Maturation of 23S ribosomal RNA requires the exoribonuclease RNase T

Abstract

Ribosomal RNAs are generally synthesized as long, primary transcripts that must be extensively processed to generate the mature, functional species. In Escherichia coli, it is known that the initial 30S precursor is cleaved during its synthesis by the endonuclease RNase III to generate precursors to the 16S, 23S, and 5S rRNAs. However, despite extensive study, the processes by which these intermediate products are converted to their mature forms are poorly understood. In this article, we describe the maturation of 23S rRNA. Based on Northern analysis of RNA isolated from a variety of mutant strains lacking one or multiple ribonucleases, we show that maturation of the 39 terminus requires the action of RNase T, an enzyme previously implicated in the end turnover of tRNA and in the maturation of small, stable RNAs. Although other exoribonucleases can participate in shortening the 39 end of the initial RNase III cleavage product, RNase T is required for removal of the last few residues. In the absence of RNase T, 23S rRNA products with extra 39 residues accumulate and are incorporated into ribosomes, with only small effects on cell growth. Purified RNase T accurately and efficiently converts these immature ribosomes to their mature forms in vitro, whereas free RNA is processed relatively poorly. In vivo, the processing defect at the 39 end has no effect on 59 maturation, indicating that the latter process proceeds independently. We also find that a portion of the 23S rRNA that accumulates in many RNase T -cells becomes polyadenylated because of the action of poly(A) polymerase I. The requirement for RNase T in 23S rRNA maturation is discussed in relation to a model in which only this enzyme, among the eight exoribonucleases present in E. coli, is able to efficiently remove nucleotides close to the double-stranded stem generated by the pairing of the 59 and 39 termini of most stable RNAs.

The pseudouridine synthase RluD is required for normal ribosome assembly and function in Escherichia coli

“…5B) complementary to residues 35-55 of mature 23S rRNA (Charollais et al 2003). Using this probe, we find that wild-type 70S and 50S particles contain mature rRNA and a +1 species, as has previously been reported (Li et al 1999;Charollais et al 2003). In the DrluDłkan mutant, 70S, 62S, and 50S particles also contain predominantly the mature and the +1 species.…”

Quality control of ribosomal RNA mediated by polynucleotide phosphorylase and RNase R

“…Based on these experiments, the apparently full-length 23S and 16S RNA molecules carried normal 5Ј termini (data not shown). To examine the 3Ј termini, chimeric oligonucleotide-directed RNase H cleavage was used (19). These analyses indicated that both the 23S and 16S rRNA species in the double mutant at 42°C have normal 3Ј termini as well (data not shown).…”

“…This technique can be used to examine the end of large RNA molecules at single nucleotide resolution. Analysis was carried out as described (19). The reaction products were resolved on a 6% polyacrylamide-8.3 M urea gel and analyzed by Northern hybridization.…”

Multiple Exoribonucleases Catalyze Maturation of the 3′ Terminus of 16S Ribosomal RNA (rRNA)

“…This includes 16S (this work), 23S (12,17), and 5S (18) rRNAs, tRNAs (9), and other small stable RNAs (19). In all cases, removal of the last precursor residues involves the action of an exoribonuclease.…”

“…MG1655 I Ϫ was used as the RNase PH Ϫ strain into which RNase R, RNase II, and PNPase mutations were introduced as described previously (10). Site-directed RNase H Cleavage-Total RNA was isolated using a hot phenol method (11) and subjected to RNase H assay followed by Northern blot analysis (5,12). The chimera C16S3 (5Ј-CCdCdGdAdAGGUUAAGCUACCU-3Ј) was used for 3Ј end cleavage of 16S rRNA, and cleavage products were visualized by using a probe complementary to residues 1502-1523.…”