Reinitiation Of RNA Synthesis Research Articles

Nucleotide sequences responsible for generation of internally deleted sendai virus defective interfering genomes

The deletion points of four internally deleted defective interfering (DI) RNA species (7a, 7b, 7c, and 7d) that reside in a single Sendai virus strain were defined by nucleotide sequencing. DI RNA 7a ( M r 1.24 × 10 6) retained the entire NP gene with the complete NP protein-coding sequence, except for the last two U residues of the polyadenylation signal, fused to an 1800-nucleotide sequence comprising 5′-terminal genome and adjacent L gene sequences. DI RNA 7b ( M r 0.70 × 10 6) consisted of 100 3′-terminal nucleotides fused to 1900 5′-terminal bases; the deletion point in the NP gene precedes the NP protein initiation codon. DI RNA 7c ( M r 0.55 × 10 6) retained 420 3′-terminal and 1150 5′-terminal nucleotides. The sequence just downstream of the sequenced deletion site is M gene specific, indicating that 7c arose from at least two deletion events and that it comprises NP, M, and L gene fragments. Transcription of RNA 7c could yield an mRNA encoding a fusion protein with a 14,000 M r (N-terminal NP sequence fused to out of frame M-specific amino acids). DI RNA 7d ( M r 0.92 × 10 6) retained 1027 3′-terminal nucleotides fused to 1600 bases from the 5′ terminus. It has an open reading frame for a 33,000 M r N-terminal NP protein fragment. Nucleotide sequences flanking each deletion and just downstream of the NP gene deletion site suggested that these DI genomes were generated by a copy-choice mechanism, involving polymerase jumping during replication of negative polarity virus genome templates. In this process, the termination and reinitiation of RNA synthesis would involve recognition of sequences that regulate virus genome transcription and replication.

Studies on the control of plasminogen activator production by cultured human embryonic lung cells: requirements for inhibition by corticosteroids.

The ability of actinomycin D to interfere with the dexamethasone-mediated inhibition of plasminogen activator (PA) production by human-embryonic lung (HuEL) cells has been examined. The enzyme produced by HuEL cells in the presence of both dexamethasone and actinomycin D appears to be the product of de novo protein synthesis, as determined by the dependence of PA production on active protein synthesis and the net increase in total PA during the course of an experiment. Inhibition of RNA synthesis must be continuous to maintain PA production in the presence of dexamethasone, since reinitiation of RNA synthesis causes an immediate loss of PA activity in the cells. Cordycepin and alpha-amanitin also prevent dexamethasone-mediated inhibition of PA in HuEL cells, indicating that the RNA whose synthesis must be prevented is of the mRNA class. These experiments imply that PA production in HuEL cells may be under translational as well as transcriptional control.

Non-random nature of 2-acetylaminofluorene-induced alterations of DNA template capacity

Male rats were fed a diet containing 0.03% (w/w) 2-acetylaminofluorene (AAF) and their hepatic DNA was isolated and transcribed with E. coli RNA polymerase. Ingestion of the carcinogen-containing diet for 4 days substantially reduced the template capacity of the isolated DNA. This reduction in template capacity was due to an apparent decreased RNA chain size (up to 50%), with no significant changes in initiation or re-initiation of RNA synthesis. This premature termination of RNA synthesis was accompanied, in some instances, by a reduced rate of RNA chain elongation. When the rats were returned to a basal diet for 7 days following 4 days of AAF ingestion, template capacity and RNA chain size returned to control values. Fractionation of hepatic chromatin on a glycerol gradient revealed that inhibition of DNA template capacity occurs on portions exhibiting characteristics of expressed, as well as those with characteristics of repressed, segments of the genome. In contrast, the DNA isolated from a small, highly condensed chromatin fraction (15% of total chromatin-DNA) showed no significant reduction in total template capacity. Analysis of the fidelity of RNA synthesis on this DNA template was performed by determining the rate of addition of individual nucleotide triphosphates to a growing RNA chain. Large reductions in the rates of adenosine and uridine polymerization were observed while no changes in guanosine or cytidine polymerization were found. This suggests the presence of functionally significant carcinogen-induced modifications of adenine. The inhibition in the rate of adenosine and uridine polymerization was reversed when the animals were placed on a basal diet after AAF ingestion.

In vitro RNA synthesis and expression of vitellogenin gene in isolated chicken liver nuclei.

Optimal conditions for prolonged in vitro synthesis of RNA in isolated chicken liver nuclei have been described. It is shown by incorporation of gamma32P-GTP into RNA, analysis of the product on sucrose density gradient, and digestion with alkaline phosphatase and ribonuclease A that there is reinitiation of RNA synthesis. Polynucleotide kinase activity has been ruled out as explanation for the incorporation of gamma32P-GTP. alpha-Amanitin inhibits RNA synthesis by about 50%. Nuclei prepared from estradiol-treated chicks have twice the RNA synthesis activity as the controls. RNA is synthesized in the presence of Hg-UTP and the mercurated product separated by affinity chromatography on sulfhydryl-Sepharose column under stringent conditions. Vitellogenin mRNA sequences are measured by hybridization with DNA complementary to vitellogenin mRNA. Estradiol treatment leads to a 10-fold increase in vitellogenin mRNA sequences.

New inhibitors of FNA synthesis in vitro isolated from Escherichia coli.

Three inhibitor proteins (I1, I2, and I3) were isolated from Escherichia coli. They depress the transcription of E. coli and T3 phage DNA by DNA‐dependent RNA polymerase from E. coli and show the following characteristics. The inhibitors I1 and I3 consist of subunits; I2, behaves as a single protein in dodecylsulfate‐polyacrylamide gel electrophoresis. Neither the initiation of the transcription of E. coli DNA nor the size of the RNA synthesized is influenced by the inhibitors. In an E. coli DNA‐dependent system reinitiation of RNA synthesis at high salt concentration seems to be blocked by the inhibitors. When inactivated, the inhibitors stimulate RNA synthesis and behave like termination factors giving rise to the production of RNA molecules of discrete size, shorter than those of control experiments. It is suggested by hybridization competition experiments that the inhibitors I1 and I3 preferentially inhibit ribosomal RNA synthesis. The inhibitors also depress the transcription of T3 phage DNA by E. coli RNA polymerase resulting in the synthesis of discrete RNA species smaller than those of control experiments without KCl as well as with 0.16 M KCl.

Pre-4S RNA made in isolated HeLa cell nuclei terminates with U

Isolated HeLa cell nuclei are capable of synthesizing a precursor to 4S RNA. The 3′ terminus of the molecule made in vitro can be determined by four separate incubations with the four iritiated ribonucleoside triphosphates. The pre-4S was found to terminate specifically with uridine and be uniformly labeled. These facts are discussed in relation to specific termination of RNA molecules and possible reinitiation of RNA synthesis in isolated nuclei.

Joint propagation of the and ' subunits of RNA polymerase in Escherichia coli.

The core enzyme of the DNA dependent RNA polymerase from E. coli consists of four subunits, 2α, 1β and 1β′ (ref. 1). The two larger subunits, β and β′, are synthesized in a coordinate manner, possibly on a polycistronic mRNA, in the sequence β before β′ in growing E. coli cells2. The bacteria also have a surplus of these two subunits and, presumably, of the core enzyme at various rates of growth, the number of RNA polymerase molecules being two to five times the number of RNA molecules synthesized by the enzyme3. Assuming a pool of dormant polymerase, the question arises whether the enzyme molecules exchange their subunits while traversing this pool between termination and reinitiation of RNA synthesis as has been described for the protein synthesizing system, the ribosome and its subunits4, or whether the enzyme, once assembled from its subunits, is stable in growing cells. The answer might shed some light on regulation of RNA polymerase activity in vivo.

Properties of the Phage f2 Replicase

Abstract Nucleotide polymerization by partially purified f2 replicase in the presence of phage f2 RNA templates is stimulated by salt and by a brief preliminary incubation of the enzyme at high ionic strength. Salt stimulation of enzyme activity is observed with both plus (viral) and minus (complementary) strand RNA templates, is independent of the composition of the salt, and is maximal at an ionic strength of 0.1. The polynucleotide products of the in vitro reaction are homologous to f2 RNAs by annealing criteria. The in vitro product made in the presence of a mixture of plus and minus strand RNAs consists largely of f2 plus strand RNA. Both plus and minus strand RNAs are synthesized in the presence of plus strand template. Less than 20% of the replicase product sediments as 27 S single-stranded viral RNA in glycerol gradients. Most of the in vitro product is present in partially double-stranded species sedimenting in a broad band at about 15 S. These results suggest that the f2 replicase is capable of limited in vitro chain termination and reinitiation of RNA synthesis.

Reinitiation of RNA synthesis on transcription of chromatin DNA by Escherichia coli RNA polymerase.

The transcription of DNA in chromatin by Escherichia coli RNA polymerase resembles that of isolated DNA in two important respects: the release of nascent RNA and reinitiation of RNA synthesis is dependent on the salt concentration, and RNA synthesis is markedly stimulated by the addition of ribosomes.