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.

SUMMARY During transcription initiation, RNA polymerase binds to promoter DNA to form an initiation complex containing a DNA bubble and enters into abortive cycles of RNA synthesis before escaping the promoter to transit into the elongation phase for processive RNA synthesis. Here we present the crystal structures of E. coli transcription initiation complexes containing a complete transcription bubble and de novo synthesized RNA oligonucleotides at about 6 Å resolution. The structures show how RNA polymerase recognizes DNA promoters that contain spacers of different lengths and reveal a bridging interaction between the 5’-triphosphate of the nascent RNA and the σ factor that may function to stabilize the short RNA-DNA hybrids during the early stage of transcription initiation. The conformation of the RNA oligonucleotides and the paths of the DNA strands in the complete initiation complexes provide insights into the mechanism that controls both the abortive and productive RNA synthesis.

SUMMARY Guanosine tetraphosphate (ppGpp) is an alarmone that enables bacterial adaptation to their environment. It has been known for years that ppGpp acts directly on RNA polymerase (RNAP) to alter the rate of transcription, but its exact target site is still under debate. Here we report a crystal structure of Escherichia coli RNAP holoenzyme in complex with ppGpp at 4.5 Å resolution. The structure reveals that ppGpp binds at an interface between the shelf and core modules on the outer surface of RNA polymerase, away from the catalytic center and the nucleic acid-binding path. Bound ppGpp connects these two pivotal modules which may restrain the opening of the RNAP cleft. A detailed mechanism of action of ppGpp is proposed, in which ppGpp prevents the closure of the active center that is induced by the binding of NTP, which could slow down nucleotide addition cycles and destabilize the initial transcription complexes.

vacB, a gene previously shown to be required for expression of virulence in Shigella and enteroinvasive Escherichia coli, has been found to encode the 3-5 exoribonuclease, RNase R. Thus, cloning of E. coli vacB led to overexpression of RNase R activity, and partial deletion or interruption of the cloned gene abolished this overexpression. Interruption of the chromosomal copy of vacB eliminated endogenous RNase R activity; however, the absence of RNase R by itself had no effect on cell growth. In contrast, cells lacking both RNase R and polynucleotide phosphorylase were found to be inviable. These data indicate that RNase R participates in an essential cell function in addition to its role in virulence. The identification of the vacB gene product as RNase R should aid in understanding how the virulence phenotype in enterobacteria is expressed and regulated. On the basis of this information we propose that vacB be renamed rnr.Exoribonucleases play an important role in RNA maturation, turnover, and degradation (for reviews, see Refs. 1 and 2). In Escherichia coli eight distinct exoribonucleases have been characterized. Most of them display a degree of overlap in their function. For example, six of the eight, including RNases II, D, BN, T, PH, and polynucleotide phosphorylase (PNPase), 1 participate in the 3Ј-maturation of tRNA precursors (3). Recently, the maturation of the small stable RNAs, M1 RNA, 10Sa RNA/tmRNA, 6S RNA and 4.5S RNA, was examined and found to involve many of the same exoribonucleases (4). It is also known that strains lacking RNases II, D, BN, T, and PH in combination are inviable, but the presence of any one of the five enzymes is sufficient to confer viability, although with varying degrees of effectiveness (5).RNase R is one of the eight exoribonucleases. It acts nonspecifically on poly(A), poly(U), and ribosomal RNAs (rRNA) in vitro (1, 6 -8). The enzyme was initially identified 20 years ago in an E. coli strain deficient in RNase II (6). Whereas RNase II accounts for more than 95% of the activity against poly(A) and poly(U) in crude cell extracts, the residual activity against these substrates and rRNA is due primarily to RNase R (1, 5, 7). Based on its gel filtration properties, RNase R is apparently a protein of ϳ85 kDa (8). However, despite all of this biochemical information, essentially nothing was known about the gene encoding RNase R other than that it mapped to the last quarter of the E. coli chromosome. 2In this paper we report the identification and characterization of the gene that encodes RNase R and show that it is the E. coli vacB gene. vacB was originally described in Shigella flexneri as a chromosomal gene required for expression of the virulence genes carried on the large plasmid of this organism (9). We were led to consider vacB as a candidate for the gene encoding RNase R because (a) sequence analysis revealed that it is homologous to the rnb gene that encodes another exoribonuclease with similar properties, RNase II (10); (b) the deduced size of the VacB protein (ϳ92 kDa...

In bacteria, multiple σ factors compete to associate with the RNA polymerase (RNAP) core enzyme to form a holoenzyme that is required for promoter recognition. During transcription initiation RNAP remains associated with the upstream promoter DNA via sequence-specific interactions between the σ factor and the promoter DNA while moving downstream for RNA synthesis. As RNA polymerase repetitively adds nucleotides to the 3′-end of the RNA, a pyrophosphate ion is generated after each nucleotide incorporation. It is currently unknown how the release of pyrophosphate affects transcription. Here we report the crystal structures of E. coli transcription initiation complexes (TICs) containing the stressresponsive σ S factor, a de novo synthesized RNA oligonucleotide, and a complete transcription bubble (σ S -TIC) at about 3.9-Å resolution. The structures show the 3D topology of the σ S factor and how it recognizes the promoter DNA, including likely specific interactions with the template-strand residues of the −10 element. In addition, σ S -TIC structures display a highly stressed pretranslocated initiation complex that traps a pyrophosphate at the active site that remains closed. The position of the pyrophosphate and the unusual phosphodiester linkage between the two terminal RNA residues suggest an unfinished nucleotide-addition reaction that is likely at equilibrium between nucleotide addition and pyrophosphorolysis. Although these σ S -TIC crystals are enzymatically active, they are slow in nucleotide addition, as suggested by an NTP soaking experiment. Pyrophosphate release completes the nucleotide addition reaction and is associated with extensive conformational changes around the secondary channel but causes neither active site opening nor transcript translocation.transcription initiation | RNA polymerase | σ S factor | promoter recognition | pyrophosphate release C ellular organisms transfer genetic information from DNA to RNA using multisubunit RNA polymerases (RNAPs) that are conserved from bacteria to humans (1, 2). In bacteria, a single fivesubunit core enzyme of RNA polymerase (α 2 ββ′ω) is responsible for all RNA synthesis, whereas multiple σ factors compete to associate with the RNAP core enzyme to form a holoenzyme that is required for initiating the process at DNA promoter sites (3, 4). RNAP remains associated with the upstream promoter DNA during transcription initiation and moves downstream for RNA synthesis, causing DNA scrunching to form a stressed and unstable initiation complex (5-8). Processive RNA synthesis happens only after the initiation complex escapes the promoter as transcription progresses from initiation to elongation (9-11).RNA synthesis in both transcription initiation and elongation involves repetitive cycles of nucleotide addition comprising translocation, NTP binding, catalysis, and pyrophosphate release steps. During this cycling process, the RNAP active site opens for NTP association and closes to align the incoming NTP with the RNA 3′ hydroxyl group for catalysis. Nucleotide addit...

RNase II is a member of the widely distributed RNR family of exoribonucleases, which are highly processive 3'-->5' hydrolytic enzymes that play an important role in mRNA decay. Here, we report the crystal structure of E. coli RNase II, which reveals an architecture reminiscent of the RNA exosome. Three RNA-binding domains come together to form a clamp-like assembly, which can only accommodate single-stranded RNA. This leads into a narrow, basic channel that ends at the putative catalytic center that is completely enclosed within the body of the protein. The putative path for RNA agrees well with biochemical data indicating that a 3' single strand overhang of 7-10 nt is necessary for binding and hydrolysis by RNase II. The presence of the clamp and the narrow channel provides an explanation for the processivity of RNase II and for why its action is limited to single-stranded RNA.

Escherichia coli RNase T, the enzyme responsible for the end-turnover of tRNA and for the 3 maturation of 5 S and 23 S rRNAs and many other small, stable RNAs, was examined in detail with respect to its substrate specificity. The enzyme was found to be a single-strandspecific exoribonuclease that acts in the 3 to 5 direction in a non-processive manner. However, although other Escherichia coli exoribonucleases stop several nucleotides downstream of an RNA duplex, RNase T can digest RNA up to the first base pair. The presence of a free 3-hydroxyl group is required for the enzyme to initiate digestion. Studies with RNA homopolymers and a variety of oligoribonucleotides revealed that RNase T displays an unusual base specificity, discriminating against pyrimidine and, particularly, C residues. Although RNase T appears to bind up to 10 nucleotides in its active site, its specificity is defined largely by the last 4 residues. A single 3-terminal C residue can reduce RNase T action by >100-fold, and 2-terminal C residues essentially stop the enzyme. In vivo, the substrates of RNase T are similar in that they all contain a doublestranded stem followed by a single-stranded 3 overhang; yet, the action of RNase T on these substrates differs. The substrate specificity described here helps to explain why the different substrates yield different products, and why certain RNA molecules are not substrates at all.RNase T, one of eight exoribonucleases present in Escherichia coli (1), was originally identified as an activity involved in the end-turnover of tRNA (2, 3). This process consists of the removal and re-addition of the 3Ј-terminal AMP and, to a very small extent, the penultimate CMP residues of tRNA. Other studies demonstrated that RNase T also is involved in other aspects of RNA metabolism, including the 3Ј maturation of tRNAs (4), 5 S and 23 S rRNAs (5, 6), and other small, stable RNAs (7). Although multiple exoribonucleases may contribute to the 3Ј maturation of these RNAs, RNase T is often the most efficient (8). In fact, RNase T is essential for generating the mature 3Ј-ends of 5 S and 23 S rRNAs (5, 6). Interestingly, all the RNase T substrates share a common sequence feature, i.e. their 5Ј-and 3Ј-ends pair with each other to form a stable, double-stranded (ds) 1 stem followed by a few unpaired 3Ј-nucleotides (7). 5 S and 23 S rRNAs differ from the other small, stable RNAs in that their mature forms contain only 1 or 2 unpaired 3Ј residues, whereas the others contain 4 unpaired residues when matured. Thus, RNase T appears to be the only exoribonuclease that can efficiently remove residues near a stable double-stranded stem. However, what determines the number of unpaired residues remaining after RNase T action is still unknown.In vitro, purified RNase T acts on a variety of tRNA-like substrates (2, 9). Of these, the preferred substrate is mature tRNA-CCA, but terminal residues also can be removed from phosphodiesterase-treated tRNA and removed slowly from tRNA-CA and tRNA-CCA-C 2-3 . In contrast, tRNA-CCp, aminoacyl...

RNase D (RND) is one of seven exoribonucleases identified in Escherichia coli. RNase D has homologs in many eubacteria and eukaryotes, and has been shown to contribute to the 3' maturation of several stable RNAs. Here, we report the 1.6 A resolution crystal structure of E. coli RNase D. The conserved DEDD residues of RNase D fold into an arrangement very similar to the Klenow fragment exonuclease domain. Besides the catalytic domain, RNase D also contains two structurally similar alpha-helical domains with no discernible sequence homology between them. These closely resemble the HRDC domain previously seen in RecQ-family helicases and several other proteins acting on nucleic acids. More interestingly, the DEDD catalytic domain and the two helical domains come together to form a ring-shaped structure. The ring-shaped architecture of E. coli RNase D and the HRDC domains likely play a major role in determining the substrate specificity of this exoribonuclease.