Publications by authors named "Apirion D"

RNA processing in Escherichia coli and some of its phages is reviewed here, with primary emphasis on rRNA and tRNA processing. Three enzymes, RNase III, RNase E and RNase P are responsible for most of the primary endonucleolytic RNA processing events. The first two are proteins, while RNase P is a ribozyme.

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The cloned rne+ gene complements temperature sensitive RNase E mutations and directs the synthesis of a polypeptide. In vitro the RNA transcribed from the rne gene directs the synthesis of a number of polypeptides, one of which is identical in size to the in vivo product of the rne gene. A rabbit reticulocyte cell free extract programmed with this RNA produced RNase E activity.

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Characterization of the maturation of precursor 10Sa RNA revealed that RNase III processed p10Sa RNA to two intermediate molecules. We showed that the intermediates are not conformers and both are larger than the mature 10Sa RNA. Cell extracts further process the RNase III products to an RNA molecule which has a different conformation than 10Sa RNA but is approximately the same size as 10Sa RNA.

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1. A precursor to small stable RNA, 10Sa RNA, accumulates in large amounts in a temperature sensitive RNase E mutant at non-permissive temperatures, and somewhat in an rnc (RNase III-) mutant, but not in an RNase P- mutant (rnp) or wild type E. coli cells.

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We recently showed that RNase III can process a small stable RNA, precursor 10Sa RNA, that accumulates in an rne (RNase E) strain at non-permissive temperatures. Precursor 10Sa (p10Sa) RNA is processed to 10Sa RNA in two steps, the first step is catalyzed by RNase III in the presence of Mn2+ but not Mg2+. It was shown that RNase III cosediments with membrane preparation from wild type as well as RNase III overexpressing cells.

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The ssrA gene, coding for the metabolically stable 10Sa RNA, affects cell growth. A mutant in which the chromosomal 10Sa RNA gene is interrupted by a cat insert does not produce detectable levels of 10Sa RNA, and it grows more slowly than the parental strain.

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Using T7 RNA polymerase and specific constructs derived from 5S rRNA and RNA I genes, we generated substrates for the RNA processing enzyme RNase E. Using these substrates we have shown that a 3.2 kb DNA fragment that complements the rne-3071 mutation can express RNase E activity.

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Cells overexpressing the RNA-processing enzymes RNase III, RNase E and RNase P were fractionated into membrane and cytoplasm. The RNA-processing enzymes were associated with the membrane fraction. The membrane was further separated to inner and outer membrane and the three RNA-processing enzymes were found in the inner membrane fraction.

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A DNA clone complementing the rne-3071 mutation has been expressed and localized in the physical map of Escherichia coli. The DNA fragment from this clone was localized to the region of the E. coli chromosome where the rne-3071 mutation has been mapped.

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RNase E is a major endonucleolytic RNA processing enzyme in Escherichia coli. We have sequenced a 3.2 kb EcoRI-BamHI fragment encoding the rne gene, and identified its reading frame.

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A precursor to 10Sa RNA accumulates in an rne mutant. However, the present studies indicate that RNase III is the enzyme that processes this RNA. Cell extracts prepared from an rne mutant failed to cleave p10Sa RNA, whereas E coli wild type, rne and rnp cell extracts processed p10Sa RNA under specific assay conditions that require the presence of Mn2+ but not under the customary conditions used for assaying RNase III.

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The gene for 10Sa RNA, which is a major small, stable RNA in Escherichia coli, is a unique gene in the E. coli chromosome. The 10Sa RNA gene (ssrA) has been located between 2,760 and 2,761 kilobases on the E.

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A gene that codes for a small stable RNA (362 nucleotides) has been sequenced. It is a monocistronic gene, with its own promoter and terminator. It produces a precursor that is about 100 nucleotides longer than the mature RNA with all the extra nucleotides at the 3' end.

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Strains carrying plasmids that code for 10Sa RNA synthesize a larger molecule when the RNA processing enzyme RNase E is inactivated. The T1 fingerprint of 10Sa RNA and the larger molecule is very similar, but the latter contains additional oligonucleotides. We show that the larger RNA is converted to the smaller, mature RNA.

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When the RNA processing enzyme RNAase E is inactivated in an Escherichia coli strain carrying derivatives of the colicin E1 plasmid, a small RNA, about 100 nucleotides long, accumulates. Structural analysis of this RNA showed that it is RNA I, the RNA that inhibits plasmid DNA synthesis. RNA I is a specific substrate for RNAase E and the cleavage takes place between the fifth and sixth nucleotides from the 5' end of the molecule.

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The 3' ends of 5-S rRNA isolated from Escherichia coli cells were analyzed and identified after different durations of labeling with 32Pi, with and without blocking of protein synthesis. These experiments suggest that the 5-S rRNA starts as a species containing 126 nucleotides, three at each end, and that the extra nucleotides are removed from the 5' and 3' ends in parallel at comparable but different rates. Inhibition of protein synthesis with chloramphenicol blocks, in addition to the 5'-end maturation, the trimming of the extra nucleotides from the 3' end.

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Infection of RNase III- (rnc) Escherichia coli cells with bacteriophage T4 delta 27, a deletion mutant missing seven out of the ten genes in the tRNA transcription unit, results in the accumulation of a tRNA precursor (10.5-S RNA) that contains the sequences of tRNAGln, tRNALeu and species 1 RNA [Pragai and Apirion (1981) J. Mol.

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A ColE1 plasmid from the Clarke and Carbon collection [Clarke, L. & Carbon, J. (1976) Cell 9, 91-99] that contains a 14.

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A precursor molecule for 10 Sb RNA, the RNA moiety of the RNA processing enzyme RNase P, was purified, characterized for enzymatic activity, and compared to 10 Sb RNA and to RNase P. In these studies the K RNA, a dimeric precursor of tRNAGln-tRNALeu, coded by bacteriophage T4, was used as a substrate. This precursor contains two RNase P cleavage sites, one at each 5' end of the two tRNAs.

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A recombinant plasmid containing the promoters, terminators and only the intact 5 S rRNA gene of rrnB is expressed efficiently in Escherichia coli cells. In strains containing a thermolabile RNAase E (rne) full-length transcripts of the rrnB region from the plasmid and a partially processed intermediate product accumulate at non-permissive temperatures. Upon addition of chloramphenicol two additional plasmid-specific RNA molecules appear.

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The 7S RNA, a precursor of 5S rRNA that contains 5S rRNA and the termination stem and loop, is a substrate for RNase E and is also a substrate for RNase III. The cleavage by RNase III is in the stem, 11 nucleotides downstream from the 3' end of the mature 5S rRNA and 8 nucleotides downstream from the RNase E cleavage site. Near the cleaved nucleotides there are three base pairs that appear in the same relative positions in most known RNase III cleavage sites.

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The recombinant plasmid pJR3 delta contains a tandem pair of promoters from rrnA followed by a hybrid 5 S rRNA gene, derived from the two 5 S rRNA genes of the rrnD transcription unit, and a terminator. Escherichia coli cells transformed with this plasmid produce 2-3-times more 5 S rRNA compared to untransformed cells. The growth of cells containing this plasmid is not affected significantly.

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