Showing papers on "RNA-dependent RNA polymerase published in 1973"

Synthesis of globin ribonucleic acid from duck-reticulocyte chromatin in vitro.

Abstract: The proteins of chromatin serve to restrict the transcription of DNA. The relevance of these findings to the control of gene expression is contingent upon the demonstration that this restriction is specific and mirrors the patterns of RNA synthesis observed in vivo. In this study we demonstrate by RNA-DNA hybridization that the vast majority of the chromatin-directed RNA is synthesized from the unique regions of the reticulocyte genome. Furthermore, by use of the DNA complement of globin mRNA as a probe in annealing reactions, de novo synthesis of globin RNA was detected in RNA transcripts from duck reticulocyte chromatin. No globin sequences were detected in similar preparations of RNA in vitro either from liver chromatin or from DNA freed of protein. These results show that the proteins of chromatin serve to restrict transcription in a very specific manner and provide convincing evidence for the existence of transcriptional control factors in eukaryotes.

Mutants of Escherichia coli Thermosensitive for the Synthesis of Transfer RNA

Abstract: Using a simple three-step procedure, we have isolated thermosensitive mutants of E. coli that are specifically defective in transfer RNA (tRNA) synthesis. Our procedure was designed to identify mutants that are unable to make su3+ tRNATyr or grow at the restrictive temperature, yet under the same conditions they retain the ability to make mRNA and protein. The mutants obtained have been analyzed, and they are defective in different steps in the synthesis of functional tRNA at the restrictive temperature. Some of them may fail to modify certain bases in the tRNA. Two mutants are unable to process the 5′ end of tRNA precursor molecules. Three are unable to cleave precursor molecules at the 3′ end. 11 Mutants can not synthesize any tRNA molecules or tRNA precursors. We speculate that these latter mutants may be defective in RNA polymerase or in an RNA polymerase factor specific for stable RNA synthesis.

Secondary Structure Maps of RNA: Processing of HeLa Ribosomal RNA

Abstract: Ribosomal RNA precursors and mature 28S ribosomal RNA from HeLa cells display a highly reproducible secondary structure of hairpin loops after they are spread and examined in an electron microscope. This structure was used to map the linear arrangement of these molecules. Partial digestion with 3′-exonuclease from ascites cell nuclei established that the 28S RNA region is located at the 5′-end of the 45S precursor. A spacer sequence follows the 28S region. This is followed by the 18S RNA region, and by another spacer sequence at the 3′-end. The 41S RNA contains both 18S and 28S regions. The 32S RNA contains only 28S RNA plus spacer, and the 20S RNA contains only 18S RNA plus spacer. Several minor nucleolar RNA components were also mapped with respect to the 45S RNA. These experiments lead to a more detailed processing pathway for HeLa ribosomal RNA which is in good agreement with earlier work.

Tissue-Specific Transcription of the Globin Gene in Isolated Chromatin

Abstract: RNA was transcribed from chrmation from mouse fetal liver or brain, by use of DNA-dependent RNA polymerase of Escherichia coli. Globin messenger RNA sequences in the transcript were measured with complementary DNA copied from globin messenger RNA with RNA-dependent DNA polymerase. Globin messenger RNA sequences were found in RNA newly transcribed from chromatin from erythropoietic tissue but not in that transcribed from brain chromatin.

L Protein Requirement for In Vitro RNA Synthesis by Vesicular Stomatitis Virus

Abstract: The endogenous transcriptase present in purified vesicular stomatitis (VS) virions was solubilized with a Triton X-100 high-salt solution. The polymerase activity was purified on glycerol gradients and by phosphocellulose column chromatography; the viral proteins present in the active enzyme fractions were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It was demonstrated that L protein, but not NS protein, was required for in vitro RNA synthesis on the VS viral nucleocapsid template. Solubilized L protein rebinds to the ribonucleoprotein template when the transcription complex is reconstituted, and the RNA synthesized in vitro by purified L protein hybridizes to virion RNA. Cyanogen bromide peptide fingerprints indicate that the large L protein is a unique polypeptide chain. It is concluded that the L protein functions as the transcriptase, and the nucleocapsid NS protein is not essential for in vitro RNA synthesis.

Loss of the sigma activity of RNA polymerase of Bacillus subtilis during sporulation.

Abstract: The activity of the sigma subunit of the RNA polymerase of Bacillus subtilis decreases markedly during the first 2 hr of sporulation. Moreover, sigma activity remains deficient throughout the sporulation process and in dormant spores. The time course of changes in RNA polymerase during sporulation indicates that alterations in the core of RNA polymerase occur after the loss of sigma activity. Core RNA polymerase purified after the second and before the ninth hour of sporulation fails to respond to vegetative sigma subunit in vitro and contains variable amounts of a 110,000-dalton polypeptide in place of the β′ subunit. Core RNA polymerase purified from dormant spores has a subunit structure indistinguishable from vegetative core enzyme. RNA polymerase purified by antibody precipitation from an extract of a mixture of sporulating and excess vegetative cells separately labeled with two different radioisotopes contains β′ subunit and no 110,000-dalton polypeptide. However, RNA polymerase purified from sporulating bacteria in the absence of excess vegetative cells progressively loses the β′ subunit at each stage of purification even in the presence of the protease inhibitor, phenylmethyl sulfonyl fluoride. These findings suggest that the alteration of the β′ subunit is due to proteolysis in vitro.

Mammalian RNA polymerases.

Abstract: Publisher Summary This chapter presents the status of RNA polymerases involved in RNA synthesis in mammalian cells, with emphasis on some recent developments in the field of nuclear RNA polymerase. The chapter discusses that the RNA polymerases of eukaryotic nuclei exist in multiple forms. These enzymes are compartmentalized within the nucleus. They have different requirements for cations and salt. The nucleolar enzymes are insensitive to α-amanitin in vitro whereas the nucleoplasmic enzymes are generally inhibited by the toxin, which binds to the enzyme and prevents RNA chain elongation. The nucleolar enzymes are more active with native DNA as the template, whereas the nucleoplasmic enzymes are more efficient with a denatured template; however, addition of a cytoplasmic protein factor can restore the ability of the latter enzyme to transcribe preferentially native DNA. The regulation of the activity of purified nucleolar RNA polymerase by hormones independent of any significant modification in the chromatin template is important, considering the emphasis in the past on template modification as the most prominent means of gene regulation. Only the early effect of hormones on the purified RNA polymerase activity has been examined with purified RNA polymerases. The chapter concludes that finally, the factors that are involved in initiation, termination and release of RNA chains in eukaryotic cells should be explored.

Evidence for 30-40S RNA as Precursor of the 60-70S RNA of Rous Sarcoma Virus

Abstract: Rous sarcoma virus harvested from cells at intervals of 3 min has the same density, sedimentation coefficient, and DNA polymerase as virus harvested at hourly intervals. The RNA of the Prague strain-C consists of a minor class of 60-70S RNA, a major class 30-40S RNA, and a 4-12S class of RNA present at variable concentration. The RNA of the Schmidt-Ruppin strain-A contains more 60-70S than 30-40S RNA. Upon incubation of virus harvested at 3-min intervals at 40° in cell growth medium or Tris-saline, most of the 30-40S RNA is converted to 60-70S RNA. The electrophoretic mobility of the 30-40S RNA of the Rous virus harvested at 3-min intervals is lower than that of the 30-40S subunits of completely dissociated 60-70S RNA; after heating, their mobilities are identical. Heating also releases some small RNAs from 30-40S RNA of virus harvested at 3-min intervals, but five times more 4S RNA is released if the 30-40S RNA is allowed to convert to 60-70S in the virus. The template activity for Rous virus DNA polymerase of the 30-40S RNA of Rous virus harvested at 3-min intervals is about five times lower than that of 60-70S RNA. It is suggested that association of 30-40S RNAs with some RNAs of the 4-12S class may take place simultaneously with their conversion to 60-70S RNA.

Detection of Avian Tumor Virus RNA in Uninfected Chicken Embryo Cells

Abstract: Uninfected chicken embryo cells were analyzed for the presence of viral ribonucleic acid (RNA) by molecular hybridization with the single-stranded deoxyribonucleic acid (DNA) product of the RNA-dependent DNA polymerase contained in avian sarcoma-leukosis virions. Viral RNA was detected in all cells which contained the avian tumor virus group-specific antigen and the virus-related helper factor. The amounts of viral RNA in these cells ranged from approximately 3 to 40 copies of viral-specific sequences per cell. In general, the viral RNA content correlated with the level of helper activity in the cells. Cells infected with Rous-associated virus 2 contained 3,000 to 4,000 copies of viral RNA per cell. RNA from these infected cells hybridized with nearly 100% of the viral 3H-DNA. By contrast, a maximum of less than 50% hybridization was obtained with RNA from the uninfected helper-positive cells, suggesting that not all of the viral RNA sequences were present in these cells. No viral RNA was detected in cells which lacked group-specific antigen and helper activity. Under the conditions used in these studies, less than 0.3 viral genome equivalents of RNA per cell would have been detected.

RNA Synthesis of Vesicular Stomatitis Virus V. INTERACTIONS BETWEEN TRANSCRIPTION AND REPLICATION

Abstract: The temperature-sensitive mutant ts114 of vesicular stomatitis virus is temperature-sensitive in both primary and secondary transcription, but not in the replication of 40S RNA. The synthesis of 40S RNA is specifically inhibited when protein synthesis is shut off. The addition of cycloheximide to cells infected by ts114, rapidly inhibits RNA replication at the permissive and nonpermissive temperatures. However, the addition of cycloheximide at the nonpermissive temperature also results in almost complete recovery of transcription to the level found at the permissive temperature. Other inhibitors of protein synthesis applied at the nonpermissive temperature result in the same recoverability of the apparent temperature-sensitive lesion. Ribonucleic acid products synthesized by ts114 at the nonpermissive temperature in the presence of cycloheximide are identical to virus-specific messenger RNA by the criteria of size and complementarity to virion RNA. When mutant-infected cells are shifted to the nonpermissive temperature, incubated for a period of time, and then treated with cycloheximide, the ratio of transcriptive to replicative activity varies depending on the time at which radioactive precursor is added to cells. This suggests an interdependence between replication and transcription during virus-specific RNA synthesis.

Relationship of replication and transcription of Simian Virus 40 DNA.

Abstract: RNA produced by the Simian Virus 40 (SV40) mutant tsA30 during lytic infection of kidney cells of African green monkeys was examined by RNA-DNA competition-hybridization. This mutant is temperature-sensitive in a function (gene A) that regulates synthesis of viral DNA. No detectable difference between mutant RNA synthesized at the permissive temperature (33°) and wild-type viral RNA was found. During continuous infection with the mutant at the restrictive temperature (41°) only early viral RNA was produced. When mutant DNA and late RNA synthesis were initiated at the permissive temperature, a shift to the restrictive temperature rapidly terminated synthesis of viral DNA but not that of late viral RNA. The data indicate that the function of gene A is required before synthesis of late viral RNA and that after initiation, the production of late RNA continues without further expression of gene A or concomittant viral DNA synthesis.

Transcription of high-molecular-weight RNA from hen-oviduct chromatin by bacterial and endogenous form-B RNA polymerases.

Abstract: 1 With the aid of the ribonuclease inhibitor, heparin, techniques were developed to isolate high-molecular-weight RNA products synthesised by Micrococcus and homologous form B RNA polymerases using hen oviduct chromatin as template. These methods are suitable for analysis of RNA transcripts synthesised in vitro by a variety of enzymes and templates. 2 A highly sensitive assay for ribonuclease, based on the ability of the latter to degrade ovalbumin mRNA, confirmed that chromatin and enzyme samples contained significant amounts of ribonuclease activity. 3 Heparin completely inhibits initiation of both bacterial and homologous enzymes, but has no effect on elongation. The average rates of elongation of RNA polymerases on chromatin DNA could therefore be determined. These values were 3–6 nucleotides per second for bacterial enzyme and 1–2 nucleotides per second for homologous enzyme. 4 By allowing initiation of bacterial enzyme prior to the addition of heparin, RNA chains approaching 2000 nucleotides in length (molecular weight 0.7 X 106) were synthesised after 1 min; at later times, these chains exceeded 5000 nucleotides in length. 5 Hen oviduct chromatin contains endogenous RNA polymerase activity which represents predominantly the elongation of form B enzymes. RNA chains ranging from 100 to 2000 nucleotides long are still attached to these enzymes in isolated chromatin. By allowing elongation of these chains in vitro, products ranging from 2000 to 10000 nucleotides in length (molecular weight 0.7 to 3.0 X 106) were synthesised. 6 Estimates of the number of endogenous RNA polymerases bound to chromatin DNA suggest that approximately 10% of the total form B RNA polymerase molecules per cell are retained in the isolated chromatin fraction.

Ribonucleic Acid Components of Murine Sarcoma and Leukemia Viruses

Abstract: Defective Kirsten murine sarcoma virus was present as leukemia virus pseudotype [Ki-MSV(MLV)] in a 10- to 100-fold excess over its helper, Kirsten murine leukemia virus (Ki-MLV), when the two viruses were propagated in an NRK rat cell line. The s(omega,20) of the fastsedimenting RNA complex of Ki-MLV and of Ki-MSV-(MLV) was 62 S and 55 S, respectively. Gel electrophoresis in buffered aqueous or formamide solution of the dissociated 62S RNA complex of Ki-MLV showed a single major peak of molecular weight about 2.5 x 10(6). Dissociated 55S RNA of Ki-MSV(MLV) was resolved into a major component with a higher electrophoretic mobility than that of Ki-MLV RNA and molecular weight about 2.3 x 10(6). Occasionally, a minor component with the same electrophoretic mobility as Ki-MLV RNA was observed in Ki-MSV(MLV) RNA; it is thought to be the RNA of Ki-MLV present as helper virus in our stocks of Ki-MSV(MLV). The RNA of an endogenous rat C-type RNA virus was electrophoretically different from both Ki-MLV RNA and Ki-MSV(MLV) RNAs. Oligonucleotide fingerprinting of the RNAs digested with RNase T1 indicated that the RNAs of Ki-MSV(MLV) and Ki-MLV are different. However, the extent of the difference between the two RNAs could not be estimated by this method. The heat-dissociated 50-70S RNAs of two other defective murine sarcoma-leukemia viruses; Harvey-MSV(MLV) and Moloney-MSV(MLV) and of defective spleen-focus-forming Friend virus were resolved electrophoretically into two components. The larger components had the same electrophoretic mobility as the RNA of Ki-MLV or Moloney MLV. The smaller were not present in leukemia virus. It is suggested that the small RNA components of the two murine viruses and of Friend virus represent specific genetic information of these replication-defective transforming viruses. Possible relationships between the RNAs of murine leukemia viruses and replication-defective murine sarcoma and Friend viruses are discussed.

Degradation of RNA in Escherichia coli. A hypothesis.

Abstract: 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.

Characterization of the Low-Molecular-Weight RNAs Associated with the 70S RNA of Rous Sarcoma Virus

Abstract: Two low-molecular-weight RNAs are associated with the 70S RNA complex of Rous sarcoma virus: a previously described 4S RNA and a newly identified 5S RNA. The 4S RNA constitutes 3 to 4% of the 70S RNA complex or the equivalent of 12 to 20 molecules per 70S RNA. It exhibits a number of structural properties characteristic of transfer RNA as revealed by two-dimensional electrophoresis of oligonucleotides obtained from a T1 ribonuclease digest of the 4S RNA species. The 5S RNA is approximately 120 nucleotides in length, constitutes 1% of the 70S RNA complex or the equivalent of 3 to 4 molecules per molecules of 70S RNA, and is identical in nucleotide composition and structure to 5S RNA from uninfected chicken embryo fibroblasts. Melting studies indicate that the 5S RNA is released from the 70S RNA complex at the same temperature required to dissociate 70S RNA into its constituent 35S subunits. In contrast, greater than 80% of the 4S RNA is released from 70S RNA prior to its conversion into subunits. The possible biological significance of these 70S-associated RNAs is discussed.

In vitro synthesis of RNA that contains polyadenylate by virion-associated RNA polymerase of vesicular stomatitis virus.

Abstract: The RNA synthesized in vitro by the virion-associated RNA-instructed RNA polymerase of purified vesicular stomatitis virus contains polyadenylate sequences. These have been demonstrated by their partial resistance to pancreatic and T1 ribonucleases and their capacity to bind to poly(U) filters and oligo(dT)-cellulose. The polyadenylate sequences range in apparent size from 50 to 200 bases, similar to the size of the poly(A) in mRNA from vesicular stomatitis virus-infected cells. Possible mechanisms of polyadenylylation of the in vitro RNA product are discussed.