Exoribonucleases play an important role in all aspects of RNA metabolism. Biochemical and genetic analyses in recent years have identified many new RNases and it is now clear that a single cell can contain multiple enzymes of this class. Here, we analyze the structure and phylogenetic distribution of the known exoribonucleases. Based on extensive sequence analysis and on their catalytic properties, all of the exoribonucleases and their homologs have been grouped into six superfamilies and various subfamilies. We identify common motifs that can be used to characterize newly-discovered exoribonucleases, and based on these motifs we correct some previously misassigned proteins. This analysis may serve as a useful first step for developing a nomenclature for this group of enzymes.

Degradation of RNA plays a central role in RNA metabolism. In recent years, our knowledge of the mechanisms of RNA degradation has increased considerably with discovery of the participating RNases and analysis of mutants affected in the various degradative pathways. Among these processes, mRNA decay and stable RNA degradation generally have been considered distinct, and also separate from RNA maturation. In this review, each of these processes is described, as it is currently understood in bacteria. The picture that emerges is that decay of mRNA and degradation of stable RNA share many common features, and that their initial steps also overlap with those of RNA maturation. Thus, bacterial cells do not contain dedicated machinery for degradation of different classes of RNA or for different processes. Rather, only the specificity of the RNase and the accessibility of the substrate determine whether or not a particular RNA will be acted upon.

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.

mRNA decay is a major determinant of gene expression. In Escherichia coli, message degradation initiates with an endoribonucleolytic cleavage followed by exoribonuclease digestion to generate 5'-mononucleotides. Although the 3' to 5' processive exoribonucleases, PNPase and RNase II, have long been considered to be mediators of this digestion, we show here that another enzyme, RNase R, also participates in the process. RNase R is particularly important for removing mRNA fragments with extensive secondary structure, such as those derived from the many mRNAs that contain REP elements. In the absence of RNase R and PNPase, REP-containing fragments accumulate to high levels. RNase R is unusual among exoribonucleases in that, by itself, it can digest through extensive secondary structure provided that a single-stranded binding region, such as a poly(A) tail, is present. These data demonstrate that RNase R, which is widespread in prokaryotes and eukaryotes, is an important participant in mRNA decay.

Escherichia coli RNase R, a 3 3 5 exoribonuclease homologous to RNase II, was overexpressed and purified to near homogeneity in its native untagged form by a rapid procedure. The purified enzyme was free of nucleic acid. It migrated upon gel filtration chromatography as a monomer with an apparent molecular mass of ϳ95 kDa, in close agreement with its expected size based on the sequence of the rnr gene. RNase R was most active at pH 7.5-9.5 in the presence of 0.1-0.5 mM Mg 2؉and 50 -500 mM KCl. The enzyme shares many catalytic properties with RNase II. Both enzymes are nonspecific processive ribonucleases that release 5-nucleotide monophosphates and leave a short undigested oligonucleotide core. However, whereas RNase R shortens RNA processively to di-and trinucleotides, RNase II becomes more distributive when the length of the substrate reaches ϳ10 nucleotides, and it leaves an undigested core of 3-5 nucleotides. Both enzymes work on substrates with a 3-phosphate group. RNase R and RNase II are most active on synthetic homopolymers such as poly(A), but their substrate specificities differ. RNase II is more active on poly(A), whereas RNase R is much more active on rRNAs. Neither RNase R nor RNase II can degrade a complete RNA-RNA or DNA-RNA hybrid or one with a 4-nucleotide 3-RNA overhang. RNase R differs from RNase II in that it cannot digest DNA oligomers and is not inhibited by such molecules, suggesting that it does not bind DNA. Although the in vivo function of RNase R is not known, its ability to digest certain natural RNAs may explain why it is maintained in E. coli together with RNase II.Escherichia coli contains eight distinct exoribonucleases that play important roles in every aspect of RNA metabolism (1, 2). One of these enzymes, now termed RNase R, was originally identified and partially purified and characterized as a nonspecific residual exoribonuclease present in strains lacking RNase II (3, 4). It was subsequently rediscovered in our laboratory as a nuclease active against rRNA and given the name RNase R (5, 6). The enzyme accounts for ϳ2% of poly(A)-degrading activity remaining in crude extracts of cells lacking RNase I and RNase II (7). Partially purified RNase R is a 3Ј 35Ј exoribonuclease that releases 5Ј-nucleoside monophosphates, and catalytically, it resembles RNase II (3, 4).RNase R is encoded by the rnr gene located at 95 min on the E. coli chromosome (7). This gene was originally termed vacB; and in Shigella and in enteroinvasive E. coli, it is necessary for expression of virulence (8). In laboratory strains of E. coli, RNase R is dispensable for cell viability (7). Moreover, multiple mutant strains lacking RNases R and T, RNases R and PH, and RNases R, II, D, and BN grow essentially normally on rich media (7). On the other hand, a double mutant strain devoid of RNase R and polynucleotide phosphorylase is inviable (7). This observation suggests that RNase R and polynucleotide phosphorylase serve some overlapping essential function(s) in E. coli that cannot be satisfied by any of the othe...

Conversion of tRNA precursors to their mature forms requires the action of both endo-and exoribonucleases. Although studies over many years identified the endoribonuclease, RNase P, and several exoribonucleases as the enzymes responsible for generating the mature 59 and 39 termini, respectively, of Escherichia coli tRNAs, relatively little is known about how tRNAs are separated from long multimeric or multifunction transcripts, or from long leader and trailer sequences. To examine this question, the tRNA products that accumulate in mutant strains devoid of multiple exoribonucleases plus one or several endoribonucleases were analyzed by northern analysis. We find that the multifunction tyrT transcript, which contains two tRNA 1Tyr sequences separated by a 209-nt spacer region plus a downstream mRNA, is cleaved at three sites in the spacer region by the endoribonuclease, RNase E. When both RNase E and RNase P are absent, a product containing both tRNAs accumulates. Two multimeric tRNA transcripts, those for tRNA Arg-His-Leu-Pro and tRNA Gly-Cys-Leu also require RNase E for maturation. For the former transcript, products with long 39 extensions on tRNA Arg , tRNA His , and tRNA Pro , as well as the primary transcript, accumulate in the absence of RNase E. For the latter transcript, RNase E cleaves downstream of each tRNA. Little processing of either multimeric transcript occurs in the absence of both RNase E and RNase P. These data indicate that RNase E is a major contributor to the initial processing of E. coli tRNA transcripts, providing substrates for final maturation by RNase P and the 39 exoribonucleases. Based on this new information, a detailed model for tRNA maturation is proposed.

RNase R is a processive, 3 to 5 hydrolytic exoribonuclease that together with polynucleotide phosphorylase plays an important role in the degradation of structured RNAs. However, RNase R differs from other exoribonucleases in that it can by itself degrade RNAs with extensive secondary structure provided that a single-stranded 3 overhang is present. Using a variety of specifically designed substrates, we show here that a 3 overhang of at least 7 nucleotides is required for tight binding and activity, whereas optimum binding and activity are achieved when the overhang is 10 or more nucleotides in length. In contrast, duplex RNAs with no overhang or with a 4-nucleotide overhang bind extremely poorly to RNase R and are inactive as substrates. A duplex RNA with a 10-nucleotide 5 overhang also is not a substrate. Interestingly, this molecule is bound only weakly, indicating that RNase R does not simply recognize single-stranded RNA, but the RNA must thread into the enzyme with 3 to 5 polarity. We also show that ribose moieties are required for recognition of the substrate as a whole since RNase R is unable to bind or degrade single-stranded DNA. However, RNA molecules with deoxyribose or dideoxyribose residues at their 3 termini can be bound and degraded. Based on these data and a homology model of RNase R, derived from the structure of the closely related enzyme, RNase II, we present a model for how RNase R interacts with its substrates and degrades RNA. The action of ribonucleases (RNases)2 is central to RNA metabolic processes such as the maturation of RNA precursors, the end-turnover of RNAs, and the degradation of RNAs that are either defective or are no longer required by the cell. Complete degradation of an RNA typically requires endoribonucleolytic cleavages followed by the action of a nonspecific 3Ј to 5Ј processive exoribonuclease (1). In Escherichia coli there are three such exoribonucleases: RNase II, RNase R, and polynucleotide phosphorylase (PNPase). RNase II and PNPase were originally thought to be responsible for mRNA decay (2). However, recent work has shown that mRNAs with extensive secondary structure, such as those containing repetitive extragenic palindromic sequences, are degraded by PNPase or by RNase R (3). Likewise, PNPase and/or RNase R is required for the degradation of rRNA (4) and tRNA (5), 3 both of which are highly structured molecules. These data suggest that PNPase and RNase R are the universal degraders of structured RNAs in vivo.Despite its role in the degradation of structured RNAs, purified PNPase is unable to digest through extensive secondary structure and stalls 6 -8 nucleotides (nt) before a stable RNA duplex (3). However, in vivo PNPase associates with an RNA helicase, RhlB, in the degradosome, which also contains the endoribonuclease, RNase E, and the glycolytic enzyme, enolase (6). A direct interaction with RhlB in the form of an ␣ 3 ␤ 2 complex has also been reported (7,8). It is likely to be the association with this helicase that allows PNPase to degrade through structure...

tRNA maturation consists of the specific removal of precursor sequences from both the 5' and 3' termini of an initial RNA transcript. How this is accomplished has heretofore not been ascertained in any system. Using Northern analysis of RNA isolated from a variety of RNase-deficient E. coli strains, we have identified the processing intermediates that accumulate in the absence of specific processing nucleases. From this information we have established the maturation pathways for 12 different E. coli tRNAs including the specific role of each of the relevant RNases in the process. The surprising conclusion from this work is that tRNA maturation is a stochastic process that lacks a defined order and that can proceed with a variety of alternative 3' processing nucleases.