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D Apirion

Publications and source records attributed to D Apirion.

At least 19 recordsLinked to original sources
PubMed1993

RNA processing in prokaryotic cells.

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. These three enzymes have unique functions and in their absence, the cleavage events they catalyze are not performed. On the other hand a relatively large number of exonucleases participate in the trimming of the 3' ends of tRNA precursor molecules and they can substitute for each other. Primary processing is the first event that happens to the nascent RNA molecule, while in secondary RNA processing, the substrate is a product of a primary processing event. Although most RNA processing occurs in RNP particles, it seems that only in secondary RNA processing is the RNP particle required for the reaction. Bacteria and especially bacteriophages contain self-splicing introns which in cases were probably acquired from other species.

Bacterial Proteins↗
PubMed1992

Characterization of the RNA processing enzyme RNase III from wild type and overexpressing Escherichia coli cells in processing natural RNA substrates.

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. 2. Since p10Sa RNA was not processed by purified RNase E and III in customary assay conditions, we purified p10Sa RNA processing activity about 700-fold from wild type E. coli cells. 3. Processing of p10Sa RNA by this enzyme shows an absolute requirement for a divalent cation with a strong preference for Mn2+ over Mg2+. Other divalent cations could not replace Mn2+. 4. Monovalent cations (NH+4, Na+, K+) at a concentration of 20 mM stimulated the processing of p10Sa RNA and a temperature of 37 degrees C and pH range of 6.8-8.2 were found to be optimal. 5. The enzyme retained half of its p10Sa RNA processing activity after 30 min incubation at 50 degrees C. 6. Further characterization of this activity indicated that it is RNase III. 7. To further confirm that the p10Sa RNA processing activity is RNase III, we overexpressed the RNase III gene in an E. coli cells that lacks RNase III activity (rnc mutant) and RNase III was purified using one affinity column, agarose.poly(I).poly(C). 8. This RNase III preparation processed p10Sa RNA in a similar way as observed using the p10Sa RNA processing activity purified from wild type E. coli cells, confirming that the first step of p10Sa RNA processing is carried out by RNase III.

Chromatography, Liquid↗
PubMed1992

10Sa RNA: processing by and inhibition of RNase III.

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. An inhibitor of p10Sa RNA processing by RNase III was identified. It is a protein, with a molecular mass of approximately 17 kDa.

Bacterial Proteins↗
PubMed1991

Sequencing and expression of the rne gene of Escherichia coli.

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. Upstream from the gene, there are appropriate consensus sequences for a putative promoter and a ribosome binding site. We have translated this gene using a T7 RNA polymerase/promoter system. We determined 25 amino acids from the N-terminal of the translated product and they are in full agreement with the DNA sequence. The translated product of the rne gene migrates in SDS containing polyacrylamide gels as a 110,000 Da polypeptide, but the open reading frame found in the sequenced DNA indicates a much smaller protein. The entity that migrates as a 110,000 Da contains RNA, which could account, at least partially, for the migration of the rne gene product in SDS containing polyacrylamide gels.

Amino Acid Sequence↗
PubMed1991

10Sa RNA, a small stable RNA of Escherichia coli, is functional.

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.

Blotting, Northern↗
PubMed1991

The gene specifying RNase E (rne) and a gene affecting mRNA stability (ams) are the same gene.

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. The position of this DNA fragment in the E. coli chromosome, the size of the product directed by this DNA fragment (110,000 Da), the restriction map of this fragment, the fact that the same clone complements the ams mutation, and the observation that the rne-3071 and the ams mutations cause similar patterns of RNA synthesis, show that the rne gene--a gene specifying the processing endonuclease RNase E--and the ams gene--a gene that affects mRNA stability--are identical.

Blotting, Southern↗
PubMed1991

Location of the RNA-processing enzymes RNase III, RNase E and RNase P in the Escherichia coli cell.

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. By assaying for these enzymatic activities we showed that even in a normal wild-type strain of Escherichia coli these enzymes fractionate primarily with the membrane. The RNA part of RNase P is found in the cytosolic fraction of cells overexpressing this RNA, while the overexpressed RNase P protein sediments with the membrane fraction; this suggests that the RNase P protein anchors the RNA catalytic moiety of the enzyme to a larger entity. The implications of these findings for the cellular organization of the RNA-processing enzymes in the cell are discussed.

Bacterial Outer Membrane Proteins↗
PubMed1991

RNA processing enzymes RNase III, E and P in Escherichia coli are not ribosomal enzymes.

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. However, the possibility of membrane preparation contamination with ribosomes could not be ruled out. Here we show that RNase III, E and P are not associated with ribosomes. E. coli cells were opened either by alumina grinding or by sonication and fractionated into cytosolic and pellet fractions. The characterization of membrane preparations was done by assaying NADH oxidase, a bona fide membrane enzyme. Ribosomes prepared by alumina grinding were found to be contaminated with small fragments of membrane which contained RNase III activity. RNase III and NADH oxidase activities were present in the ribosomal preparations which could be solubilized by reagents that dissolve the inner membrane. Isopycnic sucrose gradient centrifugation of the membrane and ribosomal preparations also confirmed that RNase III fractionated with the inner membrane. Similarly RNase P activity was found in the corresponding fractions when isopycnic centrifugation of membrane and ribosome preparations was carried out. RNase E activity was also found to be present mostly in the post-ribosomal supernatant. These findings show that RNase III, E and P are not ribosomal enzymes.

Cell Membrane↗
PubMed1990

Maturation of precursor 10Sa RNA in Escherichia coli is a two-step process: the first reaction is catalyzed by RNase III in presence of Mn2+.

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. That the p10Sa cleaving activity is solely RNase III was confirmed by comparing the increase in p10Sa and poly(A).poly(U) cleaving activities in a strain harboring a plasmid carrying an RNase III gene as compared to a normal E coli strain. It is of interest that these 2 substrates are cleaved by RNase III efficiently, but under 2 different assay conditions. In all strains tested, with normal or elevated levels of RNase III, RNase III fractionates predominantly with the membrane. Further characterization of the maturation of 10Sa RNA revealed that the processing of 10Sa RNA is a 2 step reaction involving 2 separate activities, both sensitive to heat and proteinase K treatment. The first step is catalyzed by RNase III, and results in the formation of a molecule, p10Sa', which is larger than the mature 10Sa RNA. The second activity catalyzes the conversion of p10S' to 10Sa RNA, and this step does not require a divalent cation. The second activity is not any of the known processing endoribonucleases, RNase III, E or P, but could be a new enzyme having no obligate requirement for a divalent cation.

Endoribonucleases↗
PubMed1989

A precursor for a small stable RNA (10Sa RNA) of Escherichia coli.

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. The precursor molecule starts with an adenosine triphosphate and is therefore a primary transcript. RNase E is not the enzyme that processes p10Sa (precursor 10Sa) RNA into 10Sa RNA. The cell extract contains an activity that carries out this conversion. This activity requires the dication Mn2+.

Cloning, Molecular↗
PubMed1989

The gene for a small stable RNA (10Sa RNA) of Escherichia coli.

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. The gene contains an open reading frame that corresponds to a small protein 25 amino acids long.

Base Sequence↗
PubMed1985

Processing enzyme ribonuclease E specifically cleaves RNA I. An inhibitor of primer formation in plasmid DNA synthesis.

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. This is only the second natural RNA substrate that has been found, so far, for the RNA processing enzyme ribonuclease E, the other being a precursor for 5 S ribosomal RNA. It is remarkable that nine nucleotides around the cleavage sites are identical in both substrates: (Formula: see text). Therefore, we suggest that at least part of the interaction between RNAase E and its substrate is controlled by these nine nucleotides.

Base Composition↗
PubMed1985

Maturation of the 3' end of 5-S ribosomal RNA from Escherichia coli.

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. The trimming of extra nucleotides from both ends of the 5-S rRNA is also affected by the structure of the molecular stalk of 5-S rRNA. A number of observations suggest that the trimmings from both ends are independent processes, which are carried out probably by different enzymes.

Autoradiography↗