Chain Termination of Ribosomal RNA Synthesis in vitro

Pettijohn, David E.; Stonington, Oliver G.; Kossman, Charles R.

doi:10.1038/228235a0

Cited by 59 publications

(25 citation statements)

References 23 publications

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“…Such a situation is shown clearly in Fig.2B and C. A similar interpretation could also explain the results of Pettijohn et al [30] on RNA synthesis in vitro on transcriptional complexes from Escherichia coli. These authors showed that RNA with the mobility of the precursor to 23-S rRNA, but not of the precursor to 16-S rRNA was labelled in v i m .…”

Section: Isupporting

confidence: 71%

The Synthesis of Chloroplast High-Molecular-Weight Ribosomal Ribonucleic Acid in Spinach

Hartley

Head

1979

European Journal of Biochemistry

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have located the positions of the ribosomal genes on the restriction endonuclease map of Zea mays chloroplast DNA. The two rDNA units were shown to be separated from each other by 18 500 base pairs in one direction and by 106100 base pairs in the other direction and to have an inverted orientation with respect to each other. Each unit contains one sequence for the 16-S, 23-S and 5-S rRNAs in the order given, the 16-S and 23-S sequences in each unit being separated by a 2100-base-pairs spacer. A similar arrangement has recently been reported [6] for the ribosomal genes in spinach chloroplast DNA.Although the precise pathway of rRNA synthesis in chloroplasts is not known, studies on the kinetics of labelling in vivo of chloroplast rRNAs in both algae and higher plants suggest that the mature rRNAs are synthesised via precursors of 10 -20 % higher molecular -weight [7-101. In spinach, for example, Ahhreviurions. Hepes, 4-(2-hydroxyethyl-l-piperazineethanesulphonic acid ; Mes, 2(N-morpholino)ethanesulphonic acid; tricine, N-[tris(hydroxymethyl)]methylglycine; NaCl/Cit, 0.15 M NaCI, 0.015 M trisodium citrate.

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Section: Isupporting

confidence: 71%

The Synthesis of Chloroplast High-Molecular-Weight Ribosomal Ribonucleic Acid in Spinach

Hartley

Head

1979

European Journal of Biochemistry

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“…1 (12,13); and less than 1% of the DNA polymerase activity (14) of the lysate (unpublished observations). Speed is essential in purifying the complex, since it is unstable in the crude lysate and probably also unstable in nongrowing cells.…”

Section: Methodsmentioning

confidence: 99%

“…USA The RNA of the complex consists primarily of nascent messenger and ribosomal RNA chains, the latter composing nearly half of this total population. The RNA analysis has been reported earlier (12,13) (27) that DNA supercoiling, attributable to overwinding of the double helix, is responsible for its compact structure in the cell.…”

Section: Dna Packagingmentioning

confidence: 99%

The Folded Genome of Escherichia coli Isolated in a Protein-DNA-RNA Complex

Stonington

Pettijohn

1971

Proc. Natl. Acad. Sci. U.S.A.

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The DNA of Escherichia coli has been isolated in a compact structure containing small amounts of protein and RNA and having a sedimentation coefficient of approximately 3200 S. The molecular weight of the DNA in the complex is very large (probably higher than 109); the protein is predominantly core RNA polymerase; the RNA is chiefly nascent messenger and ribosomal chains. Solutions containing the complex have low vicosities; this plus its sedimentation rate suggest that the DNA is in a tightly folded conformation. The DNA unfolds after exposure to RNase or heat; this indicates that an RNA component of the complex is involved in stabilizing the structure..The DNA in bacterial cells as seen under the electron microscope is confined in regions of the cell called "nuclear bodies," but no nuclear membrane is observed (see, for example, refs. 1 and 2). Yet these chromosome-like structures have defined boundaries, which reorganize during the nuclear segregation that accompanies cell division (3). This implies that DNA folding within the nuclear body is maintained-at least partly--by internal interactions between the elements or regions of the nuclear body. The tertiary conformation of DNA within this body is unknown; however, studies of the rates at which different operons can be transcribed and regulated suggest that a considerable portion (perhaps all) of the genome is available for free interaction with other macromolecules (for examples see refs. 4-7). The packaging of the DNA must also be compatible with the requirements of semiconservative DNA replication and the eventual segregation of daughter double-helices (8, 9). These considerations imply that the-, tertiary structure of DNA within the cell is organized; however, this structure and the interactions that stabilize it are still to be determined.We describe here the isolation from E. coli of a DNA complex in which the DNA genome remains folded into a structure which is compact and nearly homogeneous in sedimentation. The complex is approximately 80% DNA by weight; it includes a small amount of protein and also contains nascent RNA. At least some of the RNA of the complex is essential for stabilizing the packaged structure, since the DNA unfolds as a result of RNase treatment. MATERIALS AND METHODSThe E. coli strain D-10 (RNase I-, ref. 10) was used in these studies. The bacteria were grown into the log phase in M9 media supplemented with 0.2% casamino acids.Viscosities of solutions of the DNA complex were measured at 250C in a falling-ball viscometer.Proteins were obtained from a DNA complex that had been purified by a scaled-up modification of the procedure described in the legend to Fig. 1 When E. coli cells are gently opened in solutions of high ionic strength, DNA is released from the cells but the solution does not become viscous. A DNA complex, having a nearly homogeneous sedimentation profile, can be isolated from the bulk of the ultraviolet-absorbing material of the lysate ( Fig. 1; see also ref. 12). This complex contains: nearly all of the DN...

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“…A precursor of both rRNA species might be isolatable under conditions where cleavage or termination cannot occur. Pettijohn et al (23) have found an RNA species of the appropriate size which was generated during in vitro transcription of the rRNA genes of E. coli. A large stable RNA species is also formed in vivo by cultures of B. cereus grown in the presence of 8-azaguanine (24,25) (which this organism incorporates into RNA).…”

Section: Discussionmentioning

confidence: 99%

Transcriptional Organization of the Ribosomal RNA Cistrons in Escherichia Coli

Doolittle

Pace

1971

Proc. Natl. Acad. Sci. U.S.A.

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The data presented support the hypothesis that 16S, 23S, and 5S ribosomal RNAs of Escherichia coli are transcribed in vivo from transcriptional units consisting of single cistrons for these species arranged in the order 16S-23S-5S, with transcription beginning at the 16S end.In eukaryotic cells, linked 18S, 28S, and 7S ribosomal RNA cistrons are transcribed as a unit into a single polynucleotide "precursor" (45S rRNA), which is ultimately cleaved to produce one molecule of each of the three rRNA species (1, 2). The three linked cistrons together comprise a single "transcriptional unit", which we define here as a segment of DNA bounded by a single site for RNA polymerase attachment (promoter) and a single site for polymerase release (terminator). Many such rRNA transcriptional units are confined to a restricted region of the eukaryotic chromosome; adjacent units may be separated by a segment of (nonribosomal) "spacer" DNA (1).The genetic organization of the cistrons for bacterial rRNA species (16S, 23S, and 5S rRNA) is similar to that of eukaryotes in that: (a) the number of gene copies (per genome) for each RNA species is the same (3, 4), (b) these cistrons are confined to one (5) or possibly two (6, 7) small regions of the genome, (c) 16S, 23S, and 5S rRNA cistrons are tightly linked (8, 9), and (d) adjacent 16S, 23S, and 5S gene clusters are separated from each other by segments of spacer DNA (10).Data presented here support the hypothesis that the linked 16S, 23S, and 5S rRNA cistrons in Escherichia coli comprise single transcriptional units arranged in the order 16S-23S-5S, with transcription initiating at the 16S end. The fact that no "precursor" comparable to eukaryotic 45S rRNA has yet been found in bacteria may imply that cleavage into 16S and 23S (and possibly 5S) moieties occurs before transcription of the unit is complete. MATERIALS AND METHODSFor the preparation of intact 3H-labeled RNA from rifampicininhibited cells, a culture of the rifampicin-sensitive, E. coli B strain AS19 (11) Corp.), was chased by the addition of excess unlabeled orthophosphate, and after 30 min of further growth at 200C, was chilled to 00C in ice. Rifampicin (Pittman-Moore) was added to 200 Ag/ml at 2 min and [5-3H]uridine (New England Nuclear Corp.) was added to 0.04 mCi/ml (10 ;&g/ml) at 4 min after chilling. At 6 min the culture was warmed to 20'C and incubation was continued for an additional 40 min. Cultures were harvested by centrifugation and RNA was extracted and resolved on 2.8% polyacrylamide gels (13,14).For the preparation of RNA for oligonucleotide mapping,

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Chain Termination of Ribosomal RNA Synthesis in vitro

Cited by 59 publications

References 23 publications

The Synthesis of Chloroplast High-Molecular-Weight Ribosomal Ribonucleic Acid in Spinach

The Synthesis of Chloroplast High-Molecular-Weight Ribosomal Ribonucleic Acid in Spinach

The Folded Genome of Escherichia coli Isolated in a Protein-DNA-RNA Complex

Transcriptional Organization of the Ribosomal RNA Cistrons in Escherichia Coli

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