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RNase J, a prokaryotic 5'-3' exo/endoribonuclease, contributes to mRNA decay, rRNA maturation and post-transcriptional regulation. Yet the processive-exoribonucleolysis mechanism remains obscure. Here, we solved the first RNA-free and RNA-bound structures of an archaeal RNase J, and through intensive biochemical studies provided detailed mechanistic insights into the catalysis and processivity. Distinct dimerization/tetramerization patterns were observed for archaeal and bacterial RNase Js, and unique archaeal Loops I and II were found involved in RNA interaction. A hydrogen-bond-network was identified for the first time that assists catalysis by facilitating efficient proton transfer in the catalytic center. A conserved 5'-monophosphate-binding pocket that coordinates the RNA 5'-end ensures the 5'-monophosphate preferential exoribonucleolysis. To achieve exoribonucleolytic processivity, the 5'-monophosphate-binding pocket and nucleotide +4 binding site anchor RNA within the catalytic track; the 5'-capping residue Leu37 of the sandwich pocket coupled with the 5'-monophosphate-binding pocket are dedicated to translocating and controlling the RNA orientation for each exoribonucleolytic cycle. The processive-exoribonucleolysis mechanism was verified as conserved in bacterial RNase J and also exposes striking parallels with the non-homologous eukaryotic 5'-3' exoribonuclease, Xrn1. The findings in this work shed light on not only the molecular mechanism of the RNase J family, but also the evolutionary convergence of divergent exoribonucleases.

RNase P is a universal enzyme that removes 5′ leader sequences from tRNA precursors. The enzyme is therefore essential for maturation of functional tRNAs and mRNA translation. RNase P represents a unique example of an enzyme that can occur either as ribonucleoprotein or as protein alone. The latter form of the enzyme, called protein-only RNase P (PRORP), is widespread in eukaryotes in which it can provide organellar or nuclear RNase P activities. Here, we have focused on Arabidopsis nuclear PRORP2 and its interaction with tRNA substrates. Affinity measurements helped assess the respective importance of individual pentatricopeptide repeat motifs in PRORP2 for RNA binding. We characterized the PRORP2 structure by X-ray crystallography and by small-angle X-ray scattering in solution as well as that of its complex with a tRNA precursor by small-angle X-ray scattering. Of note, our study reports the first structural data of a PRORP–tRNA complex. Combined with complementary biochemical and biophysical analyses, our structural data suggest that PRORP2 undergoes conformational changes to accommodate its substrate. In particular, the catalytic domain and the RNA-binding domain can move around a central hinge. Altogether, this work provides a refined model of the PRORP–tRNA complex that illustrates how protein-only RNase P enzymes specifically bind tRNA and highlights the contribution of protein dynamics to achieve this specific interaction.

RNase II is the most active exoribonuclease in Escherichia coli cell extracts. Yet, its removal appears to have no deleterious effect on growing cells. Here, we show that RNase II is required for cell survival during prolonged stationary phase and upon starvation. The absence of RNase II leads to greatly increased rRNA degradation and to the accumulation of rRNA fragments, both of which lead to a decline in cell survival. The deleterious effects of RNase II removal can be completely reversed by the simultaneous absence of a second exoribonuclease, RNase PH, an enzyme known to be required to initiate ribosome degradation in starving cells. We have now found that the role of RNase II in this process is to regulate the amount of RNase PH present in starving cells, and it does so at the level of RNase PH stability. RNase PH normally decreases as much as 90% during starvation because the protein is unstable under these conditions; however, in the absence of RNase II the amount of RNase PH remains relatively unchanged. Based on these observations, we propose that in the presence of RNase II, nutrient deprivation leads to a dramatic reduction in the amount of RNase PH, thereby limiting the extent of rRNA degradation and ensuring cell survival during this stress. In the absence of RNase II, RNase PH levels remain high, leading to excessive ribosome loss and ultimately to cell death. These findings provide another example of RNase regulation in response to environmental stress.

International audience; RNase H is a non-specific endonuclease which degrades selectively the RNA strand in DNA/RNA duplexes. We demonstrate in the present study that 5'-boronic acid modified oligonucleotides hybridized to a RNA target sequence converts RNase H to an inactivated enzyme complex. The dynamic formation of a boronate ester upon addition of a diol moiety disrupts the enzyme-inhibitor complex and reactivates RNase H. Moreover, we show that reactivation of RNase H function can also be engineered through short RNA trimers inputs that fashion RNase H from a non-specific DNA-guided enzyme into an informational and programmable RNA-guided one. Examples of programmable RNA recognition and cleavage illustrate the potential of this new stimuli-responsive system.

RraA is a protein inhibitor of RNase E, which degrades and processes numerous RNAs in Escherichia coli. Streptomyces coelicolor also contains homologs of RNase E and RraA, RNase ES and RraAS1/RraAS2, respectively. Here, we report that, unlike other RraA homologs, RraAS1 directly interacts with the catalytic domain of RNase ES to exert its inhibitory effect. We further show that rraAS1 gene deletion in S. coelicolor results in a higher growth rate and increased production of actinorhodin and undecylprodigiosin, compared with the wild-type strain, suggesting that RraAS1-mediated regulation of RNase ES activity contributes to modulating the cellular physiology of S. coelicolor.

Gene regulation by base pairing between small noncoding RNAs (sRNAs) and their mRNA targets is an important mechanism that allows bacteria to maintain homeostasis and respond to dynamic environments. In Gram-negative bacteria, sRNA pairing and regulation are mediated by several RNA-binding proteins, including the sRNA chaperone Hfq and polynucleotide phosphorylase (PNPase). PNPase and its homolog RNase PH together represent the two 3′ to 5′ phosphorolytic exoribonucleases found in Escherichia coli ; however, the role of RNase PH in sRNA regulation has not yet been explored and reported. Here, we have examined in detail how PNPase and RNase PH interact to support sRNA stability, activity, and base pairing in exponential and stationary growth conditions. Our results indicate that these proteins facilitate the stability and regulatory function of the sRNAs RyhB, CyaR, and MicA during exponential growth. PNPase further appears to contribute to pairing between RyhB and its mRNA targets. During stationary growth, each sRNA responded differently to the absence or presence of PNPase and RNase PH. Finally, our results suggest that PNPase and RNase PH stabilize only Hfq-bound sRNAs. Taken together, these results confirm and extend previous findings that PNPase participates in sRNA regulation and reveal that RNase PH serves a similar, albeit more limited, role as well. These proteins may, therefore, act to protect sRNAs from spurious degradation while also facilitating regulatory pairing with their targets. IMPORTANCE In many bacteria, Hfq-dependent base-pairing sRNAs facilitate rapid changes in gene expression that are critical for maintaining homeostasis and responding to stress and environmental changes. While a role for Hfq in this process was identified more than 2 decades ago, the identity and function of the other proteins required for Hfq-dependent regulation by sRNAs have not been resolved. Here, we demonstrate that PNPase and RNase PH, the two phosphorolytic RNases in E. coli , stabilize sRNAs against premature degradation and, in the case of PNPase, also accelerate regulation by sRNA-mRNA pairings for certain sRNAs. These findings are the first to demonstrate that RNase PH influences and supports sRNA regulation and suggest shared and distinct roles for these phosphorolytic RNases in this process.

Ribonuclease P (RNase P) is the enzyme that endonucleolytically removes 5′-precursor sequences from tRNA transcripts in all domains of life. RNase P activities are either ribonucleoprotein (RNP) or protein-only RNase P (PRORP) enzymes, raising the question about the mechanistic strategies utilized by these architecturally different enzyme classes to catalyze the same type of reaction. Here, we analyzed the kinetics and cleavage-site selection by PRORP3 from Arabidopsis thaliana (AtPRORP3) using precursor tRNAs (pre-tRNAs) with individual modifications at the canonical cleavage site, with either Rp- or Sp-phosphorothioate, or 2′-deoxy, 2′-fluoro, 2′-amino, or 2′-O-methyl substitutions. We observed a small but robust rescue effect of Sp-phosphorothioate-modified pre-tRNA in the presence of thiophilic Cd2 + ions, consistent with metal-ion coordination to the (pro-)Sp-oxygen during catalysis. Sp-phosphorothioate, 2′-deoxy, 2′-amino, and 2′-O-methyl modification redirected the cleavage mainly to the next unmodified phosphodiester in the 5′-direction. Our findings are in line with the 2′-OH substituent at nucleotide − 1 being involved in an H-bonding acceptor function. In contrast to bacterial RNase P, AtPRORP3 was found to be able to utilize the canonical and upstream cleavage site with similar efficiency (corresponding to reduced cleavage fidelity), and the two cleavage pathways appear less interdependent than in the bacterial RNA-based system.

RNase BN, the RNase Z family member in E. coli, can participate in the processing of tRNA precursors. However, this function only becomes apparent when other processing enzymes are absent, raising the question of its primary physiological role. Here, we show that RNase BN itself is subject to growth phase-dependent regulation, because both rbn mRNA and RNase BN protein are at their highest levels in early exponential phase, but then decrease dramatically and are essentially absent in stationary phase. As a consequence of this variation, certain small RNAs, such as 6S RNA, remain low in exponential phase cells, and increase greatly in stationary phase. RNase BN affects 6S RNA abundance by decreasing its stability in exponential phase. RNase BN levels increase rapidly as cells exit stationary phase and are primarily responsible for the decrease in 6S RNA that accompanies this process. Purified RNase BN directly cleaves 6S RNA as shown by in vitro assays, and the 6S RNA:pRNA duplex is an even more favorable substrate of RNase BN. The exoribonuclease activity of RNase BN is unnecessary because all its action on 6S RNA is due to endonucleolytic cleavages. These data indicate that RNase BN plays an important role in determining levels of the global transcription regulator, 6S RNA, throughout the growth cycle.

Background: The Ribonuclease III (RNase III) enzymatic class is involved in many important biological processes from bacteria to higher eukaryotes. Consequently, they have been useful as drug-target candidates for drug development. Despite their high molecular diversity, RNases III share common structural and catalytic features and some degree of enzymatic activity. However, the role of accessory domains as key determinants of substrate selectivity and over the biological function of each RNase III type is still under study. Results: The in vitro enzymatic activity of three RNase III members from class I (Escherichia coli RNase III, Schizosaccharomyces pombe Pac1 and Saccharomyces cerevisiae Rntp1) and the human Drosha placed in class II was revisited against four different substrates. These two RNase III classes comprise members showing different domain organization. Enzymatic activity differences were found among members of the class I, which were even higher when the human Drosha (class II) was tested. The substrate promiscuity of the E. coli RNase III was corroborated in vivo through its expression in S. cerevisiae, as reported previously, but was extended here to Pichia pastoris. The putative molecular mechanisms contributing for the lethal effect of the heterologous RNase III on the yeast lives were deeply discussed. Conclusion: The new generated biochemical data integrated with previous available information affirmed that RNases III substrate specificity as well as their cellular biological role is highly influenced by its protein structure architecture. This fact also allowed drawing evolutionary links between RNase III members from their structural and substrate specificity differences.

RNase E (Rne) is an essential enzyme involved in the processing and degradation of a large portion of RNAs in Escherichia coli. The enzymatic activity of RNase E is controlled by regulators of ribonuclease activity, namely, RraA and RraB. Gram-positive bacterium Streptomyces coelicolor also contains homologs of Rne and RraA, designated as RNase ES (Rns), RraAS1, and RraAS2. In the present study, we investigated the effect of S. coelicolor RraAS1 on the ribonucleolytic activity of RNase E in E. coli. Coexpression of RraAS1 with Rne resulted in the decreased levels of rpsO, ftsZ, and rnhB mRNAs, which are RNase E substrates, and augmented the toxic effect of Rne overexpression on cell growth. These in vivo effects appeared to be induced by the binding of RraAS1 to Rne, as indicated by the results of co-immunoprecipitation analysis. These results suggested that RraAS1 induces ribonucleolytic activity of RNase E in E. coli.

SAMHD1 plays diverse roles in innate immunity, autoimmune diseases and HIV restriction, but the mechanisms involved are still unclear. SAMHD1 has been reported to have both dNTPase and RNase activities. However, whether SAMHD1 possesses RNase activity remains highly controversial. Here, we found that, unlike conventional hydrolytic exoribonucleases, SAMHD1 requires inorganic phosphate to degrade RNA substrates and produces nucleotide diphosphates rather than nucleoside monophosphates, which indicated that SAMHD1 is a phosphorolytic but not hydrolytic 3'-5' exoribonuclease. Furthermore, SAMHD1 preferentially cleaved single-stranded RNAs comprising A20 or U20, whereas neither C20 nor G20 was susceptible to SAMHD1-mediated degradation. Our findings will facilitate more advanced studies into the role of the SAMHD1 RNase function in the cellular pathogenesis implicated in nucleic acid-triggered inflammatory responses and the anti-retroviral function of SAMHD1.

Persistent R-loops lead to replicative stress due to RNA polymerase stalling and DNA damage. RNase H enzymes facilitate the organisms to survive in the hostile condition by removing these R-loops. MS_RHII-RSD was previously identified to be the second (p)ppGpp synthetase in Mycobacterium smegmatis. The unique presence of an additional RNase HII domain raises an important question regarding the significance of this bifunctional protein. In this report, we demonstrate its ability to hydrolyze R-loops in Escherichia coli exposed to UV stress. MS_RHII-RSD gene expression was upregulated under UV stress, and this gene deleted strain showed increased R-loop accumulation as compared to the wild type. The domains in isolation are known to be inactive, and the full length protein is required for its function. Domain interdependence studies using active site mutants reveal the necessity of a hexamer form with high alpha helical content. In previous studies, bacterial RNase type HI has been mainly implicated in R-loop hydrolysis, but in this study, the RNase HII domain containing protein showed the activity. The prospective of this differential RNase HII activity is discussed. This is the first report to implicate a (p)ppGpp synthetase protein in R-loop-induced stress response.

Among all enzymes in nature, RNase P is unique in that it can use either an RNA- or a protein-based active site for its function: catalyzing cleavage of the 5′-leader from precursor tRNAs (pre-tRNAs). The well-studied catalytic RNase P RNA uses a specificity module to recognize the pre-tRNA and a catalytic module to perform cleavage. Similarly, the recently discovered proteinaceous RNase P (PRORP) possesses two domains – pentatricopeptide repeat (PPR) and metallonuclease (NYN) – that are present in some other RNA processing factors. Here, we combined chemical modification of lysines and multiple-reaction monitoring mass spectrometry to identify putative substrate-contacting residues in Arabidopsis thaliana PRORP1 (AtPRORP1), and subsequently validated these candidate sites by site-directed mutagenesis. Using biochemical studies to characterize the wild-type (WT) and mutant derivatives, we found that AtPRORP1 exploits specific lysines strategically positioned at the tips of it's V-shaped arms, in the first PPR motif and in the NYN domain proximal to the catalytic center, to bind and cleave pre-tRNA. Our results confirm that the protein- and RNA-based forms of RNase P have distinct modules for substrate recognition and cleavage, an unanticipated parallel in their mode of action.

We describe the first human RNase 6 crystal structure in complex with sulfate anions. Kinetic analysis, site-directed mutagenesis and molecular dynamics simulations identified novel substrate recognition and cleavage sites., Human RNase 6 is a cationic secreted protein that belongs to the RNase A superfamily. Its expression is induced in neutrophils and monocytes upon bacterial infection, suggesting a role in host defence. We present here the crystal structure of RNase 6 obtained at 1.72 Å (1 Å=0.1 nm) resolution, which is the first report for the protein 3D structure and thereby setting the basis for functional studies. The structure shows an overall kidney-shaped globular fold shared with the other known family members. Three sulfate anions bound to RNase 6 were found, interacting with residues at the main active site (His15, His122 and Gln14) and cationic surface-exposed residues (His36, His39, Arg66 and His67). Kinetic characterization, together with prediction of protein–nucleotide complexes by molecular dynamics, was applied to analyse the RNase 6 substrate nitrogenous base and phosphate selectivity. Our results reveal that, although RNase 6 is a moderate catalyst in comparison with the pancreatic RNase type, its structure includes lineage-specific features that facilitate its activity towards polymeric nucleotide substrates. In particular, enzyme interactions at the substrate 5′ end can provide an endonuclease-type cleavage pattern. Interestingly, the RNase 6 crystal structure revealed a novel secondary active site conformed by the His36–His39 dyad that facilitates the polynucleotide substrate catalysis.

RNase R, a ubiquitous 3' exoribonuclease, plays an important role in many aspects of RNA metabolism. In contrast to other exoribonucleases, RNase R can efficiently degrade highly structured RNAs, but the mechanism by which this is accomplished has remained elusive. It is known that RNase R contains an unusual, intrinsic RNA helicase activity that facilitates degradation of duplex RNA, but how it stimulates the nuclease activity has also been unclear. Here, we have made use of specifically designed substrates to compare the nuclease and helicase activities of RNase R. We have also identified and mutated several residues in the S1 RNA-binding domain that are important for interacting with duplex RNA and have measured intrinsic tryptophan fluorescence to analyze the conformational changes that occur upon binding of structured RNA. Using these approaches, we have determined the relation of the RNA helicase, ATP binding, and nuclease activities of RNase R. This information has been combined with a structural analysis of RNase R, based on its homology to RNase II, whose structure has been determined, to develop a detailed model that explains how RNase R digests structured RNA and how this differs from its action on single-stranded RNA.

RNase E and RNase G are homologous endonucleases that play important roles in RNA processing and decay in Escherichia coli and related bacterial species. Rapid mRNA degradation is facilitated by the preference of both enzymes for decay intermediates whose 5′ end is monophosphorylated. In this report we identify key characteristics of RNA that influence the rate of 5′-monophosphate-assisted cleavage by these two ribonucleases. In vitro, both require at least two and prefer three or more unpaired 5′-terminal nucleotides for such cleavage; however, RNase G is impeded more than RNase E when fewer than four unpaired nucleotides are present at the 5′ end. Each can tolerate any unpaired nucleotide (A, G, C, or U) at either of the first two positions, with only modest biases. The optimal spacing between the 5′ end and the scissile phosphate appears to be eight nucleotides for RNase E but only six for RNase G. 5′-Monophosphate-assisted cleavage also occurs, albeit more slowly, when that spacing is greater or at most one nucleotide shorter than the optimum, but there is no simple inverse relationship between increased spacing and the rate of cleavage. These properties are also manifested during 5′-end-dependent mRNA degradation in E. coli.

RNase 4, a member of the RNase A superfamily with substrate preference for uridine, has roles in host defence, angiogenesis and neurodegenerative diseases. It also exhibits the highest interspecies amino acid sequence similarity amongst RNase A family members. However, compared to other members of the RNase A family, including eosinophil-derived neurotoxin, eosinophil cationic protein and angiogenin, little is known about the molecular basis of substrate specificity in RNase 4. Here we report high to medium resolution structures of native porcine RNase 4 (PL3), a ‘substrate-specificity’ determining mutant D80A and their respective complexes with deoxyuridine 5′-monophosphate (dUMP) and deoxycytidine 5′-monophosphate (dCMP). These structures provide insight into the structural basis of the uridine versus cytosine substrate specificity in RNase 4: in the D80A mutant (D80A•dCMP), the side chain of Arg101 is positioned further away from the substrate-binding pocket due to the loss of the Asp80 side chain, reducing the repulsion force on the less favoured dCMP from Arg101 and allowing the ligand to occupy the binding pocket. This can also explain the observation that the ligand in the D80A•dCMP complex is stabilized only by a small number of hydrogen bonds. Compared to the previously reported structure of the human RNase 4•2′-deoxyuridine 3′-phosphate complex, the structure of PL3•dUMP complex shows additional hydrogen bonds between the ligand and the protein. In addition, the interaction between Arg101 and the dUMP ligand is absent. These observed differences are probably the result of the flexibility and different ‘positioning’ of the phosphate group among the mononucleotide ligands. Database The atomic coordinates and structure factors for PL3 (5AR6), D80A (5ARJ), PL3∙dUMP (5ARK) and D80A∙dCMP (5ARL) complexes have been deposited with the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ, USA (http://www.rcsb.org/).

RNase II, a 3' to 5' processive exoribonuclease, is the major hydrolytic enzyme in Escherichia coli accounting for ∼90% of the total activity. Despite its importance, little is actually known about regulation of this enzyme. We show here that one residue, Lys501, is acetylated in RNase II. This modification, reversibly controlled by the acetyltransferase Pka, and the deacetylase CobB, affects binding of the substrate and thus decreases the catalytic activity of RNase II. As a consequence, the steady-state level of target RNAs of RNase II may be altered in the cells. We also find that under conditions of slowed growth, the acetylation level of RNase II is elevated and the activity of RNase II decreases, emphasizing the importance of this regulatory process. These findings indicate that acetylation can regulate the activity of a bacterial ribonuclease.

The recent findings that the narrow-specificity endoribonuclease RNase III and the 5′ exonuclease RNase J1 are not essential in the Gram-positive model organism, Bacillus subtilis, facilitated a global analysis of internal 5′ ends that are generated or acted upon by these enzymes. An RNA-Seq protocol known as PARE (Parallel Analysis of RNA Ends) was used to capture 5′ monophosphorylated RNA ends in ribonuclease wild-type and mutant strains. Comparison of PARE peaks in strains with RNase III present or absent showed that, in addition to its well-known role in ribosomal (rRNA) processing, many coding sequences and intergenic regions appeared to be direct targets of RNase III. These target sites were, in most cases, not associated with a known antisense RNA. The PARE analysis also revealed an accumulation of 3′-proximal peaks that correlated with the absence of RNase J1, confirming the importance of RNase J1 in degrading RNA fragments that contain the transcription terminator structure. A significant result from the PARE analysis was the discovery of an endonuclease cleavage just 2 nts downstream of the 16S rRNA 3′ end. This latter observation begins to answer, at least for B. subtilis, a long-standing question on the exonucleolytic versus endonucleolytic nature of 16S rRNA maturation.

Protein-only RNase Ps (PRORPs) are a recently discovered class of RNA processing enzymes that catalyze maturation of the 5′ end of precursor tRNAs (pre-tRNAs) in Eukaryotes. PRORPs are found in the nucleus and/or organelles of most eukaryotic organisms. Arabidopsis thaliana is a representative organism that contains PRORP enzymes (PRORP1, PRORP2, PRORP3) in both its nucleus and organelles; PRORP2 and 3 localize to the nucleus, PRORP1 to the chloroplast and the mitochondria. Apart from their identification, almost nothing is known about the structure and function of PRORPs that act in the nucleus. Here we use a combination of biochemical assays and X-ray crystallography to characterize A. thaliana PRORP2. We solved the crystal structure of PRORP2 (3.2 Å) revealing an overall V-shaped protein and conserved metallonuclease active site structure. Our biochemical studies indicate that PRORP2 requires Mg2+ for catalysis and catalyzes the maturation of nuclear encoded substrates up to 10-fold faster than mitochondrial encoded precursor nad6 t-element under single turnover conditions. We also demonstrate that PRORP2 preferentially binds pre-tRNAs containing short 5′ leaders and 3′ trailers; however, leader and trailer length do not significantly alter the observed rate constants of PRORP2 in single turnover cleavage assays. Our data provide a biochemical and structural framework to begin understanding how nuclear localized PRORPs recognize and cleave their substrates.

IMPACT (Imprinted and Ancient)-like proteins are known to be regulators of GCN2 (General control non-derepressible 2) kinases involved in translation regulation. Here, we report on cloning and characterization of an IMPACT-like protein, LdIMPACT from Leishmania donovani which harbours two domains. 'RWD domain' at the N-terminal end that mediates GCN2 regulation, while a conserved 'ancient domain' lies at the C-terminal end whose function remains elusive. Interestingly, our observations indicated that LdIMPACT has a novel non-specific nuclease activity. In silico analysis further revealed the resemblance of ancient domain of LdIMPACT to RNase PH domain (known to bind to nucleic acids). The recombinant LdIMPACT exhibited a Mg2+-dependent nuclease activity. Moreover, thermostability and pH stability assays of the protein suggest it to be a stress-responsive protein. Circular dichroism studies elucidated the conformational transitions of the enzyme in response to various temperature and pH conditions which correlated well with the activity profiles. Thus, the current study highlights the structural and functional characteristics of LdIMPACT which interestingly also possesses a novel nuclease activity. With its physiological relevance unresolved, the multifaceted LdIMPACT might therefore lie in a hitherto unknown network, whose perturbation could be an attractive therapeutic approach for treating leishmaniasis.

YbeY is a highly conserved, multifunctional endoribonuclease that plays a significant role in ribosome biogenesis and has several additional roles. Here we show that overexpression of the conserved GTPase Era in Escherichia coli partially suppresses the growth defect of a ΔybeY strain while improving 16S rRNA processing and 70S ribosome assembly. This suppression requires both the ability of Era to hydrolyze GTP and the function of three exoribonucleases, RNase II, RNase R, and RNase PH, suggesting a model for the action of Era. Overexpression of Vibrio cholerae Era similarly partially suppresses the defects of an E. coli ΔybeY strain, indicating that this property of Era is conserved in bacteria other than E. coli., National Institutes of Health (U.S.) (Grant GM31030), National Institutes of Health (U.S.) (Grant P30ES002109)

tRNA maturation and quality control are crucial for proper functioning of these transcripts in translation. In several organisms, defective tRNAs were shown to be tagged by poly(A) or CCACCA tails and subsequently degraded by 3′-exonucleases. In a deep-sequencing analysis of tRNA 3′-ends, we detected the CCACCA tag also in Escherichia coli. However, this tag closely resembles several 3′-trailers of tRNA precursors targeted for maturation and not for degradation. Here, we investigate the ability of two important exonucleases, RNase R and RNase T, to distinguish tRNA precursors with a native 3′-trailer from tRNAs with a CCACCA tag. Our results show that the degrading enzyme RNase R breaks down both tRNAs primed for degradation as well as precursor transcripts, indicating that it is a rather nonspecific RNase. RNase T, a main processing exonuclease involved in trimming of 3′-trailers, is very inefficient in converting the CCACCA-tagged tRNA into a mature transcript. Hence, while both RNases compete for trailer-containing tRNA precursors, the inability of RNase T to process CCACCA tails ensures that defective tRNAs cannot reenter the functional tRNA pool, representing a safeguard to avoid detrimental effects of tRNAs with erroneous integrity on protein synthesis. Furthermore, these data indicate that the RNase T-mediated end turnover of the CCA sequence represents a means to deliver a tRNA to a repeated quality control performed by the CCA-adding enzyme. Hence, originally described as a futile side reaction, the tRNA end turnover seems to fulfill an important function in the maintenance of the tRNA pool in the cell.

Ribosomal RNA (rRNA) is a major component of ribosomes and is fundamental to the process of translation. In bacteria, 16S rRNA is a component of the small ribosomal subunit and plays a critical role in mRNA decoding. rRNA maturation entails the removal of intervening spacer sequences contained within the pre-rRNA transcript by nucleolytic enzymes. Enzymatic activities involved in maturation of the 5'-end of 16S rRNA have been identified, but those involved in 3'-end maturation of 16S rRNA are more enigmatic. Here, we investigate molecular details of 16S rRNA maturation using purified in vivo-formed small subunit (SSU) assembly intermediates (pre-SSUs) from wild-type Escherichia coli that contain precursor 16S rRNA (17S rRNA). Upon incubation of pre-SSUs with E. coli S100 cell extracts or purified enzymes implicated in 16S rRNA processing, the 17S rRNA is processed into additional intermediates and mature 16S rRNA. These results illustrate that exonucleases RNase R, RNase II, PNPase, and RNase PH can process the 3'-end of pre-SSUs in vitro. However, the endonuclease YbeY did not exhibit nucleolytic activity with pre-SSUs under these conditions. Furthermore, these data demonstrate that multiple pathways facilitate 16S rRNA maturation with pre-SSUs in vitro, with the dominant pathways entailing complete processing of the 5'-end of 17S rRNA prior to 3'-end maturation or partial processing of the 5'-end with concomitant processing of the 3'-end. These results reveal the multifaceted nature of SSU biogenesis and suggest that E. coli may be able to escape inactivation of any one enzyme by using an existing complementary pathway.

RNase PH, encoded by the rph gene, is a 3′→5′ exoribonuclease that in E. coli participates primarily in the 3′ maturation of pre-tRNAs and the degradation of rRNA in stationary-phase cells. Interestingly, the routinely used laboratory strains of MG1655 and W3110 have naturally acquired the rph-1 allele, encoding a truncated catalytically inactive RNase PH protein which is widely assumed to be benign. Contrary to this assumption, we show that the rph-1 -encoded Rph-1 protein inhibits RNase P-mediated 5′-end maturation of primary pre-tRNAs with leaders of rph-1 strain and for any tRNAs in Δ rph mutant or rph + strains. We propose that the Rph-1 protein bound to the 3′ end of the substrate creates a steric hindrance that in the presence of a triphosphate at the 5′ end reduces the ability of RNase P to bind to the pre-tRNA. IMPORTANCE In this paper, we demonstrate that the rph-1 mutation found in commonly used E. coli strains leads to the synthesis of a truncated functionally inactive RNase PH protein that interferes with the 5′-end maturation of specific tRNAs with short 5′ leaders by RNase P in the absence of RppH, an RNA pyrophosphohydrolase that converts primary 5′ triphosphates into 5′ monophosphates. The data presented indicate that the presence of the triphosphate interferes with RNase P binding to the pre-tRNA.

Ribonuclease E (RNase E) of Escherichia coli, which is the founding member of a widespread family of proteins in bacteria and chloroplasts, is a fascinating enzyme that still has not revealed all its secrets. RNase E is an essential single-strand specific endoribonuclease that is involved in the processing and degradation of nearly every transcript in E. coli. A striking enzymatic property is a preference for substrates with a 5' monophosphate end although recent work explains how RNase E can overcome the protection afforded by the 5' triphosphate end of a primary transcript. Other features of E. coli RNase E include its interaction with enzymes involved in RNA degradation to form the multienzyme RNA degradosome and its localization to the inner cytoplasmic membrane. The N-terminal catalytic core of the RNase E protomer associates to form a tetrameric holoenzyme. Each RNase E protomer has a large C-terminal intrinsically disordered (ID) noncatalytic region that contains sites for interactions with protein components of the RNA degradosome as well as RNA and phospholipid bilayers. In this review, RNase E homologs have been classified into five types based on their primary structure. A recent analysis has shown that type I RNase E in the γ-proteobacteria forms an orthologous group of proteins that has been inherited vertically. The RNase E catalytic core and a large ID noncatalytic region containing an RNA binding motif and a membrane targeting sequence are universally conserved features of these orthologs. Although the ID noncatalytic region has low composition and sequence complexity, it is possible to map microdomains, which are short linear motifs that are sites of interaction with protein and other ligands. Throughout bacteria, the composition of the multienzyme RNA degradosome varies with species, but interactions with exoribonucleases (PNPase, RNase R), glycolytic enzymes (enolase, aconitase) and RNA helicases (DEAD-box proteins, Rho) are common. Plasticity in RNA degradosome composition is due to rapid evolution of RNase E microdomains. Characterization of the RNase E-PNPase interaction in α-proteobacteria, γ-proteobacteria and cyanobacteria suggests that it arose independently several times during evolution, thus conferring an advantage in control and coordination of RNA processing and degradation.

The ribonuclease P (RNase P) proteins TkoPop5 and TkoRpp30, homologs of human Pop5 and Rpp30, respectively, in the hyperthermophilic archaeon Thermococcus kodakarensis were prepared and characterized with respect to pre-tRNA cleavage activity using the reconstitution system of the well-studied Pyrococcus horikoshii RNase P. The reconstituted particle containing TkoPop5 in place of the P. horikoshii counterpart PhoPop5 retained pre-tRNA cleavage activity comparable to that of the reconstituted P. horikoshii RNase P, while that containing TkoRpp30 instead of its corresponding protein PhoRpp30 had slightly lower activity than the P. horikoshii RNase P. Moreover, we determined crystal structures of TkoRpp30 alone and in complex with TkoPop5. Like their P. horikoshii counterparts, whose structures were solved previously, TkoRpp30 and TkoPop5 fold into TIM barrel and RRM-like fold, respectively. This finding demonstrates that RNase P proteins in T. kodakarensis and P. horikoshii are interchangeable and that their three-dimensional structures are highly conserved.

Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome.

The complex posttranscriptional regulation mechanism of the Escherichia coli pnp gene, which encodes the phosphorolytic exoribonuclease polynucleotide phosphorylase (PNPase), involves two endoribonucleases, namely, RNase III and RNase E, and PNPase itself, which thus autoregulates its own expression. The models proposed for pnp autoregulation posit that the target of PNPase is a mature pnp mRNA previously processed at its 5′ end by RNase III, rather than the primary pnp transcript (RNase III-dependent models), and that PNPase activity eventually leads to pnp mRNA degradation by RNase E. However, some published data suggest that pnp expression may also be regulated through a PNPase-dependent, RNase III-independent mechanism. To address this issue, we constructed isogenic Δ pnp rnc + and Δ pnp Δ rnc strains with a chromosomal pnp-lacZ translational fusion and measured β-galactosidase activity in the absence and presence of PNPase expressed by a plasmid. Our results show that PNPase also regulates its own expression via a reversible RNase III-independent pathway acting upstream from the RNase III-dependent branch. This pathway requires the PNPase RNA binding domains KH and S1 but not its phosphorolytic activity. We suggest that the RNase III-independent autoregulation of PNPase occurs at the level of translational repression, possibly by competition for pnp primary transcript between PNPase and the ribosomal protein S1. IMPORTANCE In Escherichia coli , polynucleotide phosphorylase (PNPase, encoded by pnp ) posttranscriptionally regulates its own expression. The two models proposed so far posit a two-step mechanism in which RNase III, by cutting the leader region of the pnp primary transcript, creates the substrate for PNPase regulatory activity, eventually leading to pnp mRNA degradation by RNase E. In this work, we provide evidence supporting an additional pathway for PNPase autogenous regulation in which PNPase acts as a translational repressor independently of RNase III cleavage. Our data make a new contribution to the understanding of the regulatory mechanism of pnp mRNA, a process long since considered a paradigmatic example of posttranscriptional regulation at the level of mRNA stability.

Mitochondrial RNA polymerase produces long polycistronic precursors that contain the mRNAs, rRNAs and tRNAs needed for mitochondrial translation. Mitochondrial RNase P (mt-RNase P) initiates the maturation of the precursors by cleaving at the 5′ ends of the tRNAs. Human mt-RNase P is only active as a tripartite complex (mitochondrial RNase P proteins 1–3; MRPP1-3), whereas plant and trypanosomal RNase Ps (PRORPs)—albeit homologous to MRPP3—are active as single proteins. The reason for this discrepancy has so far remained obscure. Here, we present the crystal structure of human MRPP3, which features a remarkably distorted and hence non-productive active site that we propose will switch to a fully productive state only upon association with MRPP1, MRPP2 and pre-tRNA substrate. We suggest a mechanism in which MRPP1 and MRPP2 both deliver the pre-tRNA substrate and activate MRPP3 through an induced-fit process.

RNase J is a conserved ribonuclease that belongs to the β-CASP family of nucleases. It possesses both endo- and exo-ribonuclease activities, which play a key role in pre-rRNA maturation and mRNA decay. Here we report high-resolution crystal structures of Deinococcus radiodurans RNase J complexed with RNA or uridine 5′-monophosphate in the presence of manganese ions. Biochemical and structural studies revealed that RNase J uses zinc ions for two-metal-ion catalysis. One residue conserved among RNase J orthologues (motif B) forms specific electrostatic interactions with the scissile phosphate of the RNA that is critical for the catalysis and product stabilization. The additional manganese ion, which is coordinated by conserved residues at the dimer interface, is critical for RNase J dimerization and exonuclease activity. The structures may also shed light on the mechanism of RNase J exo- and endonucleolytic activity switch.

Ribonuclease P (RNase P) is an endonuclease that catalyzes the essential removal of the 5′ end of tRNA precursors. Until recently, all identified RNase P enzymes were a ribonucleoprotein with a conserved catalytic RNA component. However, the discovery of protein-only RNase P (PRORP) shifted this paradigm, affording a unique opportunity to compare mechanistic strategies used by naturally evolved protein and RNA-based enzymes that catalyze the same reaction. Here we investigate the enzymatic mechanism of pre-tRNA hydrolysis catalyzed by the NYN (Nedd4-BP1, YacP nuclease) metallonuclease of Arabidopsis thaliana, PRORP1. Multiple and single turnover kinetic data support a mechanism where a step at or before chemistry is rate-limiting and provide a kinetic framework to interpret the results of metal alteration, mutations, and pH dependence. Catalytic activity has a cooperative dependence on the magnesium concentration (nH = 2) under kcat/Km conditions, suggesting that PRORP1 catalysis is optimal with at least two active site metal ions, consistent with the crystal structure. Metal rescue of Asp-to-Ala mutations identified two aspartates important for enhancing metal ion affinity. The single turnover pH dependence of pre-tRNA cleavage revealed a single ionization (pKa ∼ 8.7) important for catalysis, consistent with deprotonation of a metal-bound water nucleophile. The pH and metal dependence mirrors that observed for the RNA-based RNase P, suggesting similar catalytic mechanisms. Thus, despite different macromolecular composition, the RNA and protein-based RNase P act as dynamic scaffolds for the binding and positioning of magnesium ions to catalyze phosphodiester bond hydrolysis. Background: A protein-only ribonuclease P (PRORP) has been recently discovered. Results: PRORP activity has a single ionization (pKa ∼ 8.7) important for catalysis and a cooperative dependence on Mg2+ (nH = 2). Conclusion: PRORP uses catalytic strategies similar to RNA-dependent RNase P. Significance: These results provide evidence for the mechanistic convergence of two different enzymatic macromolecules (RNA and protein) that perform the same biological function.

Pathways for tolerating and repairing DNA-protein crosslinks (DPCs) are poorly defined. We used transposon mutagenesis and candidate gene approaches to identify DPC-hypersensitive Escherichia coli mutants. DPCs were induced by azacytidine (aza-C) treatment in cells overexpressing cytosine methyltransferase; hypersensitivity was verified to depend on methyltransferase expression. We isolated hypersensitive mutants that were uncovered in previous studies (recA, recBC, recG, and uvrD), hypersensitive mutants that apparently activate phage Mu Gam expression, and novel hypersensitive mutants in genes involved in DNA metabolism, cell division, and tRNA modification (dinG, ftsK, xerD, dnaJ, hflC, miaA, mnmE, mnmG, and ssrA). Inactivation of SbcCD, which can cleave DNA at protein-DNA complexes, did not cause hypersensitivity. We previously showed that tmRNA pathway defects cause aza-C hypersensitivity, implying that DPCs block coupled transcription/translation complexes. Here, we show that mutants in tRNA modification functions miaA, mnmE and mnmG cause defects in aza-C-induced tmRNA tagging, explaining their hypersensitivity. In order for tmRNA to access a stalled ribosome, the mRNA must be cleaved or released from RNA polymerase. Mutational inactivation of functions involved in mRNA processing and RNA polymerase elongation/release (RNase II, RNaseD, RNase PH, RNase LS, Rep, HepA, GreA, GreB) did not cause aza-C hypersensitivity; the mechanism of tmRNA access remains unclear.

Thermotoga maritima RNase H1 and Bacillus stearothermophilus RNase H2 have an N-terminal substrate binding domain, termed hybrid binding domain (TmaHBD), and N-terminal domain (BstNTD), respectively. HIV-1 reverse transcriptase (RT) is a heterodimer consisting of a P66 subunit and a P51 subunit. The P66 subunit contains a C-terminal RNase H domain, which exhibits RNase H activity either in the presence of Mg(2+) or Mn(2+) ions. The isolated RNase H domain of HIV-1 RT (RNH(HIV)) is inactive, possibly due to the lack of a substrate binding ability, disorder of a loop containing His539, and increased flexibility. To examine whether the activity of RNH(HIV) is restored by the attachment of TmaHBD or BstNTD to its N-terminus, two chimeric proteins, TmaHBD-RNH(HIV) and BstNTD-RNH(HIV), were constructed and characterized. Both chimeric proteins bound to RNA/DNA hybrid more strongly than RNH(HIV) and exhibited enzymatic activity in the presence of Mn(2+) ions. They did not exhibit activity or exhibited very weak activity in the presence of Mg(2+) ions. These results indicate that TmaHBD and BstNTD function as an RNA/DNA hybrid binding tag, and greatly increase the substrate binding affinity and Mn(2+)-dependent activity of RNH(HIV) but do not restore the Mg(2+)-dependent activity of RNH(HIV).

RNase He1 from Hericium erinaceus, a member of the RNase T1 family, has high identity with RNase Po1 from Pleurotus ostreatus with complete conservation of the catalytic sequence. However, the optimal pH for RNase He1 activity is lower than that of RNase Po1, and the enzyme shows little inhibition of human tumor cell proliferation. Hence, to investigate the potential antitumor activity of recombinant RNase He1 and to possibly enhance its optimum pH, we generated RNase He1 mutants by replacing 12 Asn/Gln residues with Asp/Glu residues; the amino acid sequence of RNase Po1 was taken as reference. These mutants were then expressed in Escherichia coli. Using site-directed mutagenesis, we successfully modified the optimal pH for enzyme activity and generated a recombinant RNase He1 that inhibited the proliferation of cells in the human leukemia cell line. These properties are extremely important in the production of anticancer biologics that are based on RNase activity.

RNase E is a major intracellular endoribonuclease in many bacteria and participates in most aspects of RNA processing and degradation. RNase E requires a divalent metal ion for its activity. We show that only Mg2+and Mn2+will support significant rates of activityin vitroagainst natural RNAs, with Mn2+being preferred. Both Mg2+and Mn2+also support cleavage of an oligonucleotide substrate with similar kinetic parameters for both ions. Salts of Ni2+and Zn2+permitted low levels of activity, while Ca2+, Co3+, Cu2+, and Fe2+did not. A mutation to one of the residues known to chelate Mg2+, D346C, led to almost complete loss of activity dependent on Mg2+; however, the activity of the mutant enzyme was fully restored by the presence of Mn2+with kinetic parameters fully equivalent to those of wild-type enzyme. A similar mutation to the other chelating residue, D303C, resulted in nearly full loss of activity regardless of metal ion. The properties of RNase E D346C enabled a test of the ionic requirements of RNase Ein vivo. Plasmid shuffling experiments showed that bothrneD303C(i.e., thernegene encoding a D-to-C change at position 303) andrneD346Cwere inviable whether or not the selection medium was supplied with MnSO4, implying that RNase E relies on Mg2+exclusivelyin vivo.

RNase P is an essential tRNA-processing enzyme in all domains of life. We identified an unknown type of protein-only RNase P in the hyperthermophilic bacterium Aquifex aeolicus: Without an RNA subunit and the smallest of its kind, the 23-kDa polypeptide comprises a metallonuclease domain only. The protein has RNase P activity in vitro and rescued the growth of Escherichia coli and Saccharomyces cerevisiae strains with inactivations of their more complex and larger endogenous ribonucleoprotein RNase P. Homologs of Aquifex RNase P (HARP) were identified in many Archaea and some Bacteria, of which all Archaea and most Bacteria also encode an RNA-based RNase P; activity of both RNase P forms from the same bacterium or archaeon could be verified in two selected cases. Bioinformatic analyses suggest that A. aeolicus and related Aquificaceae likely acquired HARP by horizontal gene transfer from an archaeon.

Ribonuclease P (RNase P) is an endoribonuclease that catalyzes the processing of the 5′-leader sequence of precursor tRNA (pre-tRNA) in all phylogenetic domains. We have found that RNase P in the hyperthermophilic archaeon Pyrococcus horikoshii OT3 consists of RNase P RNA (PhopRNA) and five protein cofactors designated PhoPop5, PhoRpp21, PhoRpp29, PhoRpp30, and PhoRpp38. Biochemical characterizations over the past 10 years have revealed that PhoPop5 and PhoRpp30 fold into a heterotetramer and cooperate to activate a catalytic domain (C-domain) in PhopRNA, whereas PhoRpp21 and PhoRpp29 form a heterodimer and function together to activate a specificity domain (S-domain) in PhopRNA. PhoRpp38 plays a role in elevation of the optimum temperature of RNase P activity, binding to kink-turn (K-turn) motifs in two stem-loops in PhopRNA. This review describes the structural and functional information on P. horikoshii RNase P, focusing on the structural basis for the PhopRNA activation by the five RNase P proteins.

Ribonucleases play an important role in the RNA metabolism which is critical for the localization, stability and function of mature RNA transcripts. More and more ribonucleases were discovered in recent years with the progress of technology. In the present study, we found that the uncharacterized C19orf43, a novel interacting protein of human telomerase RNA (hTR), digested T7 transcribed RNA, total cellular RNA and RNA oligos but not DNA. Thus we named this new RNase as hTRIR (human telomerase RNA interacting RNase). Genetic analysis showed that hTRIR is conserved among eukaryotic species and widely expressed in different cell lines. The RNase activity of hTRIR works in a broad temperature and pH range while divalent cations are not required. The conserved C-terminus of C19orf43 is necessary for its activity. Finally, we found that hTRIR cleaves all four unpaired RNA nucleotides from 5′ end or 3′ end with higher efficiency for purine bases, which suggested that hTRIR is an exoribonuclease. Taken together, our study showed the first evidence of the novel function of hTRIR in vitro, which provides clue to study the regulatory mechanism of hTR homeostasis in vivo.

Bacterial ribonuclease E (RNase E) plays a crucial role in the processing and decay of RNAs. A small protein named RraA negatively regulates the activity of RNase E via protein-protein interaction in various bacteria. Recently, RraAS1 and RraAS2, which are functional homologs of RraA from Escherichia coli, were identified in the Gram-positive species Streptomyces coelicolor. RraAS1 and RraAS2 inhibit RNase ES ribonuclease activity in S. coelicolor. RraAS1 and RraAS2 have a C-terminal extension region unlike typical bacterial RraA proteins. In this study, we present the crystal structure of RraAS2, exhibiting a hexamer arranged in a dimer of trimers, consistent with size exclusion chromatographic results. Importantly, the C-terminal extension region formed a long α-helix at the junction of the neighboring subunit, which is similar to the trimeric RraA orthologs from Saccharomyces cerevisiae. Truncation of the C-terminal extension region resulted in loss of RNase ES inhibition, demonstrating its crucial role. Our findings present the first bacterial RraA that has a hexameric assembly with a C-terminal extension α-helical region, which plays an essential role in the regulation of RNase ES activity in S. coelicolor.

Double-stranded RNA (dsRNA)-specific ribonuclease III (RNase III) proteins are required for RNA maturation and gene regulation. The mechanism of prokaryotic RNase IIIs has been well characterized, but how eukaryotic RNase IIIs (exemplified by Rnt1p, Drosha, and Dicer) work is less clear. Recently, we reported the crystal structure of Rnt1p in complex with RNA, revealing a double-ruler mechanism for substrate selection. Here, we present more structures of Rnt1p, either RNA-free or RNA-bound, featuring two major conformations of the enzyme. Using these structures with existing data, we describe the functional cycle of Rnt1p in five steps, selecting, loading, locking, cleavage, and releasing. We also describe atomic details of the two-Mg2+-ion catalytic mechanism that is applicable to all eukaryotic RNase III enzymes. Overall, our results indicate that substrate selection is achieved independent of cleavage, allowing the recognition of substrates with different structures while preserving the basic mechanism of cleavage.

RNA decay and RNA maturation are important steps in the regulation of bacterial gene expression. RNase J, which is present in about half of bacterial species, has been shown to possess both endo- and 5′ to 3′ exo-ribonuclease activities. The exonucleolytic activity is clearly involved in the degradation of mRNA and in the maturation of at least the 5′end of 16S rRNA in the two Firmicutes Staphylococcus aureus and Bacillus subtilis. The endoribonuclease activity of RNase J from a number of species has been shown to be weak in vitro and 3-D structural data of different RNase J orthologs has not provided a clear explanation for the molecular basis of this activity. Here, we show that S. aureus RNase J1 is a manganese dependent homodimeric enzyme with strong 5′ to 3′ exo-ribonuclease as well as endo-ribonuclease activity. In addition, we demonstrated that SauJ1 can degrade efficiently 5′triphosphorylated RNA. Our results highlight RNase J1 as an important player in RNA turnover in S. aureus.