Characteristics of synthesized RNA in the isolated and perfused rat liver

The incorporation of 3H-orotic acid into nuclear and microsomal RNA from isolated perfused rat liver has been studied. The specific radioactivity of nuclear RNA indicates that the efficiency for RNA synthesis in the perfused liver is similar to that of the liver 'in vivo'. In contrast, the microsomal RNA specific radioactivity is well below that observed 'in vivo'. This may indicate a slower transport of the labelled RNA from the nucleus to the cytoplasm. Labelling pattern of total nuclear RNA, nuclear poly(A) containing RNA and microsomal RNA appear to be in line with these assumptions.     

Specific labelling of the active site of T7 RNA polymerase.

We describe a method for specifically labelling T7 RNA polymerase at (or near) the active site. Enzyme molecules that have been modified by covalent attachment of a benzaldehyde nucleotide derivative in the presence of template DNA are subsequently incubated with radioactively labelled nucleoside triphosphates. Labelling of the enzyme occurs as a result of the formation of the first phosphodiester bond. The labelling is template-directed and the expected specificity of initiation at individual T7 promoters is observed. The label has been localized to an 80 kd tryptic fragment that contains the carboxy-terminal portion of the enzyme.     

The dynamics of the specific activity of newly synthesized RNA during loach embryo development]

The transport of the stable form of newly synthesized RNA from the nucleus to the cytoplasm has been studied in the loach (Misgurnus fossilis) development. Following the pulse labelling with 3H-uridine, the embryos were cultivated in the medium with non-labelled uridine and actinomycin D. The cell homogenate was fractionated and the specific activity of nuclear and cytoplasmic RNAs was determined. It was shown that a great part of newly synthesized RNA degraded within the nucleus and its insignificant part was preserved in the nucleus for several hours. The exit of stable RNA in the cytoplasm depends on the developmental stage. This part of RNA was found to stay in the nucleus at the stages of early--midblastula and leave it in the beginning of gastrulation. At the later developmental stages the newly synthesized RNA passes in the cytoplasm immediately.     

Increased RNA labelling in boron-deficient root-tip segments

The rate of incorporation of radioactive precursors into RNA of root-tip segments has been shown to increase during the early stages of boron deficiency in 6- to 8-day-old Phaseolus aureus seedlings. This effect is observed well before morphological changes occur, and before both the decrease in level of total RNA and increase in ribonuclease activity can be detected. The nucleotide pool in root tips was too small to measure directly on the rapidly growing seedlings, although it was easily measurable in the slower growing 12- to 15-day-old plants. The available indirect evidence suggests, however, that the early effect is not due to changes within the total nucleotide pool.     

Highly selective affinity labelling of RNA polymerase B (II) from wheat germ

DNA-dependent RNA polymerase B (II) from wheat germ was modified by incubation with 4-[N-(β-hydroxyethyl)-N-methyl]benzaldehyde esters of AMP, ADP or ATP, followed by reduction with NaBH₄. Reaction of the modified enzyme with [-³²P]UTP in the presence of various DNA templates led to a highly selective affinity labelling of the subunit with M_r 140000 by covalently linked ApU. Labelling was inhibited by 1μg/ml -amanitin.     

Incorporation of 13C,15N-labeled nucleosides and measurement of RNA synthesis and turnover in sea urchin embryos

The uptake of nucleosides into sea urchin embryos and their subsequent incorporation into RNA increases with increasing external nucleoside concentration. When embryos are incubated with high concentrations of ¹³C,¹⁵N-labeled nucleosides, newly synthesized RNA becomes sufficiently labeled with heavy isotope to be separated from unlabeled RNA on cesium formate equilibrium gradients. High concentrations of nucleosides do not affect development of embryos or rates of RNA synthesis. The extent of density-labeling of precursor pools increases with incubation time, and only levels off after many hours. During incubations with high concentrations of nucleosides, ATP pools expand up to twofold. Using density-labeling to circumvent precursor pool measurements, a method is presented to study the synthesis and decay of pulse-labeled RNA. The instantaneous rate of synthesis of total RNA at the blastula stage is 9.3 × 10^?15 mol of total nucleotide/embryo per minute and the average half-life of total RNA is 23 minutes.     

Activity of the endogenous RNA polymerase on fixed polytene chromosomes.

Fixed polytene chromosomes can serve as templates for RNA synthesis in situ, using the endogenous chromosomal DNA-dependent RNA polymerase. Labelling is mainly localized in band regions. However, radioactivity can also be found in interbands and puffs similar to that which occurs in vivo. It is also found by this technique that the nucleolar RNA polymerase appears to be active in these preparations and requires Mg²⁺ for activity. Since the pattern of the RNA transcribed in situ with the DNA-dependent RNA polymerase from E. coli of native chromosomes differs from that with endogenous RNA polymerase and resembles the one obtained with heat-treated chromosomes, it is suggested that the polymerase from E. coli does not act specifically on eukaryotic chromosomes.     

An embarrassment of riches: the enzymology of RNA modification

The maturation of transfer RNA (tRNA) involves extensive chemical modification of the constituent nucleosides and results in the introduction of significant chemical diversity to tRNA. Many of the pathways to these modified nucleosides are characterized by chemically complex transformations, some of which are unprecedented in other areas of biology. To illustrate the scope of the field, recent progress in understanding the enzymology leading to the formation of two distinct classes of modified nucleosides, the thiouridines and queuosine, a 7-deazaguanosine, is reviewed. In particular, recent data validating the involvement of several proposed intermediates in the formation of thiouridines are discussed, including two key enzyme intermediates and the activated tRNA intermediate. The discovery and mechanistic characterization of a new enzyme activity in the queuosine pathway is discussed.     

A new class of small nuclear RNA molecules synthesized by a type I RNA polymerase in HeLa cells

A new class of previously undetected small RNA molecules with a range of discrete sizes between 6S and 10S has been identified in HeLa cell nuclei. They differ in size and location from the previously described small nuclear RNA species (snRNA). These RNA molecules were initially found by selective RNA labeling in vitro in isolated nuclei. The in vitro products migrate in gel electrophoresis in the region from 6–10S with predominant components between 8S and 10S. They are labeled in the presence of very high concentrations of α-amanitin (150–400 μg/ml), suggesting they are synthesized by a type I polymerase. Unlike the major polymerase I product, ribosomal precursor RNA, however, these molecules are found in the nucleoplasm and their labeling is not affected by pretreatment of cells with low concentrations of actinomycin D (0.04 μg/ml). Their formation by a presumptive polymerase I type of enzyme is the basis of their tentative designation as small nuclear polymerase I (snPI) RNAs.The snPI RNA molecules appear to be associated with chromatin and the nuclear matrix. They can be selectively eluted from nuclei leaving most of hnRNA behind. This association is used as the basis of fractionation procedures which separate these molecules from hnRNA and permit the demonstration of the synthesis of at least the most predominant of these RNA molecules in vivo. w     

Genetic Modulation of RNA Metabolism in Drosophila. IV. Measurement of Rates of RNA Synthesis by Density Labeling

Accumulation of RNA was measured in adult males of two genotypes: car bb/Ybb- and car bb/YbbSuVar-5. The two genotypes have similar amounts of rDNA, which is reduced in comparison to wild type (CLARK, STRAUSBAUGH and KIEFER 1977). Although genotypically bobbed, car bb/YbbSuVar-5 flies have a wild-type phenotype; car bb/Ybb- flies are both phenotypically and genotypically bobbed (CLARK, STRAUSBAUGH and KIEFER 1977). The wild-type phenotype observed in the car bb/YbbSuVar-5 flies is thought to be the result of an increased rate of rRNA synthesis due to the presence of the YbbSuVar-5 chromosome (SHERMOEN and KIFFER 1975; CLARK, STRAUSBAUGH and KIEFER 1977; CLARK and KIEFER 1977). To further define this phenomenon, the absolute accumulation of RNA was measured in the two genotypes, using density labeling methods. The accumulation of RNA is 1.4 to 1.8 times higher in car bb/YbbSuVar-5 flies than in car bb/Ybb- flies, demonstrating that there is genetic regulation of synthesis in this genotype. The use of density-labeled nucleosides has clearly shown that there is no difference in precusor pool sizes or use between the two genotypes studied.     

Impact of 3-deazapurine nucleobases on RNA properties

Deazapurine nucleosides such as 3-deazaadenosine (c³A) are crucial for atomic mutagenesis studies of functional RNAs. They were the key for our current mechanistic understanding of ribosomal peptide bond formation and of phosphodiester cleavage in recently discovered small ribozymes, such as twister and pistol RNAs. Here, we present a comprehensive study on the impact of c³A and the thus far underinvestigated 3-deazaguanosine (c³G) on RNA properties. We found that these nucleosides can decrease thermodynamic stability of base pairing to a significant extent. The effects are much more pronounced for 3-deazapurine nucleosides compared to their constitutional isomers of 7-deazapurine nucleosides (c⁷G, c⁷A). We furthermore investigated base pair opening dynamics by solution NMR spectroscopy and revealed significantly enhanced imino proton exchange rates. Additionally, we solved the X-ray structure of a c³A-modified RNA and visualized the hydration pattern of the minor groove. Importantly, the characteristic water molecule that is hydrogen-bonded to the purine N3 atom and always observed in a natural double helix is lacking in the 3-deazapurine-modified counterpart. Both, the findings by NMR and X-ray crystallographic methods hence provide a rationale for the reduced pairing strength. Taken together, our comparative study is a first major step towards a comprehensive understanding of this important class of nucleoside modifications.     

Anti-VPg antibody precipitation of product RNA synthesized in vitro by the poliovirus polymerase and host factor is mediated by VPg on the poliovirion RNA template.

Antibody to the poliovirus genome-linked protein, VPg, specifically immunoprecipitated the product RNA synthesized in vitro by the poliovirus RNA polymerase and HeLa cell host factor when VPg-linked poliovirion RNA was used as a template. The largest product RNA that was immunoprecipitated was twice the size of the template RNA. The complete denaturation of the product RNA with CH3HgOH had no effect on the immunoprecipitation reaction. In contrast, CH3HgOH denaturation prevented the immunoprecipitation of the oligo(U)-primed product RNA. Immunoprecipitation of the product RNA synthesized in the host-factor-dependent reaction was prevented if VPg was removed from the template RNA by pretreatment with proteinase K or if an RNA template without VPg was used in the reaction. The results support our previous evidence that a covalent linkage exists between the labeled negative-strand product RNA and the VPg-linked template RNA and suggest that the purified polymerase and host factor initiated RNA synthesis in vitro in the absence of VPg or a VPg-precursor protein.     

Autoradiographic study of RNA synthesis in adrenal cells of the young rat in primary culture

Primary cultures of young rat adrenal cells were used in a radioautographic study of the effect of ACTH on RNA synthesis. Control and ACTH-stimulated cultures were incubated with 5.6 [3H]-uridine for 30 min and chased for 15 and 30 min. Labelling over nucleolar and extranucleolar areas was always lower in the ACTH-stimulated animals at all experimental times. At 30 min chase-time a relatively high labelling of nuclei in both experimental conditions suggest the slow metabolism of RNA in adrenal. The results confirm the inhibitor effect of adrenocorticotrophin on [3H]-uridine incorporation.     

The nature of the changes in the pattern of RNA synthesis by the juvenile hormone analogue altosid

The effect of various concentrations of Altosid and actinomycin D under defined conditions on housefly metamorphosis was investigated with three strains of houseflies. The morphogenetic response varied with the strains and the length of time which the larvae were exposed to the juvenile hormone analogue. De novo RNA synthesis was studied with (2-¹⁴C)-glycine. Methods were developed for the isolation of nuclear, soluble, and ribosomal RNA. The procedure presented provides a DEAE-cellulose chromatographic method for the removal of high molecular weight RNA from DEAE at a neutral pH. Labelling of the RNAs was increased in the presence of the juvenile hormone analogue indicating an increase in the rate of RNA synthesis. The higher incorporation of the labelled precursor into nuclear RNA demonstrates that cytoplasmic RNA is derived from the nuclei.