Initiation of tRNA maturation by RNase E is essential for cell viability in E. coli

Ribonuclease E (RNase E) of Escherichia coli was first characterized in a temperature-sensitive mutant (rne-3071) that accumulated 9S precursors of the 5S rRNA at 42°C (Ghora and Apirion 1978). Independently, the ams (altered mRNA stability) gene was identified because of its ability to affect the decay of total pulse-labeled RNA at elevated temperatures (Ono and Kuwano 1979). Subsequently, both loci were shown to encode RNase E (Mudd et al. 1990; Babitzke and Kushner 1991; Taraseviciene et al. 1991). The rne gene encodes a 1061-amino-acid protein (Casaregola et al. 1992, 1994) that has now been characterized as a 5′-end-dependent endoribonuclease (Mackie 1998). In addition, it has also been shown that RNase E is part of a multiprotein complex, called the degradosome, that includes polynucleotide phosphorylase (PNPase), the RhlB RNA helicase, and the glycolytic enzyme enolase (Carpousis et al. 1994; Py et al. 1994, 1996; Miczak 1996). In vivo experiments with either the rne-1 or rne-3071 temperature-sensitive alleles have shown the accumulation of unprocessed 5S rRNA intermediates (Ghora and Apirion 1978; Babitzke et al. 1993) and a general slowing in the decay of specific mRNA transcripts (Arraiano et al. 1988; Mackie 1991; Regnier and Hajnsdorf 1991). As such it was assumed that the inviability associated with the inactivation of RNase E resulted from a defect in either 9S rRNA processing or mRNA decay. However, experiments by Lopez et al. (1999) and Ow et al. (2000) suggest that this hypothesis is not correct. For example, using rne deletion mutations, both laboratories showed that 9S rRNA processing was almost normal under conditions in which mRNA decay was significantly impaired (Lopez et al. 1999; Ow et al. 2000). Furthermore, Ow et al. (2000) characterized an extensive RNase E C terminus truncation mutation (rneΔ610) that was missing 609 amino acids, including the ARRBS (arginine-rich RNA-binding site) and the degradosome scaffolding region (Fig. ​(Fig.1).1). This protein was able to support cell viability at 37°C but not at 44°C. More importantly, decay of specific mRNAs was more defective at 37°C than in an rne-1 mutant shifted to the nonpermissive temperature. Therefore, it was concluded that inviability in the absence of RNase E was not associated with defects in either mRNA decay or 9S rRNA processing (Ow et al. 2000). Figure 1 Schematic representation of the rne+, rneΔ610, and rneΔ645 alleles. The rneΔ610 mutation encodes a truncated RNase E protein encompassing the first 427 amino acids of the N terminus plus the last 25 amino acids of the C ... Accordingly, we have sought to determine what other aspect of RNA metabolism requires the activity of this enzyme. One possibility was a defect in the processing of the M1 RNA subunit of RNase P (Gurevitz et al. 1983; Lundberg and Altman 1995). Because this is the only enzyme in E. coli that can generate the mature 5′ termini of tRNAs, the loss of its activity leads to cell inviability. However, because M1 RNA containing extra nucleotides at its 3′ terminus still retains catalytic activity (Liu and Altman 1995), this did not seem to be a likely explanation. A more attractive candidate was tRNA processing. The E. coli genome contains 86 tRNAs, many of which exist in polycistronic operons (Berlyn 1998). Although these transcripts could be processed by a combination of endonucleolytic cleavage by RNase P at the 5′ end (Altman et al. 1995) and exonucleolytic degradation at the 3′ end by RNase II, RNase BN, RNase PH, RNase D, RNase T, and PNPase (Li and Deutscher 1996), it is also possible that RNase E is required to cleave within the intercistronic regions. Failure to process tRNAs properly would lead to a cessation of protein synthesis and concomitantly cell growth. In fact, Ono and Kuwano (1979) observed a drop-off in the rate of protein synthesis when an rne-1 strain (called ams-1 at that time) was shifted to the nonpermissive temperature. In addition, when Ray and Apirion (1981b) isolated small RNAs from an rne-3071 mutant shifted to 42°C, they found 9S rRNA precursors as well as molecules that contained both tRNALeu and tRNAHis. These two tRNAs are part of a four-gene tRNA transcription unit, argX hisR leuT proM (Berlyn 1998). Subsequently, they showed that if such tRNA precursors were treated in vitro with RNase E, then RNase P could cleave at the 5′ end (Ray and Apirion 1981a). Using a series of RNase E mutants, we have examined the relationships among RNase E function, tRNA processing, and cell viability. Of particular importance was the fortuitous isolation of a temperature-resistant revertant of the rneΔ610 allele (Ow et al. 2000), called rneΔ645. The rneΔ645 mutation encodes an RNase E protein of only 417 amino acids, but unlike rneΔ610, permits cell growth at both 37°C and 44°C. Whereas the processing of both polycistronic and monocistronic tRNAs was impaired at 44°C in rne-1, rneΔ610, and rneΔ645 strains, transcripts matured 2.9- to 3.7-fold faster in the rneΔ645 mutant compared with the rne-1 and rneΔ610 strains. There was a direct correlation among tRNA processing rates, the steady-state levels of mature tRNAs, and cessation of cell growth. In addition, RNase P cleavage at the 5′ end of tRNA transcripts was dependent on prior RNase E processing at the 3′ terminus.