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Biomedical subjects

D Apirion

Publications and source records attributed to D Apirion.

At least 73 records · Page 4Linked to original sources
PubMed1980

Precursors to 16S and 23S ribosomal RNA from a ribonuclear III-strain of Escherichia coli contain intact RNase III processing sites.

Escherichia coli cells lacking the ribosomal RNA processing enzyme RNase III do no excise the normal RNA precursors p16a (17S) and p23a from nascent rRNA transcripts. These cells produce, instead, slightly larger p16b and p23b precursors. Digestion of p16b or p23b rRNA with RNases A plus T1 yields double-stranded fragments composed of sequences, located at both the 5' and the 3' end regions of the molecules. The terminal duplex, or stem, of p16b contains sequences surrounding the site of RNase III processing which is wild-type cells produces p16a rRNA: the p23b stem likewise contains an intact RNase III cleavage site. The results confirm our earlier prediction for the structure of rRNA transcripts, and also yield a definite secondary structure for the p16 stem, which was not uniquely determined by the corresponding DNA sequence. These experiments demonstrate the absence of significant RNase III processing activity in rnc-105 strains of E. coli, and implicate the participation of another endonuclease(s) in rRNA processing in mutant and wild-type cells.

Base Sequence↗
PubMed1980

Decay of RNA in RNA processing mutants of Escherichia coli.

Stability of RNA was tested in strains of Escherichia coli carrying single, double, or triple mutations in the RNA processing enzymes RNase III, RNase E and RNase P. Tests were carried out for total pulse labeled RNA, beta-galactosidase mRNA and for the decay of preexisting RNA during carbon starvation. Decay of RNA was measured at permissive and nonpermissive temperatures, and in no case were significant differences between mutants and non-mutant strains found. Therefore, we conclude that the three processing enzymes; RNase III, E and P do not contribute significantly to turnover of RNA IN Escherichia coli.

Escherichia coli↗
PubMed1980

Cloning the gene for ribonuclease E, an RNA processing enzyme.

A transducing bacteriophage lambda Ch25rne+, which codes for ribonuclease E of E. coli, has been isolated. To achieve this a random library of Escherichia coli HindIII fragments was cloned in the lambda Charon 25 vector (prepared in F.R. Blattner's laboratory), and lambda Ch25rne+ was selected by its ability upon lysogenization to enable a temperature-sensitive (ts) rne-3071 mutant to grow and to exhibit normal RNA processing at the nonpermissive temperature of 45 degrees C. The level of RNase E was doubled in an rne+ strain lysogenized with lambda Ch25rne+. lambda Ch25rne+ directs the synthesis of a polypeptide of 71 000 m.wt., which is the size of RNase E. Restriction analysis and electron micrography of heteroduplexes suggested that the size of the host DNA insert is about 1.9 kb.

Bacteriophage lambda↗
PubMed1980

Genetic mapping and some characterization of the rnpA49 mutation of Escherichia coli that affects the RNA-processing enzyme ribonuclease P.

A mutant defective in the enzyme RNase P was isolated by P. SCHEDL and P. PRIMAKOFF (1973). The mutation rnpA49 found in this strain, which confers temperature sensitivity on carrier strains, was mapped by conjugation and transduction experiments and located around minute 82 of the E. coli map, with the suggested order rnpA bglB phoS rbsP ilv. As expected, the rnpA49 mutation is recessive. Even though this mutation is conditional, it is manifested at temperatures at which the carrier strains can grow.

Chromosome Mapping↗
PubMed1979

RNase E, an RNA processing enzyme from Escherichia coli.

An activity, RNase E, was purified about 100-fold from Escherichia coli cells, it can process p5 rRNA from a 9 S RNA molecule which accumulates in a mutant of E. coli defective in the maturation of 5 S rRNA. The enzyme requires Na+, K+, or NH4+, and Mg2+ or Mn2+. The molecular weight of the enzyme is about 70,000 and its pH optimum is 7.6 to 8.0. Its temperature optimum is around 30 degrees C, and it can be irreversibly inactivated at 50 degrees C. It has a very high degree of specificity but the reaction can be inhibited by nonspecific RNAs. We interpret its mode of action in producing p5 RNA as being accomplished in two steps, 9 S RNA is first processed to 7 S and 4 S, and subsequently 7 S is further processed to p5.

Ammonia↗
PubMed1979

Characterization of 10S RNA: a new stable rna molecule from Escherichia coli.

When cells of Escherichia coli are labeled with 32Pi for long periods of time and the cell content is subjected to electrophoresis in polyacrylamide gels, an RNA band appears which is about 10S in size. This band seems to contain three conformers. After treatment with formamide only a single band appears in this region of the gel, which contains 550 nucleotides as determined from its mobility. The complexity of the fingerprint of this material, after digestion with T1-RNase, is in agreement with the size as determined by the mobility, this confirming that indeed it is a single molecule. Composition of the T1-oligonucleotides was determined by digesting the T1-generated oligonucleotides with pancreatic RNase and T2-RNase. The quantitative and qualitative analysis of these digestions suggest that 10S RNA contains 609 nucleotides. The molecule contains, besides the four regular bases, one copy per molecule of the modified base pseudouridine. 10S RNA cannot be processed by cell extracts to tRNA-sized molecules and does not bind significantly to ribosomes, hence it is unlikely to be a tRNA precursor or an mRNA.

Base Sequence↗
PubMed1979

Identification of a novel RNA molecule in a new RNA processing mutant of Escherichia coli which contains 5 S rRNA sequences.

A temperature-sensitive mutant of Escherichia coli at the nonpermissive temperature fails to produce normal levels of 5 S rRNA. Instead, a number of larger RNA molecules are accumulated. One of these molecules, a 9 S RNA, contains 5 S rRNA sequences. When the strain is shifted from a nonpermissive to a permissive temperature, radioactive label is lost from the 9 S RNA and appears in 5 S rRNA. The identification of this 5 S rRNA-containing molecule indicates the participation of a new processing ribonuclease (RNase E) in the maturation of rRNA in E. coli. The 9 S RNA was not detected in a wild type strain, indicating that the processing step(s) involved in the formation of 5 S rRNA might be performed before the growing rRNA transcript is terminated.

Base Sequence↗
PubMed1979

Repetitive DNA in Escherichia coli: multiple sequences complementary to small stable RNAs.

Radioactively labeled 4.5S, 6S, and 10S RNAs from Escherichia coli were hybridized to EcoRI fragments from the E. coli genome. Each of these molecules bound to more than one DNA fragment. Cot curve analysis of the kinetics of the annealing of these RNAs to denatured E. coli DNA suggests that the DNA corresponding to each of these molecules is reiterated in the genome. These experiments also suggest that these reiterated sequences are non adjacent.

DNA, Bacterial↗
PubMed1978

Characterization of an endoribonuclease, RNase N, from Escherichia coli.

The properties of the enzyme ribonuclease N were investigated. By comparing the distribution in the cell of RNase N with the bonafide intracellular beta-galactosidase, and the periplasmic alkaline phosphatase enzymes, we showed that RNase N is an intracellular enzyme. Since previous studies suggested that it is an endoribonuclease, it was compared to RNase III, the only other known intracellular endoribonuclease in Escherichia coli. Using homopolymers and co-polymers we found that, while RNase III could digest double-stranded RNA only, RNase N digested single-stranded and double-stranded RNA with similar efficiency. Furthermore, all RNAs used, natural as well as synthetic, were substrates for the enzyme. Using 5 S rRNA as a substrate it was confirmed that the enzyme is an endonuclease. The final products of the reaction of this enzyme are 5'-mononucleotides. The molecular weight of the enzyme is about 120,000 and it seems to contain two subunits which are similar in size. These properties thus differentiate this enzyme from all other known ribonucleases in E. coli.

Alkaline Phosphatase↗
PubMed1978

A conditional lethal mutant of Escherichia coli which affects the processing of ribosomal RNA.

A temperature-sensitive mutant strain was isolated from an RNase III-(rnc) strain of Escherichia coli. At the permissive temperature it behaves like the parental strain, but at the nonpermissive temperature it fails to produce normal levels of 23 S and 5 S rRNA, while instead the 25 S rRNA species becomes very prominent. (The 25 S molecule appears in rnc cells and contains 23 S rRNA sequences). When an rnc+ mutation was introduced to such a strain, or when the rnc mutation was replaced by an rnc+ allele, the strain remained temperature-sensitive. At the permissive temperature such strains synthesized rRNA like any other E. coli strain, but at the nonpermissive temperature they remained unable to synthesize normal levels of 5 S rRNA, and instead a larger molecule was accumulated. The simplest interpretation of theses findings is that the mutant strain contains a temperature-sensitive processing endoribonuclease, RNase E, which normally introduces a cut in the growing rRNA chain somewhere between the 23 S and the 5 S rRNA cistrons. These findings help also to explain the nature and origin of the various rRNA species observed in RNase III- cells and add to our understanding of processing of ribosomal RNA in normal cells of Escherichia coli.

Alleles↗
PubMed1978

Processing of rRNA by RNAase P: spacer tRNAs are linked to 16S rRNA in an RNAase P RNAase III mutant strain of E. coli.

To determine which enzymes are responsible for the processing cleavages of ribosomal RNA transcripts in Escherichia coli, we constructed a mutant strain lacking RNAase III and containing a thermolabile RNAase P. At the nonpermissive temperature, this strain accumulates a novel "19S" RNA species which contains 17S precursor rRNA sequences covalently linked to tRNA sequences transcribed from the ribosomal RNA spacer region between the 16S and the 23S rRNA cistrons. In vitro treatment of 19S RNA with cell extracts releases tRNA2Glu and other tRNA species. These "spacer" tRNA sequences are apparently not contained with the 18S RNA species found in an RNAase III- RNAase P+ cell. RNAase P-deficient extracts are incapable of cleaving space tRNA from 19S RNA, indicating that RNAase P is required for the release of spacer tRNAs from rRNA transcripts of E. coli cells.

Escherichia coli↗
PubMed1978

Structural analysis and in vitro processing to p5 rRNA of a 9S RNA molecule isolated from an rne mutant of E. coli.

A temperature-sensitive mutant of E. coli, rne (RNAase E-), accumulates a number of relatively small RNA molecules at the nonpermissive temperature. One of these molecules, 9S, contains 5S rRNA sequences. The 9S RNA can be processed in vitro to 5S (p5) and a 4S-sized molecule. The 9S and the p5 and 4S RNAs processed from it were all characterized by RNAase T1 fingerprinting and pancreatic RNAase redigestion analysis. Unique oligonucleotides found in the p5 and the 4S RNA are present as unique oligonucleotides in the 9S RNA, suggesting that each 9S molecule contains one 5S and one 4S RNA moiety. Since the 9S RNA contains about 420 nucleotides, and intervening sequences are not known to exist in E. coli, the 5S an 4S RNAs cannot be more than 200 nucleotides apart in the genome. E. coli contains at least three different sequence variants of 5S rRNA, all of which can be identified in the 9S transcript, indicating that 9S RNA is transcribed from most, if not all, of the active rRNA genes, and that RNAase E processes transcripts derived from all these rRNA genes. No modified bases were found in the 4S RNA, nor was its sequence compatible with the known sequences of tRNAs found near the 5S rRNA. We therefore conclude that the 9S RNA doses not contain tRNA, and that the tRNA molecules located near the 5S rRNA are distal to it.

Escherichia coli↗
PubMed1978

Metabolism of ribosomal RNA in mutants of Escherichia coli doubly defective in ribonuclease III and the transcription termination factor rho.

To determine if proteins RNase III and rho, both of which can determine the 3' ends of RNA molecules, can complement each other, double mutants defective in these two factors were constructed. In all cases (four rho mutations tested) the double mutants were viable at lower temperatures, but were unable to grow at higher temperatures at which both of the parental strains grew. Genetic analyses suggested that the combinations of the rnc rho (RNase III-Rho-) mutations was necessary and probably sufficient to confer temperature sensitivity on carrier strains. Physiological studies showed that synthesis and maturation of rRNA, which is greatly affected by RNase III, as well as other RNAs, was indistinguishable in rnc rho strains as compared to rnc rho+ strains, thus suggesting that RNase III and rho do not complement one another in determining the 3' ends of RNA molecules. In rnc rho strains, however, the newly synthesized rRNA failed to accumulate. Thus, decay of rRNA could be the reason for the temperature sensitivity of the double mutant strains. These experiments suggest that RNase III and rho can both protect rRNA from degradation by cellular ribonucleases. They also point to the possibility that the nucleotide sequences involved in the determination of the 3' ends of RNA molecules by these two factors are not identical.

Escherichia coli↗