RNase G (CafA protein) and RNase E are both required for the 5′ maturation of 16S ribosomal RNA

Abstract

In Escherichia coli, rRNA operons are transcribed as 30S precursor molecules that must be extensively processed to generate mature 16S, 23S and 5S rRNA. While it is known that RNase III cleaves the primary transcript to separate the individual rRNAs, there is little information about the secondary processing reactions needed to form their mature 3' and 5' termini. We have now found that inactivation of the endoribonuclease RNase E slows down in vivo maturation of 16S RNA from the 17S RNase III cleavage product. Moreover, in the absence of CafA protein, a homolog of RNase E, formation of 16S RNA also slows down, but in this case a 16.3S intermediate accumulates. When both RNase E and CafA are inactivated, 5' maturation of 16S rRNA is completely blocked. In contrast, 3' maturation is essentially unaffected. The 5' unprocessed precursor that accumulates in the double mutant can be assembled into 30S and 70S ribosomes. Precursors also can be processed in vitro by RNase E and CafA. These data indicate that both RNase E and CafA protein are required for a two step, sequential maturation of the 5' end of 16S rRNA, and that CafA protein is a new ribonuclease. We propose that it be renamed RNase G.

Transfer RNA instability as a stress response inEscherichia coli: Rapid dynamics of the tRNA pool as a function of demand

“…24 It is also a major endoribonuclease in tRNA 49 and rRNA 50 maturation. Furthermore, RNase E is a core member of the degradosome which is a multiprotein complex involved in RNA degradation 48 assembled on RNase E through PNPase, RhlB, and enolase binding to the C-terminal half of RNase E. 51-53 The degradosome is effective in structured RNA decay from duplex unwinding by the helicase RhIB.…”

Identification of the gene encoding the 5S ribosomal RNA maturase in Bacillus subtilis: Mature 5S rRNA is dispensable for ribosome function

“…With the completion of the sequencing project in Bacillus subtilis, it became clear that the genes for several of the key enzymes involved in RNA maturation and decay in Escherichia coli are absent from its Grampositive counterpart+ Neither the RNase E gene, rne, nor that of oligoribonuclease, orn, both essential for E. coli viability (Apirion & Lassar, 1978;Ono & Kuwano, 1979;Ghosh & Deutscher, 1999), is present in B. subtilis+ Also missing are the genes encoding a battery of exonucleases involved in tRNA maturation, namely RNase T, RNase BN, and RNase D (Li & Deutscher, 1994)+ RNase E is a key enzyme of RNA metabolism in E. coli (Ono & Kuwano, 1979)+ It is responsible for the initial rate-limiting cleavage in the decay of many mRNAs (reviewed in Grunberg-Manago, 1999), as well as playing an important role in 16S and 5S ribosomal RNA processing (Ghora & Apirion, 1978;Li et al+, 1999)+ In 1974, Pace and coworkers described a ribosomeassociated activity called RNase M5, responsible for 5S rRNA maturation in B. subtilis (Sogin & Pace, 1974)+ We therefore wondered whether this enzyme could fulfill an RNase E-like role in B. subtilis and undertook to repurify this activity and identify the gene responsible+ The enzyme that Pace described had a molecular weight of about 24 kDa on SDS-polyacrylamide gels (Pace et al+, 1984)+ It required a protein cofactor, identified as ribosomal protein L18 (called BL16 at the time), for pre-5S to 5S maturation (Stahl et al+, 1984)+ The function provided by L18, one of three proteins known to bind 5S rRNA in E. coli, along with L25 and L5 (Gray et al+, 1972;Douthwaite et al+, 1979;Egebjerg et al+, 1989; Fig+ 1), can be replaced by addition of 25% DMSO+ Thus, L18 is thought to ensure the correct conformation of the substrate for processing, rather than playing a direct role in the cleavage reaction itself (Pace et al+, 1984)+ B. subtilis L18 can also be replaced by its E. coli counterpart (Stahl et al+, 1984)+ L5, which binds to the 5S processing stalk, inhibits the cleavage reaction at high concentrations (Stahl et al+, 1984)+ RNase M5 cleaves the 5S precursor in a doublestranded region that extends from the L5 arm (Fig+ 1)+ The specificity determinants of the cleavage reaction were well characterized by Pace+ Although the natural substrate is double-stranded for three further nucleotides beyond the 39 end of the mature 5S rRNA, it was shown that base pairing of the 39-terminal residue was sufficient for cleavage to occur Stahl et al+, 1980)+ Although this residue is naturally a cytosine in B. subtilis, cleavage still occurred when it was replaced by any of the other 3 nt, provided b...…”

Structure of Escherichia coli RNase E catalytic domain and implications for RNA turnover

“…Such coordination may be partly achieved at the level of messenger RNA stability 1 , in which the targeted destruction of subsets of transcripts generates the potential for cross-regulating metabolic pathways. In Escherichia coli, the balance and composition of the transcript population is affected by RNase E, an essential endoribonuclease that not only turns over RNA but also processes certain key RNA precursors [2][3][4][5][6][7][8][9][10] . RNase E cleaves RNA internally, but its catalytic power is determined by the 5 0 terminus of the substrate, even if this lies at a distance from the cutting site [11][12][13][14] .…”

“…coli RNase E is one of the largest members of a highly conserved RNase family 15 . A catalytic core is contained within the aminoterminal half [16][17][18] of RNase E, which in length and sequence closely resembles its paralogue, RNase G 9,19 (Fig. 1a and Supplementary Fig.…”

“…The question naturally arises as to why RNase E does not indiscriminately cleave all RNA with a 5 0 monophosphate, including the structured substrates that it processes, such as ribosomal RNA 9 and RNase P precursors 7 . The answer may lie in the mouse-trap mechanism, which requires that the 5 0 end is engaged in the sensing site and that the portion to be cleaved is simultaneously engaged at the active site: structured RNA segments may not be able to satisfy all of these requirements simultaneously.…”