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D Apirion

Publications and source records attributed to D Apirion.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1985

The ribonuclease-III-processing site near the 5' end of an RNA precursor of bacteriophage T4 and its effect on termination.

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. Biol. 153, 619-630]. In vitro studies, using partially purified RNase III or cell extracts and 10.5-S RNA as substrate, have revealed a cleavage site at the 5' side of the molecule. A computerized secondary structure suggests that the RNase III cleavage site can be placed in a small bulge which could be part of a duplex structure and is adjacent to A-A-G and its complementary sequence U-U-U in the same relative relationships found for most RNase III cleavage sites were the adjacent sequences are (A-A-G/U-U-C). Under normal processing conditions (presence of RNase III) the 3' end of the processed intermediate precursors, 10.1-S and p2Sp1 RNAs, is C-U-U-(U1-2)-UOH, which is determined by a stem and loop structure that could serve as a rho-independent termination signal site. However, in the absence of RNase III, the accumulated 10.5-S precursor RNA does not terminate at the same site and its 3' end is shifted a few nucleotides downstream. Thus, RNase III, besides playing a role in processing of 10.5-S RNA, also affects the termination of that molecule, even though both sites, the RNase III cleavage site and the termination site, are about 390 nucleotides apart.

Binding Sites↗
PubMed1985

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

A ColE1 plasmid from the Clarke and Carbon collection [Clarke, L. & Carbon, J. (1976) Cell 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-105 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 III. 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.

Cloning, Molecular↗
PubMed1984

A site in a tRNA precursor that can be processed by the whole RNase P enzyme but not by the RNA alone.

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. The precursor 10 Sb and 10 Sb RNAs have the capacity to cleave the precursor tRNA molecule but only at the 5' end of tRNALeu, not at the 5' end of tRNAGln. Even when a substrate was prepared that contained only one site for RNase P (the one next to tRNAGln), this substrate was not cleaved by the RNA alone while the whole enzyme was effective in processing this substrate. The possible function of the protein of RNase P in the enzymatic reaction is discussed.

Base Sequence↗
PubMed1984

Synthesis and processing of 5 S rRNA from an rrnB minigene in a plasmid.

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. They are shorter than the full-length transcripts. These species contain the 3'-end region of the full-length transcripts. Even though the 5' ends of these RNAs were most likely produced by degradative enzymes these 5' ends are not ragged. All these plasmid-specific RNAs are specific substrates for the two endonucleolytic RNA processing enzymes, RNAase E and RNAase III.

Base Sequence↗
PubMed1984

7S RNA, containing 5S ribosomal RNA and the termination stem, is a specific substrate for the two RNA processing enzymes RNase III and RNase E.

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. The large product of the RNase III cleavage reaction, which is a 5S rRNA that contains 11 extra nucleotides at the 3' end, is a substrate for RNase E. This suggests that the information for the 3'-end cleavage by RNase E resides mainly in the 5S rRNA itself. Using rnc rne strains, carrying the plasmid that leads to the accumulation of 7S RNA, we showed that the 7S RNA does not result from an RNase III cleavage but is apparently a proper transcription termination product.

Base Sequence↗
PubMed1984

Hybrid 5 S ribosomal RNA encoded by a multicopy plasmid is incorporated into ribosomes of Escherichia coli.

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. Although the sequence and the secondary structure of the plasmid-specific 5 S rRNA differ from those of its counterparts (e.g., from 5 S rRNA species encoded by chromosomal genes), it is processed properly and is incorporated into ribosomes.

Base Sequence↗
PubMed1984

Self cleavage of a precursor RNA from bacteriophage T4.

We found that a precursor of an RNA molecule from T4-infected Escherichia coli cells (p2Spl; precursor of species 1) has the capacity to cleave itself in a specific position. This cleavage is similar to a cleavage carried out by the aid of a protein, RNase F, that has been previously identified. This cleavage could lead to the maturation of an RNA (species 1) found in T4-infected E. coli cells. The reaction is time and temperature-dependent and is relatively slow as compared to the protein-dependent reaction. It requires at least a monovalent cation and is aided by non-ionic detergents. In the absence of detergent the cleavage can occur but at a reduced rate. The substrate does not contain hidden nicks and a variety of experiments suggest that it does not contain a protein. Moreover, we found no indication that the cleavage is due to contaminating nucleases in the substrate or in the reagents. The intact secondary and tertiary structures of the molecule are necessary for the cleavage to occur. The finding of a self cleaving RNA molecule has interesting evolutionary implications.

Base Sequence↗
PubMed1983

Precursor nucleotides at the 5' end are not required for processing by RNase E at the 3' end of 5-S rRNA.

7-S RNA, a single-site substrate for the processing enzyme RNase E of Escherichia coli, consists of p5 rRNA (the precursors of 5-S RNA) and the 3'-end region of the rRNA transcript which is mainly a termination stem and loop. The 7-S RNA studied here was derived mainly from the rRNA gene cluster rrnD, carried in a multicopy plasmid. It contains four different populations of molecules that differ from each other at their 5' ends only: the shortest species has the 5' end of the mature 5-S rRNA, while the others are one, two and three nucleotides longer. The four different 5'-end forms were separated in a long sequencing gel. Processing of these forms and unfractionated 7-S RNA by RNase E in vitro, showed that all forms, even the shortest one, can be processed to p5 rRNA. Since the extra nucleotides at the 5' end of the molecule could be base-paired with the region of 7-S RNA where RNase E cuts, it is concluded that this double-stranded structure is not required for the action of RNase E in separating the 3' end of p5 rRNA from the termination stem.

Base Composition↗
PubMed1983

Purification and properties of ribonuclease E, an RNA-processing enzyme from Escherichia coli.

The Escherichia coli RNA-processing enzyme RNAase E was purified through a number of steps including isoelectrofocusing. The final fraction contained mainly a single polypeptide of 66 kDa. However, while all the steps in the purification yielded the same qualitative activity, the specific activity of fractions was decreased in the last steps of the purification. By combining the most-purified enzyme with earlier fractions from the purification, we could show that the cells could contain a factor that enhances RNAase E activity. The purified enzyme showed the same characteristics with respect to temperature optimum, pH and ionic requirements as less-purified preparations. Testing specific inhibitors we concluded that the enzyme requires SH groups, free amino groups, and either of the amino acids tryptophan, tyrosine, histidine or methionine for its activity.

Base Sequence↗
PubMed1983

Interplay among processing and degradative enzymes and a precursor ribonucleic acid in the selective maturation and maintenance of ribonucleic acid molecules.

In order to understand why the first tRNA (tRNAGln) in the T4 tRNA gene cluster is not produced when T4 infects an RNase III- mutant of Escherichia coli, RNA metabolism was analyzed in RNase III- RNase P- (rnc, rnp) cells infected with bacteriophage T4. After such an infection a new dimeric precursor RNA molecule of tRNAGln and tRNALeu has been identified and analyzed. This molecule is structurally very similar to K band RNA that accumulates in rnc+ rnp strains. It is four nucleotides shorter than K RNA at the 5' end. This molecule like K RNA contains two RNase P processing sites at the 5' ends of each tRNA. Both sites are accessible to RNase P. However, while in the K RNA the site at the 5' end of tRNALeu (the site in the middle of the substrate) is more efficiently cleaved than the other site, this differential is even increased in the Ks (K like) molecule. This difference is sufficiently large that in vivo in the RNase III- strain the smaller precursor of tRNAGln is degraded rather than being matured to tRNAGln by RNase P. This information contributes to the elucidation of the key role of RNase III in the processing of T4 tRNA. It shows the dependence of RNase P activity at the 5' end of tRNAGln on a correct and specific cleavage by RNase III at a position six nucleotides proximal to the RNase P site, and it explains why in the absence of RNase III the first tRNA in the T4 tRNA cluster, tRNAGln, does not accumulate.

Base Sequence↗
PubMed1983

Initiation, processing and termination of ribosomal RNA from a hybrid 5 S ribosomal RNA gene in a plasmid.

Transformation of an RNA-processing mutant (rne, RNase E-) of Escherichia coli with a recombinant plasmid containing the promoter region of the ribosomal cluster rrnA and portions from the 3' region of the rrnD cluster results in the accumulation of the precursors to 5 S ribosomal RNAs at the permissive as well as that of two full-length transcripts and a processing intermediate at the nonpermissive temperature. The two full-length transcripts start from the two rrnA promoters, which are about 120 nucleotides apart. This plasmid, pJR3 delta, contains an intact 5 S rRNA gene and portions from the 16 S and 23 S rRNA genes. Analysis of the major plasmid-specific RNA species revealed that RNA molecules initiated in vivo from the first promoter (P1) start with pppA, while transcripts from the second promoter (P2) contain either pppG or pppC at their 5' ends. Termination occurs mainly at the first available termination site. Full-length transcripts initiated from both promoters are processed to precursors of 5 S rRNAs in vivo at the permissive temperature, but only about 20% of these transcripts are processed to mature 5 S rRNA. RNA1 and RNA2 (the transcripts initiated from P1 and P2, respectively) and RNA3 (an RNA-processing intermediate containing the entire 5 S region and the 3' end of the transcripts) can be cleaved in vitro by cell extracts of wild type strains resulting in precursor and mature 5 S rRNAs in a reaction that is RNase E dependent but not ribosome dependent. The 5' end of the processed 5 S rRNA can correspond to the 5' end of mature 5 S rRNA or it can contain one to three additional nucleotides.

Base Composition↗
PubMed1983

Processing of bacteriophage T4 tRNAs: a precursor of species 1 RNA.

A precursor molecule of species 1 RNA, p2Sp1, that accumulates when an rne (RNase E-) mutant is infected with a T4 deletion mutant (delta 27) is also found after infection of an rne host mutant by different deletion mutants or wild type bacteriophage T4. Low levels of this molecule were also found in a wild-type host infected with a wild-type T4. This precursor molecule accumulates at higher concentrations at 43 degrees C as compared to 30 degrees C or 37 degrees C. Structural analysis of the precursor molecules from the different sources has shown a complete identity of p2Sp1 RNA isolated from the different sources. Therefore, we suggest that this precursor is a normal intermediate in processing of T4 tRNAs, and that it is unrelated to a particular T4 deletion strain. Since RNase E does not process this precursor, its accumulation in an rne mutant reflects an interaction between RNase E and the enzyme that processes this intermediate.

Endoribonucleases↗
PubMed1983

7 S RNA: a single site substrate for the RNA processing enzyme ribonuclease E of Escherichia coli.

7 S RNA accumulates at non-permissive temperatures in an RNAase E strain containing the recombinant plasmid pJR3 delta which carries a single 5 S rRNA gene and expression sequences. 7 S RNA is a processing intermediate that contains the complete sequence of 5 S rRNA as well as a stem-and-loop structure encoded by the terminator of rrnD. 7 S RNA can be processed in vitro by RNAase E. Structural analysis of the products (5 S rRNA and the stem) of in vitro processing of 7 S RNA revealed that the cleavage site of RNAase E in 7 S RNA is 3 nucleotides downstream from the 3' end of the mature 5 S rRNA. The cleavage generates 3'-hydroxyl and 5'-phosphate termini.

Base Sequence↗
PubMed1983

RNA processing: new mutants that affect endonucleolytic processing of RNA.

A strain of Escherichia coli carrying the rne-3071 mutation that affects the RNA processing enzyme ribonuclease E, was mutagenized, and double mutants deficient in RNA processing were isolated. The isolation was based on the appearance of a particular RNA precursor molecule upon infection of an rne mutant with a specific bacteriophage T4 deletion strain. From one of the double mutants the rne mutation was removed, and the new single mutant, designated rng, was examined. In this mutant the maturation of host RNA as well as of bacteriophage T4 RNA is affected. The effect of the rng mutation on RNA synthesis is unique and can be distinguished from the effects of the other established mutations in RNA processing. The effects of the rng mutation can be recognized in vivo and in vitro.

Coliphages↗
PubMed1983

Maturation of 5-S rRNA: ribonuclease E cleavages and their dependence on precursor sequences.

9-S RNA is a processing intermediate that accumulates in an RNase E- strain of Escherichia coli. It spans from the RNase III 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 as seven nucleotides from the 5' end of 8-S RNA rendered it almost completely unsuitable as a substrate for RNase E.

Base Sequence↗
PubMed1983

Identification of a precursor molecular for the RNA moiety of the processing enzyme RNase P.

A precursor molecule for 10Sb (M1) RNA, the RNA moiety of the RNA processing enzyme ribonuclease P (EC 3.1.26.5), is accumulated transiently in an Escherichia coli strain containing a plasmid that carries the 10Sb RNA gene. The same RNA precursor molecule is accumulated, in relatively large quantities, in a temperature-sensitive RNase E- mutant at the nonpermissive temperature. The RNA precursor includes 10Sb RNA and an extra 3' fragment that contains a termination stem and loop. It can be processed in vitro to a molecule the size of 10Sb RNA. None of the four endoribonucleases of E. coli--RNase III, RNase E, RNase F, or RNase P--takes part in this cleavage reaction. Therefore, we suggest that the processing of the precursor-10Sb RNA to 10Sb RNA is carried out by a thus-far unidentified endoribonuclease. The accumulation of a RNA molecule in a RNase E- mutant that does not contain a cleavage site for RNase E has been encountered previously and can be explained by assuming the existence of a RNA processing complex in the E. coli cell.

Base Sequence↗