATP or of [gamma-32P]GTP in control and captan-treated samples indicated that initiation was inhibited, but the length of RNA chains being synthesized was not altered by captan treatment. Limited-substrate assays in which re-initiation of RNA chains did not occur also showed that captan had no effect on the elongation reaction. Studies which measured the interaction of RNA polymerase with template DNA revealed that the binding of enzyme to DNA was inhibited by captan. Glycerol-gradient sedimentation of the captan-treated RNA polymerase indicated that the inhibition of the enzyme was irreversible and did not result in dissociation of its subunits. These data are consistent with a mechanism in which RNA polymerase activity was irreversibly altered by captan, resulting in an inability of the enzyme to bind to the template. This interaction was probably at the DNA-binding site on the polymerase and did not involve reaction of captan with the DNA template.     

DNA primase-DNA polymerase alpha from simian cells. Modulation of RNA primer synthesis by ribonucleoside triphosphates

DNA primase-DNA polymerase alpha, purified 53,000-fold from CV-1 cells, synthesized predominantly (p)ppA(pA)6-primed DNA on a poly(dT) template. About 80% of the RNA primers synthesized on an M13 DNA template were (p)ppA/G(pN)5-7, and 20% were (p)ppA/G(pN)0-4. RNA primer size was determined by gel electrophoresis after removing nascent DNA with phage T4 DNA polymerase 3'-5' exonuclease, leaving a single dNMP at the 3'-end of the RNA primer, and the terminal 5'-(p)ppN residue was determined by "capping" with [alpha-32P]GTP using vaccinia guanylyl-transferase. The processivity of DNA synthesis initiated by de novo synthesis of RNA primers was the same as that initiated on pre-existing RNA primers (10-15 dNMPs), although initiation on pre-existing primers was strongly preferred. Primers always began with A or G, even at high levels of CTP or UTP, although the ratio of A to G varied from 4:1 to 1:1 depending on the relative concentrations of ATP and GTP in the assay. ATP and GTP had no effect on primer length, but the fraction of shorter RNA primers increased 2-fold with higher concentrations of CTP or UTP. Nearest-neighbor analysis revealed a preference for purine ribonucleotides at RNA covalently linked to the 5'-end of DNA (RNA-p-DNA) junctions, and increasing the concentration of a single rNTP increased slightly its presence at RNA-p-DNA junctions. Thus, the base composition and size of RNA primers synthesized by DNA primase-DNA polymerase alpha is modulated by the relative concentrations of ribonucleoside triphosphates.     

Full 1H NMR assignment of a 24-nucleotide RNA hairpin: Application of the 1H 3D-NOE/2QC experiment

The subject RNA models the binding site for the coat protein of the R17 virus, as well as the ribosome recognition sequence for the R17 replicase gene. With an RNA of this size, overlaps among the sugar protons complicate assignments of the ¹H NMR spectrum. The cross peaks that overlap significantly in 2D-NOE spectra can frequently be resolved by introducing a third, in our approach the double-quantum, frequency axis. In particular the planes in a 3D-NOE/2QC spectrum perpendicular to the 2Q axis are extremely useful, showing a highly informative repeating NOE-2Q pattern. In this experiment substantial J-coupling confers special advantages. This always occurs for geminal pairs (H5/H5 for RNA plus H2/H2 for DNA), as well as for H5/H6, for H3/H4 in sugars with substantial populations of the N-pucker, for H1/H2 for S-puckered sugars, and usually for H2/H3. For the 24-mer RNA hairpin the additional information from the 3D-NOE/2QC spectrum allowed assignment of all of the non-exchangeable protons, eliminating the need for stable-isotope labeling.     

Characterization of ribonucleic acid synthesis by nuclei isolated from Zea mays

Nuclei were isolated from the shoots of Zea mays and assayed for endogenous RNA polymerase activity in vitro. Maximum incorporation from radioactive precursors (70 pmol [³H]uridine 5 monophosphate/100 g DNA) was reached after incubation for 1 h at 25°C. The RNA product, analysed by polyacrylamide gel electrophoresis, was polydisperse in size with an upper limit of 2x10⁶ daltons. Discrete peaks of rRNA were not detected, probably because of endogenous ribonuclease activity. The inclusion of -amanitin (4 g/ml) in the incubation reduced the total incorporation by approximately 40% but did not significantly alter the size of the RNA product. Although 40% of the total activity could be attributed to RNA polymerase II, [³H]RNA synthesised in vitro was found not to contain long sequences of poly (A).Abbreviations oligo (dT) oligo (deoxythymidylic acid) - poly (A) poly (adenylic acid) - GTP guanosine 5 triphosphate - ATP adenosine 5 triphosphate - CTP cytidine 5 triphosphate - UTP utidine 5 triphosphate - UMP uridine 5 monophosphate - PPO 2,5-diphenyloxazole - POPOP 1,4-di-2-(5-phenyloxazolyl) benzene     

RNA folding and catalysis

Catalysis in RNA is intimately connected to the folding. The small nucleolytic ribozymes function by a nucleophilic attack of the 2-oxygen on the 3-phosphate, in an S_N2 mechanism. This requires an alignment of the 2-O, 3-P and 5-O, that does not occur in normal A-form RNA. It is therefore likely that structural distortion plays a major role in the enhancement of the reaction rate, facilitating the trajectory into the in-line transition state. Given the polyelectrolyte nature of nucleic acids, metal ions are critical to folding processes in RNA. We have shown that two small nucleolytic ribozymes, the hammerhead and hairpin ribozymes, undergo metal ion-induced folding processes. The hammerhead ribozyme folds in two stages, each of which is induced by the binding of a single structural ion. The first corresponds to the formation of the ribozyme scaffold, while the second is the formation of the catalytic core of the ribozyme. By contrast, the hairpin ribozyme undergoes a single folding event induced by the binding of at least two metal ions, and involves the close interaction between two internal loops to form the active ribozyme.This revised version was published online in October 2005 with corrections to the Cover Date.     

Structure,mechanism and evolution of chloroplast transfer RNA processing systems

Chloroplasts of land plants have an active transfer RNA processing system, consisting of an RNase P-like 5 endonuclease, a 3 endonuclease, and a tRNA:CCA nucleotidyltransferase. The specificity of these enzymes resembles more that of their eukaryotic counterparts than that of their cyanobacterial predecessors. Most strikingly, chloroplast RNase P activity almost certainly resides in a protein, rather than in an RNA protein complex as in Bacteria, Archaea, and Eukarya. The chloroplast enzyme may have evolved from a preexisting chloroplast NADP-binding protein. Chloroplast RNase P cleaves pre-tRNA by a reaction mechanism in which at least one of the Mg²⁺ ions utilized by the bacterial ribozyme RNase P is replaced by an amino acid side chain.Abbreviations pre-tRNA precursor to tRNA - pCp cytidine 5, 3-bisphosphate - IC₅₀ inhibitor concentration giving 50% inhibition - GAPDH glyceraldehyde 3-phosphate dehydrogenase     

MAAP: a versatile and universal tool for genome analysis

Multiple arbitrary amplicon profiling (MAAP) uses one or more oligonucleotide primers (5 nt) of arbitrary sequence to initiate DNA amplification and generate characteristic fingerprints from anonymous genomes or DNA templates. MAAP markers can be used in general fingerprinting as well as in mapping applications, either directly or as sequence-characterized amplified regions (SCARs). MAAP profiles can be tailored in the number of monomorphic and/or polymorphic products. For example, multiple endonuclease digestion of template DNA or the use of mini-hairpin primers can enhance detection of polymorphic DNA. Comparison of the expected and actual number of amplification products produced with primers differing in length, sequence and GC content from templates of varying complexity reveal severe departures from theoretical formulations with interesting implications in primer-template interaction. Extensive primer-template mismatching can occur when using templates of low complexity or long primers. Primer annealing and extension appears directed by an 8 nt 3-terminal primer domain, requires sites with perfect homology to the first 5–6 nt fom the 3 terminus, and involves direct physical interaction between amplicon annealing sites.     

Degradation of RNA in Escherichia coli. A hypothesis

Summary A hypothesis to explain RNA degradation in Escherichia coli is proposed. In this hypothesis all classes of RNA are potentially degradable unless they are protected. The proposed mechanism for mRNA degradation requires a combination of endonuclease(s) and exonuclease(s) which degrades RNA in the 3 to 5 direction. Ribosomes attached to the newly synthesized 5 end of an mRNA molecule protect it from being attacked endonucleolytically; a delay in attachment of ribosomes to this end exposes it to endonucleolytic cleavage, followed by exonucleolytic digestion from the newly exposed 3 end to the 5 end. This mechanism is consistent with an overall 5 to 3 direction of degradation for mRNA. Exoribonucleases that degrade polyribonucleotides from the 5 end to 3 end are not required. The 3 end of the messenger is protected by its association with DNA. In order to enable the mRNA to remain anchored to the DNA while serving as an efficient template for protein synthesis, a special region near the 3 end of the mRNA is envisaged. This hypothetical region would not be translated.     

Den 1, Den2 and Den3, ATP-inhibited deoxyribonucleases from Dropsophila embryonic nuclei

Three Drosophila embryonic deoxyribonucleases, designated den1, den2 and den3, are identified in nuclear extracts separated by glycerol density gradient centrifugation. Den1, removes short products from the 5-ends of single-stranded DNA or double-stranded DNA with either blunt or 5-recessed termini. Den2 is inactive with single-stranded DNA and acts as 3-exonuclease with double-stranded DNA possessing either blunt or 3-recessed termini. Den3 preferentially uses partial duplex DNA containing single-stranded gap and it catalyzes hydrolysis, in 3-5 direction, of one of the shorter strands that flank the gap. Nucleolytic activities of den1, den2 and den3 are inhibited with ATP.     

RNA editing in trypanosomes

Guide RNAs are encoded in maxicircle and minicircle DNA of trypanosome mitochondria. They play a pivotal role in RNA editing, a process during which the nucleotide sequence of mitochondrial RNAs is altered by U-insertion and deletion. Guide RNAs vary in length from 35 to 78 nucleotides, which correlates with the variation in length of the three functionally important regions of which they are composed: (i) a 4–14 nucleotide anchor sequence embedded in the 5 region, which is complementary to a target sequence on the pre-edited RNA downstream of an editing domain, (ii) a middle part containing the editing information, which ranges from guiding the insertion of just one U into one site to that of the insertion of 32 Us into 10 sites, and (iii) a 5–24 nucleotide 3 terminal oligo [U] extension. Moreover, a variable uridylation site creates gRNAs containing a varying segment of editing information for the same domain. Comparison of different guide RNAs demonstrates that, besides the U-tail, they have no obvious common primary and secondary sequence motifs, each particular sequence being unique. The occurrence in vivo and the synthesis in vitro of chimeric molecules, in which a guide RNA is covalently linked through its 3 U-tail to an editing site of a pre-edited RNA, suggests that RNA editing occurs by consecutive transesterification reactions and is evidence that the guide RNAs not only provide the genetic information, but also the Us themselves.Abbreviations gRNA guide RNA     

Effects of ATP and cyclic AMP on the in vitro assembly and stability of mammalian brain microtubules

The relevance of protein phosphorylation, transphosphorylation and binding phenomena in the kinetics of the ATP-induced assembly of cycle-purified microtubule protein from mammalian brain were studied. ATP was able to induce the polymerization of microtubules of normal appearance. However, the assembled structures, were unstable and microtubules depolymerized after achievement of a transitory maximum. Cyclic AMP reduced the amplitude of the polymerization maximum in a concentration-dependent manner, correlating with the stimulation of the endogenous phosphorylation reaction. When microtubule assembly was induced by GTP, in the presence of various concentrations of ATP, the slope of the depolymerization phase was found to depend on the concentration of ATP. Fluoride ion inhibited the endogenous phosphorylation reaction and reduced the disassembly rate, in a concentration-dependent manner. Evidence is also presented indicating that ATP did not bind to phosphocellulose-purified tubulin. These results further contribute to indicate that ATP and cyclic AMP, acting coordinately to control the phosphorylation extent of microtubule proteins are important factors to determine microtubule stability within the cell. Some implications of this mechanism for the regulation by cAMP of the initiation of DNA synthesis and mitosis are considered.Abbreviations MAPs microtubule-associated proteins - MAP2 microtubule-associated protein 2, Mes-4-morpholinoethanesulfonic acid - EGTA ethylene-glycol bis (-aminoethyl ether)N,N,N,N-tetraacetic acid - PMSF phenylmethylsulfonyl fluoride - PEI polyethyleneimine - PC phosphocellulose - DEAE Diethylaminoethyl - SDS-PAGE polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate     

The yeast,Saccharomyces cerevisiae,RNase P/MRP ribonucleoprotein endoribonuclease family

Ribonuclease P (RNase P) is a ribonucleoprotein responsible for the endonucleolytic cleavage of the 5-termini of tRNAs. Ribonuclease MRP (RNase MRP) is a ribonucleoprotein that has the ability to cleave both mitochondrial RNA primers presumed to be involved in mitochondrial DNA replication and rRNA precursors for the production of mature rRNAs. Several lines of evidence suggest that these two ribonucleoproteins are related to each other, both functionally and evolutionarily. Both of these enzymes have activity in the nucleus and mitochondria. Each cleave their RNA substrates in a divalent cation dependent manner to generate 5-phosphate and 3-OH termini. In addition, the RNA subunits of both complexes can be folded into a similar secondary structure. Each can be immunoprecipitated from mammalian cells with Th antibodies. In yeast, both have been found to share at least one common protein. This review will discuss some of the recent advances in our understanding of the structure, function and evolutionary relationship of these two enzymes in the yeast,Saccharomyces cerevisiae.Abbreviations LRI long range interaction - mt mitochondrial - MRP mitochondrial RNA processing - NME nuclear mitochondrial endonuclease - POP processing of precursor - RNase ribonuclease - SNM suppressor of NME - RNP ribonucleoprotein     

Characterization of the guanosine 3′∶5′-monophosphate-dependent protein kinase from silkworm eggs and analysis of the endogenous protein substrates

Summary In a previous paper, two types of cyclic nucleotide-dependent protein kinases, namely cGMP dependent G-kinase and cAMP dependent A-kinase, in silkworm eggs has been reported (Takahashi et al. 1975; Takahashi 1976). One of these, G-kinase, has now been purified 2400-fold by means of ammonium sulfate fractionation, chromatography on hydroxylapatite, DEAE cellulose, and gel filtration.Some of the properties of the enzyme are described. The enzyme is highly dependent on cGMP; it is strongly inhibited by GTP in a noncompetitive manner not only for ATP but also for cGMP. GTP was found to be highly inhibitory on G-kinases from various tissues of the silkworm, but did not inhibit the A-kinase.Incubation of the egg extract with [-³²P]ATP and Mg²⁺ led to the formation of three major³²P-labelled proteins, with molecular weights of 42.000, 70.000 and 180.000 as analyzed by SDS polyacrylamide gel electrophoresis. Two of them corresponded to the subunits of vitellin.The silkworm vitellin was effectively phosphorylated both by the highly purified G-kinase and by the A-kinase. It is concluded that the G-kinase is involved in the phosphorylation of vitellin in developing silkworm eggs.Abbreviations cAMP adenosine 35-monophosphate - cGMP guanosine 35-monophosphate - A-kinase adenosine 35-monophosphate-dependent protein kinase - G-kinase guanosine 35-monophosphate-dependent protein kinase - MIX 1-methyl-3-isobutylxanthine     

Macromolecular synthesis and cell division during morphogenesis of Arthrobacter crystallopoietes

The sphere-rod-sphere morphology cycle of Arthrobacter crystallopoietes was accompanied by changes in the rate of growth and the rates of DNA, RNA and protein synthesis. The patterns of macromolecule synthesis resembled those found in other bacteria during a step-up followed by a step-down in growth rate. During the step-up in growth spherical cells grew into rods and macromolecules were synthesized in the absence of cell division. During stepdown, successive rounds of septation produced progressively smaller cells which did not separate and remained in chains. The morphology of the cells was dependent on the growth rate and could be altered by changing the dilution rate in a malate-limited chemostat. Gradual transitions in morphology and gradual increases in macromolecule content of the cells occurred as the growth rate was increased in the chemostat. Sphere to rod morphogenesis occurred when DNA synthesis was inhibited by treatment with mitomycin C or by thymine starvation. The DNA-deficient rods did not divide and eventually lysed. DNA, RNA and protein synthesis were continuously required for the reductive division of rods to spheres.Abbreviations MS mineral salts - GS mineral salts plus glucose - CA casamino acids - GSCA mineral salts plus glucose plus casamino acids - cAMP cyclic adenosine-3,5-monophosphate - RNA ribonucleic acid - DNA deoxyribonucleic acid     

Purification and properties of the ATP: adenylylsulphate 3′-phosphotransferase from Chlamydomonas reinhardii

APS-kinase (ATP: adenylylsulphate 3-phosphotransferase, EC 2.7.1.25) has been purified from the alga Chlamydomonas reinhardii, strain CW 15 by means of chromatofocussing and affinity chromatography. The isolated protein showed an apparent molecular mass of 44,000 upon sodium dodecylsulphate polyacrylamide gel electrophoresis. The transfer of phosphate groups from ATP onto APS required a pH of 6.8, the presence of Mg²⁺ ions and a reducing thiol. Its catalytical activity was destroyed by sulphhydryl group inhibitors (phenyl-mercuri compounds, dithiopyridine) and alkylating reagents.The purified enzyme attained a V _max of 360 pkat under optimal reaction conditions declining to v _limit of 260 pkat in the presence of excess substrate APS. This sensitivity towards changes in substrate concentrations was parallelled by a high affinity and specificity: apparent K _m APS: 2 · 10^-6 mol · l^-1, and K _m ATP: 7 · 10^-6 mol · l^-1. The enzyme was found specific for ATP, d-ATP and CTP, while UTP, ITP and GTP showed marginal activity. The Hill coefficients suggested 4 binding sites for APS and 1 for ATP. Excessive APS resulted in a negative slope indicating 3 inhibiting sites of the substrate.Abbreviations APS Adenosine 5-phosphosulphate - dATP 2-deoxyadenosine 5-triphosphate - p-CMB p-chloromercuribenzoate - DTE dithioerythritol - DTT dithiothreitol - -MSH -mercaptoethanol - PAPS 3-phosphoadenosine 5-phosphosulphate - PAP 3-phosphoadenosine 5-phosphate - SDS sodium dodecyl sulphate This work is part of a dissertation submitted by H. G. J., Bochum 1982     

De novo purine biosynthesis in the crustacean Artemia: Influence of salinity and geographical origin

Summary In vivo studies of the incoporation of [U-¹⁴C]glycine into purine nucleotides have established the de novo pathway for purine biosynthesis in Artemia sp. during the early period of larval development. This pathway can be modified by the salt concentration of the incubation media. In addition, Artemia of different geographical origins may differ with respect to the detection, functionality and variability of this metabolical pathway.Abbreviations ADP adenosine, diphosphate - ASN acid soluble nucleotides - ATP adenosine triphosphate - DNA desoxyribonucleic acid - GDP guanosine diphosphate - GP₄G pl, p4-diguanosine 5-tetraphosphate - HPLC high performance liquid chromatography - PCA perchloric acid - RNA ribonucleic acid