Processing of Rna in Bacteria

Apirion, David; Ghora, Basanta K.; Plautz, Greg; Misra, Tapan K.; Geqenheimer, Peter

doi:10.1016/b978-0-12-604450-8.50035-3

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“…Within the past decade the importance of RNA processing has emerged in a wide variety of biological systems (1)(2)(3). The cloning of genes for precursor RNAs and RNA-processing enzymes could greatly assist the study of RNA processing.…”

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confidence: 99%

Molecular cloning of the gene for the RNA-processing enzyme RNase III of Escherichia coli.

Watson¹,

Apirion²

1985

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

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A ColEl plasmid from the Clarke and Carbon collection [Clarke, L. & Carbon, J. (1976) CeU 9, 91-99] that contains a 14.4-kilobase Escherichia coli DNA insert complements the rnc-105 mutation, which destroys the activity of the RNA-processing enzyme RNase III. This insert and smaller restriction endonuclease fragments derived from it were cloned into the plasmid pBR329. A number of these recombinant plasmids complemented the rnc-lOS mutation in a recA genetic background. The smallest cloned fragment that compensated for the rnc-105 mutation was 1.3 kilobase in size. This fragment led to the synthesis of two polypeptides. One of these polypeptides was 25,300 daltons and corresponded in size to the subunit of RNase HI. Fragments cloned in opposite orientations led to synthesis of RNase III, indicating that the cloned fragments contained an endogenous promoter. Extracts of an rnc' E. coli strain containing an rnc' plasmid had at least 10 times more RNase III activity than did an analogous strain containing the pBR329 plasmid.Within the past decade the importance of RNA processing has emerged in a wide variety of biological systems (1-3). The cloning of genes for precursor RNAs and RNA-processing enzymes could greatly assist the study of RNA processing. Although the genes for many RNA precursors have been cloned, thus far cloning has contributed only marginally to the study of the proteins that mediate many of the RNAprocessing reactions that occur in the cell (4-6). Since the endonuclease RNase III introduces specific cleavages in a number of RNA (rRNA, tRNA, and mRNA) precursors in Escherichia coli and bacteriophages that parasitize it (1-3), we set out to clone the gene for this enzyme. The work described here depended on the existence of the rnc-105 mutation (7) and was aided by the previous purification of RNase III (8,9) and the demonstration that this enzyme is a dimer of two homologous polypeptides, 25,000 daltons each (9). MATERIALS AND METHODSBacterial Strains. All strains were E. coli K-12. JC10240 was srlC30::TnlO, recA56 obtained from Alvin J. Clark (10). X1411 (11) was a minicell-producing strain provided by Roy Curtiss III. N2076 was wild type for RNase III and has been described (12). N2100 was an rnc-105, arg41, purI66, str-9 strain prepared similarly to the previously described N2077 (12) with selection for NAD independence (Nad+). N3589 was an rnc-105, rne-3071 double mutant constructed similarly to N3520 (13). N3441 was constructed by a prolonged mating between an rne-3071 mutant, N3431 (14), and N7060 (12), with selection for streptomycin resistance (Strr) and tyrosine independence (Tyr'). N4021 was an recA56, rnc-105 strain constructed as follows. A Pl-mediated transduction (15) was carried out with JC10240 as donor and N2100 as recipient with selection for tetracycline resistance (Tetr). A resulting Rec-recombinant, N4019, was then used as a recipient in a P1-mediated transduction with D10 (16) as a donor and selection for sorbitol independence (Srl+). A resulting recombinant which wa...

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confidence: 99%

Molecular cloning of the gene for the RNA-processing enzyme RNase III of Escherichia coli.

Watson¹,

Apirion²

1985

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

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“…While all seven ribosomal RNA clusters (named A through G) contain one or two tRNAs between the 16-S and 23-S rRNAs (called spacer tRNAs), only three of these clusters, namely rrnC, rrnD and rrnF, contain tRNA(s) after the 5-S rRNA gene (named distal tRNAs [3,5,6]). The production of the individual RNAs is achieved by a series of processing reactions which are performed before the transcript is terminated [4,7]. …”

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Maturatio of 5‐S rRNA: Ribonuclease E Cleavages and Their Dependence on Precursor Sequences

Roy

Singh

Ray

et al. 1983

European Journal of Biochemistry

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(Received September 21, 1982) -EJB 6026 9-S RNA is a processing intermediate that accumulates in an RNase E -strain of Eschcridziu coli. It spans from the RNaseIII cleavage site, after 23-S rRNA, to the 3' end of the transcript and is derived from rRNA genes which do not contain tRNAs distal to 5-S rRNA. Here, we have studied the processing of 9-S RNA with ribonuclease E. RNase E cleaves 9-S RNA in two sites: one of these is three nucleotides upstream from the 5' end of 5-S rRNA, the other downstream from its 3' end. Both cleavages are probably introduced by the same enzyme, since both cleavages are thermolabile when an extract of a temperature-sensitive RNase E mutant was used for processing in vitro.In order to asses the role of 5' and 3' end precursor-specific sequences in the RNase E reaction, we isolated the molecules lacking nucleotides at the 5' or 3' end. Molecules having the 5' end of 9-S RNA but missing nucleotides from the 3' end (called 8-S RNA) were as good a substrate for RNase E as 9-S, RNA itself. However, molecules having the 3' end of 9-S RNA but the 5' end of p5 (called 7-S RNA), were less efficient substrates for RNase E. Finally, the removal of as little a s seven nucleotides from the 5' end of 8-S RNA rendered it almost completely unsuitable as a substrate for RNase E A number of RNAs in prokaryotic as well as eukaryotic cells are synthesized as larger precursors. Precursor-specific RNA sequences are then removed from the termini or from the interior of these RNA molecules by slicing/splicing enzymes. Ribosomal RNA synthesis in E.schrrichiu coli provides a very useful system for studying a number of specific processing reactions. The primary transcripts of ribosomal R NAs include P -16-S-rRNA -tRNA(s) -23-S-rRNA -5-SrRNA-tRNA(s)-T; where P denotes promoter and T terminator [I -41. While all seven ribosomal RNA clusters (named A through G) contain one or two tRNAs between the 16-S and 23-S rRNAs (called spacer tRNAs), only three of these clusters, namely rrnC, rrnD and rrnF, contain tRNA(s) after the 5-S rRNA gene (named distal tRNAs [3,5,6]). The production of the individual RNAs is achieved by a series of processing reactions which are performed before the transcript is terminated [4,7].

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“…These findings support the assumption that the degradation step in mRNA turnover is catalyzed by PNPase rather than by a nuclease. Recently Apirion (16) has shown that PNPase is at least one of the enzymes involved in the process of mRNA turnover.…”

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Pyrophosphate-condensing activity linked to nucleic acid synthesis

1979

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In some preparations of DNA dependent RNA polymerase a new enzymatic activity has been found which catalyzes the condensation of two pyrophosphate molecules, liberated in the process of RNA synthesis, to one molecule of orthophosphate and one molecule of Mg (or Mn) -chelate complex with trimetaphosphate. This activity can also cooperate with DNA-polymerase, on condition that both enzymes originate from the same cells. These results point to two general conclusions. First, energy is conserved in the overall process of nucleic acid synthesis and turnover, so that the process does not require an energy influx from the cell's general resources. Second, the synthesis of nucleic acids is catalyzed by a complex enzyme system which contains at least two separate enzymes, one responsible for nucleic acid polymerization and the other for energy conservation via pyrophosphate condensation.

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Processing of Rna in Bacteria

Cited by 3 publications

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Molecular cloning of the gene for the RNA-processing enzyme RNase III of Escherichia coli.

Molecular cloning of the gene for the RNA-processing enzyme RNase III of Escherichia coli.

Maturatio of 5‐S rRNA: Ribonuclease E Cleavages and Their Dependence on Precursor Sequences

Pyrophosphate-condensing activity linked to nucleic acid synthesis

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