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

Szeberényi, József; Apirion, David; Miller, J. Ross

doi:10.1016/s0022-2836(83)80300-3

Cited by 23 publications

(15 citation statements)

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“…(Spots a and b, A-COH and A-C-NOH, respectively, are usually present in our fingerprints regardless of the RNA analyzed; they are probably generated by contaminating nucleases present in the RNase TI preparations; see also [2,31.) The 5' ends were also analyzed and, in agreement with previous studies [4,8,10, 141, we found the four forms (see Fig. 1 and Fig.…”

Section: ' Pa-u-u-u-a-u-c-a-aohsupporting

confidence: 90%

“…A pair at these positions. Furthermore, a hybrid 5-S rRNA, synthesized from a 5-S rRNA gene that consists of the 5' end region of the first and the 3' end region of the second 5-S rRNA gene of rrnD [4], is trimmed even more slowly (J. Szeberenyi and D. Apirion, unpublished observations). Owing to the recombination event between the two 5-S rRNA genes of r m D , the hybrid 5-S rRNA synthesized from this gene has an imperfect molecular stalk: it contains nucleosides C and A at positions 6 and 120 respectively.…”

Section: Discussionmentioning

confidence: 99%

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

1985

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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. 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.The primary processing of 5-S rRNA (m5) from the transcripts of chromosomal rrn gene clusters as well as from precursors (p5) synthesized from a recombinant plasmid containing a 5-S rRNA gene is performed by RNase E [l -41. The final product of RNase E action is p5 rRNA, which is six nucleotides longer than the mature 5-S rRNA by three nucleotides at each end [I, 3-51, The secondary processing of p5 to m5 rRNA, i.e. the removal of the extra nucleotides from the termini, is accomplished by unidentified nuclease(s) (for reviews see [6, 71). The three different 5'-end forms of p5 rRNA (i.e. one, two or three nucleotides longer than the 5' end of rn5 rRNA) have been identified earlier [8,9], and it was also found that the trimming at the 5' end is inhibited by chloramphenicol [lo]. However, there is almost no information concerning the 3'-end maturation of 5-S rRNA in Escherichia coli.Here we report the identification of the different 3'-end forms of p5 rRNA, the inhibition of 3'-end maturation in the absence of protein synthesis, and the impact of the structure of the molecular stalk of 5-S rRNA on the rate of trimming of the 5' and 3' ends of the precursors of 5-S rRNA. MATERIALS AND METHODS Labeling of cells and isolation of 5-S rRNAStrain N3433 (wild type with respect to the known RNAprocessing enzymes) has been described earlier [l 11. Cells were grown in low-phosphate medium at 30°C and 1-ml portions of the cultures were labeled with 32Pi (1 -5 mCi/ml) at an A560 of 0.4 unit. For chloramphenicol treatment, the drug was added to the culture in a final concentration of 200-300 pg/ml 20 min prior to the addition of 32Pi. After the desired time (see Table 2) cells were harvested and lysed as described earlier 1127. RNAs were fractionated in a 5%/%%

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Section: ' Pa-u-u-u-a-u-c-a-aohsupporting

confidence: 90%

Section: Discussionmentioning

confidence: 99%

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

1985

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“…Escherichia coli cells with thermolabile RNase E (an me mutant) transformed by the recombinant plasmid pJR3d [17] accumulate a plasmid-specific RNA (7-S RNA) at nonpermissive temperatures [15,161. The plasmid contains the promoter region of the ribosomal cluster rrnA, the terminator of rrnD and a hybrid 5-S rRNA gene that arose from a recombination event between the two 5-S rRNA genes in the rrnD gene cluster [15,17,251.…”

Section: Separation Of the 5'-end Forms Of 7-s Rnamentioning

confidence: 99%

“…The plasmid contains the promoter region of the ribosomal cluster rrnA, the terminator of rrnD and a hybrid 5-S rRNA gene that arose from a recombination event between the two 5-S rRNA genes in the rrnD gene cluster [15,17,251. 7-S RNA is produced by the incomplete processing of the primary transcripts of this hybrid ribosomal gene cluster.…”

Section: Separation Of the 5'-end Forms Of 7-s Rnamentioning

confidence: 99%

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Precursor nucleotides at the 5′ end are not required for processing by RNase E at the 3′ end of 5‐S rRNA

Szeberényi

Roy

Apirion

1983

European Journal of Biochemistry

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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 doublsstranded structure is not required for the action of RNase E in separating the 3' end of p5 rRNA from the termination stem.The awareness of the importance of RNA processing for the accumulation and maintenance of specific RNA molecules in the cell is continuously increasing. RNA processing is a feature common to all cells. In prokaryotic cells much has been learned about post-transcriptional endonucleolytic cleavages and trimming reactions in the preparation of RNA molecules for their function [I -61. In the Escherichia coli cell three endoribonuceolytic RNA-processing enzymes were identified, RNase 111, RNase E and RNase P.RNase E is a processing endoribonuclease that plays a role in the processing of 5-S rRNA in E. coli . In a mutant that contains a thermolabile RNase E, the production of 5-S rRNA is blocked at nonpermissive temperatures [7], while the accumulation of different precursors containing 5-S rRNA can be observed [8].One of the major precursors accumulating in me cells after RNase E is inactivated is 9-S RNA [8, 91. This precursor is produced by the RNase 111 cleavage of ribosomal transcripts encoded by rrn clusters lacking trailer tRNA gene(s) [lo, 121. It contains the 23-S -5-S spacer (between the cleavage sites of RNase I11 and RNase E), a complete p5 sequence and the termination stemofthe ribosomal transcripts [lo, 121.9-S RNA can be processed in vitro to p5 rRNA by cellular extracts of me+ (RNase E') cells [9] or by partially purified RNase E preparations [lo, 111. The reaction proceeds in two steps: first the 5' end and then the 3' end of p5 rRNA is produced [lo, 111. Analysis of the secondary structure of 9-S RNA and the p5 processed from it revealed that the two cleavages by RNase E are introduced in a double-stranded stem consisting of the complementary sequences around the 5' and 3' ends of 5-S rRNA [I21 (see also Fig. 5). However, since the cleavage at the 5' end of p5 and the subsequent trimming producing the 5' end of m5 rRNA could disturb the integrity of the stem at the RNase E cleavage site at the 3' end of 5-S RNA, it was not clear whether the intactness of th...

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The 5 S Ribosomal and Other Small RNAs

Dillon

1987

The Gene

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

Cited by 23 publications

References 39 publications

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

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

Precursor nucleotides at the 5′ end are not required for processing by RNase E at the 3′ end of 5‐S rRNA

The 5 S Ribosomal and Other Small RNAs

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