Construction and characterization of an absolute deletion mutant of Escherichia coli ribonuclease II

Abstract The degradation of mRNA plays a central role in the control of protein synthesis. In Escherichia coli , the rnb gene encodes ribonuclease II (RNase II), one of the two main exonucleases involved in mRNA decay. We have constructed strain CMA201, in which the rnb promoter region and the gene were deleted from the chromosome and replaced by a tet^tr cassette. This is the first rnb absolute deletion mutant that shows the complete absence of rnb -specific mRNA. This strain has growth characteristics similar to the wild-type, even though it has no RNase II activity, and it should be useful in studies of mRNA metabolism.     

The role of endonucleases in the expression of ribonuclease II in Escherichia coli

Abstract Ribonuclease II (RNase II), encoded by the rnb gene, is one of the two major Escherichia coli exonucleases involved in mRNA degradation. Some of the ribonucleases implicated in this process have recently been shown to be inter-regulated. In this paper we studied the effects of the endonucleases RNase E and RNase III in rnb expression. We have shown that RNase E cleaves the rnb message internally: when this ribonuclease is inactivated rnb mRNA accumulates with a concomitant increase in RNase II activity. RNase III also affects RNase II expression but in an indirect way. We discuss these implications for the regulation of mRNA degradation.     

Nucleotide sequence and stability of the RNA component of RNase P from a temperature-sensitive mutant of E. coli.

The gene coding for the RNA component of RNase P was cloned from a temperature-sensitive mutant of Escherichia coli defective in RNase P activity (ts709) and its parental wild-type strain (4273), and the complete nucleotide sequences of the gene and its flanking regions were determined. The 5'- and 3'-terminal sequences of the RNA component were determined and mapped on the DNA sequence. The mutant gene has GC-to-AT substitutions at positions corresponding to 89 and 365 nucleotides downstream from the 5' terminus of the RNA sequence. Comparing to the wild-type RNA, the mutant RNA is less stable and rapidly degraded in vivo and in vitro.     

Characterization of the functional domains of Escherichia coli RNase II

RNase II is a single-stranded-specific 3'-exoribonuclease that degrades RNA generating 5'-mononucleotides. This enzyme is the prototype of an ubiquitous family of enzymes that are crucial in RNA metabolism and share a similar domain organization. By sequence prediction, three different domains have been assigned to the Escherichia coli RNase II: two RNA-binding domains at each end of the protein (CSD and S1), and a central RNB catalytic domain. In this work we have performed a functional characterization of these domains in order to address their role in the activity of RNase II. We have constructed a large set of RNase II truncated proteins and compared them to the wild-type regarding their exoribonucleolytic activity and RNA-binding ability. The dissociation constants were determined using different single- or double-stranded substrates. The results obtained revealed that S1 is the most important domain in the establishment of stable RNA-protein complexes, and its elimination results in a drastic reduction on RNA-binding ability. In addition, we also demonstrate that the N-terminal CSD plays a very specific role in RNase II, preventing a tight binding of the enzyme to single-stranded poly(A) chains. Moreover, the biochemical results obtained with RNB mutant that lacks both putative RNA-binding domains, revealed the presence of an additional region involved in RNA binding. Such region, was identified by sequence analysis and secondary structure prediction as a third putative RNA-binding domain located at the N-terminal part of RNB catalytic domain.     

Purification and some novel properties of Escherichia coli RNase II.

RNase II of Escherichia coli (EC 3.1.4.23) has been purified to apparent homogeneity. The K+-activated diesterase activity against poly(U), which defines RNase II, cochromatographs with activity against T4 mRNA or pulse-labeled E. coli RNA successively on DEAE-cellulose, hydroxyapatite or phosphocellulose, and Sephadex G-150 columns. Activities with both substrates are selectively reduced to less than 2% of the wild type level in a newly isolated mutant strain, S296, or after thermal inactivation in a mutant strain with temperature-sensitive RNase II. RNase II releases 5'-XMP without a lag as its only detectable alcohol-soluble produce from all substrates and has an apparent molecular weight of 80,000 to 90,000 in both nondissociating and sodium dodecyl sulfate-polyacrylamide gels. The pure enzyme shows the standard K+ activation against poly(A), poly(U), or poly(C), but only a slight preference for K+ over Na+ ions with T4 mRNA or pulse labeled E. coli RNA as substrate. Uniformly labeled E. coli rRNA or tRNA is degraded little if at all.     

Eukaryotic RNase P: role of RNA and protein subunits of a primordial catalytic ribonucleoprotein in RNA-based catalysis

Ribonuclease P (RNase P) is an essential enzyme that processes the 5' leader sequence of precursor tRNA. Eubacterial RNase P is an RNA enzyme, while its eukaryotic counterpart acts as catalytic ribonucleoprotein, consisting of RNA and numerous protein subunits. To study the latter form, we reconstitute human RNase P activity, demonstrating that the subunits H1 RNA, Rpp21, and Rpp29 are sufficient for 5' cleavage of precursor tRNA. The reconstituted RNase P precisely delineates its cleavage sites in various substrates and hydrolyzes the phosphodiester bond. Rpp21 and Rpp29 facilitate catalysis by H1 RNA, which seems to require a phylogenetically conserved pseudoknot structure for function. Unexpectedly, Rpp29 forms a catalytic complex with M1 RNA of E. coli RNase P. The results uncover the core components of eukaryotic RNase P, reveal its evolutionary origin in translation, and provide a paradigm for studying RNA-based catalysis by other nuclear and nucleolar ribonucleoprotein enzymes.     

Investigating the structure of human RNase H1 by site-directed mutagenesis

In this study we examine for the first time the roles of the various domains of human RNase H1 by site-directed mutagenesis. The carboxyl terminus of human RNase H1 is highly conserved with Escherichia coli RNase H1 and contains the amino acid residues of the putative catalytic site and basic substrate-binding domain of the E. coli RNase enzyme. The amino terminus of human RNase H1 contains a structure consistent with a double-strand RNA (dsRNA) binding motif that is separated from the conserved E. coli RNase H1 region by a 62-amino acid sequence. These studies showed that although the conserved amino acid residues of the putative catalytic site and basic substrate-binding domain are required for RNase H activity, deletion of either the catalytic site or the basic substrate-binding domain did not ablate binding to the heteroduplex substrate. Deletion of the region between the dsRNA-binding domain and the conserved E. coli RNase H1 domain resulted in a significant loss in the RNase H activity. Furthermore, the binding affinity of this deletion mutant for the heteroduplex substrate was approximately 2-fold tighter than the wild-type enzyme suggesting that this central 62-amino acid region does not contribute to the binding affinity of the enzyme for the substrate. The dsRNA-binding domain was not required for RNase H activity, as the dsRNA-deletion mutants exhibited catalytic rates approximately 2-fold faster than the rate observed for wild-type enzyme. Comparison of the dissociation constant of human RNase H1 and the dsRNA-deletion mutant for the heteroduplex substrate indicates that the deletion of this region resulted in a 5-fold loss in binding affinity. Finally, comparison of the cleavage patterns exhibited by the mutant proteins with the cleavage pattern for the wild-type enzyme indicates that the dsRNA-binding domain is responsible for the observed strong positional preference for cleavage exhibited by human RNase H1.     

tRNA nucleotidyltransferase is not essential for Escherichia coli viability.

The role of tRNA nucleotidyltransferase in Escherichia coli has been uncertain because all tRNA genes studied in this organism already encode the -C-C-A sequence. Examination of a cca mutant, originally thought to contain 1-2% enzyme activity, indicated that it actually produces an inactive fragment of 40 kd compared to 47 kd for the wild-type enzyme due to a nonsense mutation in its cca gene. To confirm that the residual activity in extracts of this strain is due to another enzyme, and that tRNA nucleotidyltransferase is non-essential, we have interrupted the cca gene in vitro, and transferred this mutant gene to a variety of strains. In all cases mutant strains are viable, although as much as 15% of the tRNA population contains defective 3' termini, and no tRNA nucleotidyltransferase is detectable. Mutant strains grow slowly, but can be restored to more normal growth by a relA mutation or by a decrease in RNase T activity. In the latter case the amount of defective tRNA decreases dramatically. These findings indicate that tRNA nucleotidyltransferase is not essential for E. coli viability, and therefore, that all essential tRNA genes in this organism encode the -C-C-A sequence.     

The structure and enzymatic properties of a novel RNase II family enzyme from Deinococcus radiodurans

Exoribonucleases are vital in nearly all aspects of RNA metabolism, including RNA maturation, end-turnover, and degradation. RNase II and RNase R are paralogous members of the RNR superfamily of nonspecific, 3'→5', processive exoribonucleases. In Escherichia coli, RNase II plays a primary role in mRNA decay and has a preference for unstructured RNA. RNase R, in contrast, is capable of digesting structured RNA and plays a role in the degradation of both mRNA and stable RNA. Deinococcus radiodurans, a radiation-resistant bacterium, contains two RNR family members. The shorter of these, DrR63, includes a sequence signature typical of RNase R, but we show here that this enzyme is an RNase II-type exonuclease and cannot degrade structured RNA. We also report the crystal structure of this protein, now termed DrII. The DrII structure reveals a truncated RNA binding region in which the N-terminal cold shock domains, typical of most RNR family nucleases, are replaced by an unusual winged helix-turn-helix domain, where the "wing" is contributed by the C-terminal S1 domain. Consistent with its truncated RNA binding region, DrII is able to remove 3' overhangs from RNA molecules closer to duplexes than do other RNase II-type enzymes. DrII also displays distinct sensitivity to pyrimidine-rich regions of single-stranded RNA and is able to process tRNA precursors with adenosine-rich 3' extensions in vitro. These data indicate that DrII is the RNase II of D. radiodurans and that its structure and catalytic properties are distinct from those of other related enzymes.     

Mutational analysis of vaccinia virus nucleoside triphosphate phosphohydrolase II, a DExH box RNA helicase.

Vaccinia virus nucleoside triphosphate phosphohydrolase II (NPH-II), a 3'-to-5' RNA helicase, displays sequence similarity to members of the DExH family of nucleic acid-dependent nucleoside triphosphatases (NTPases). The contributions of the conserved GxGKT and DExH motifs to enzyme activity were assessed by alanine scanning mutagenesis. Histidine-tagged versions of NPH-II were expressed in vaccinia virus-infected BSC40 cells and purified by nickel affinity and conventional fractionation steps. Wild-type His-NPH-II was indistinguishable from native NPH-II with respect to RNA helicase, RNA binding, and nucleic acid-stimulated NTPase activities. The K-191-->A (K191A), D296A, and E297A mutant proteins bound RNA as well as wild-type His-NPH-II did, but they were severely defective in NTPase and helicase functions. The H299A mutant was active in RNA binding and NTP hydrolysis but was defective in duplex unwinding. Whereas the NTPase of wild-type NPH-II was stimulated > 10-fold by polynucleotide cofactors, the NTPase of the H299A mutant was nucleic acid independent. Because the specific NTPase activity of the H299A mutant in the absence of nucleic acid was near that of wild-type enzyme in the presence of DNA or RNA and because the Km for ATP was unaltered by the H299A substitution, we regard this mutation as a "gain-of-function" mutation and suggest that the histidine residue in the DExH box is required to couple the NTPase and helicase activities.     

The role of the S1 domain in exoribonucleolytic activity: substrate specificity and multimerization

RNase II is a 3'-5' exoribonuclease that processively hydrolyzes single-stranded RNA generating 5' mononucleotides. This enzyme contains a catalytic core that is surrounded by three RNA-binding domains. At its C terminus, there is a typical S1 domain that has been shown to be critical for RNA binding. The S1 domain is also present in the other major 3'-5' exoribonucleases from Escherichia coli: RNase R and polynucleotide phosphorylase (PNPase). In this report, we examined the involvement of the S1 domain in the different abilities of these three enzymes to overcome RNA secondary structures during degradation. Hybrid proteins were constructed by replacing the S1 domain of RNase II for the S1 from RNase R and PNPase, and their exonucleolytic activity and RNA-binding ability were examined. The results revealed that both the S1 domains of RNase R and PNPase are able to partially reverse the drop of RNA-binding ability and exonucleolytic activity resulting from removal of the S1 domain of RNase II. Moreover, the S1 domains investigated are not equivalent. Furthermore, we demonstrate that S1 is neither responsible for the ability to overcome secondary structures during RNA degradation, nor is it related to the size of the final product generated by each enzyme. In addition, we show that the S1 domain from PNPase is able to induce the trimerization of the RNaseII-PNP hybrid protein, indicating that this domain can have a role in the biogenesis of multimers.     

Metal ion binding and enzymatic mechanism of Methanococcus jannaschii RNase HII

RNase H degrades the RNA moiety in DNA:RNA hybrid in a divalent metal ion dependent manner. It is essential to understand the role of metal ion in enzymatic mechanism. One of the key points in this study is how many metal ions are involved in the enzyme catalysis. Accordingly, either one-metal binding mechanism or two-metal binding mechanism is proposed. We have studied the thermodynamic properties of four metal ions (Mg(2+), Mn(2+), Ca(2+), and Ba(2+)) binding to Methanococcus jannaschii RNase HII using isothermal titration calorimetry. All of the four metal ions were found to bind Mj RNase HII with 1:1 stoichiometry in the absence of substrate. Together with enzymatic activity assay data, we propose that only one metal ion binding to the enzyme in catalytic process. We also studied the pH dependence of metal binding and enzyme activity and found that at pH 6.5, Mg(2+) did not bind to the enzyme without the substrate but still activated the enzyme to about 2% of its maximum activity (in 10 mM Mn(2+) at pH 8). This implies that the substrate may also be incorporated in metal ion binding and help to position the metal ion. To find which acidic residues correspond to metal ion binding, we also studied the binding thermodynamics and enzymatic activity assay of four mutants: D7N, E8Q, D112N, and D149N in the presence of Mn(2+). The thermodynamic parameters are least affected for the D149N mutant, which has a very low enzymatic activity. This indicates that Asp149 is essential for the enzymatic activity. On the basis of all these observations, we suggest a metal binding model in which D7, E8, and D112 bind the metal ion and D149 activates a water molecule to attack the P-O bond in the RNA chain of the substrate.     

Swapping the domains of exoribonucleases RNase II and RNase R: conferring upon RNase II the ability to degrade ds RNA

RNase II and RNase R are the two E. coli exoribonucleases that belong to the RNase II super family of enzymes. They degrade RNA hydrolytically in the 3' to 5' direction in a processive and sequence independent manner. However, while RNase R is capable of degrading structured RNAs, the RNase II activity is impaired by dsRNAs. The final end-product of these two enzymes is also different, being 4 nt for RNase II and 2 nt for RNase R. RNase II and RNase R share structural properties, including 60% of amino acid sequence similarity and have a similar modular domain organization: two N-terminal cold shock domains (CSD1 and CSD2), one central RNB catalytic domain, and one C-terminal S1 domain. We have constructed hybrid proteins by swapping the domains between RNase II and RNase R to determine which are the responsible for the differences observed between RNase R and RNase II. The results obtained show that the S1 and RNB domains from RNase R in an RNase II context allow the degradation of double-stranded substrates and the appearance of the 2 nt long end-product. Moreover, the degradation of structured RNAs becomes tail-independent when the RNB domain from RNase R is no longer associated with the RNA binding domains (CSD and S1) of the genuine protein. Finally, we show that the RNase R C-terminal Lysine-rich region is involved in the degradation of double-stranded substrates in an RNase II context, probably by unwinding the substrate before it enters into the catalytic cavity.     

Reassessment of the in vivo functions of DNA polymerase I and RNase H in bacterial cell growth

A major factor in removing RNA primers during the processing of Okazaki fragments is DNA polymerase I (Pol I). Pol I is thought to remove the RNA primers and to fill the resulting gaps simultaneously. RNase H, encoded by rnh genes, is another factor in removing the RNA primers, and there is disagreement with respect to the essentiality of both the polA and rnh genes. In a previous study, we looked for the synthetic lethality of paralogs in Bacillus subtilis and detected several essential doublet paralogs, including the polA ypcP pair. YpcP consists of only the 5'-3' exonuclease domain. In the current study, we first confirmed that the polA genes of both Escherichia coli and B. subtilis could be completely deleted. We found that the 5'-3' exonuclease activity encoded by either polA or ypcP xni was required for the growth of B. subtilis and E. coli. Also, the 5'-3' exonuclease activity of Pol I was indispensable in the cyanobacterium Synechococcus elongatus. These results suggest that a 5'-3' exonuclease activity is essential in these organisms. Our success in constructing a B. subtilis strain that lacked all RNase H genes indicates that the enzymatic activity is dispensable, at least in the wild type. Increasing the 5'-3' exonuclease activity partially compensated for a defective phenotype of an RNase H-deficient mutant, suggesting cooperative functions for the two enzyme systems. Our search for the distribution of the 5'-3' exonuclease domain among 250 bacterial genomes resulted in the finding that all eubacteria, but not archaea, possess this domain.     

Mycoplasma fermentans simplifies our view of the catalytic core of ribonuclease P RNA.

The catalytic RNA moiety of (eu)bacterial RNase P is responsible for cleavage of the 5' leader sequence from precursor tRNAs. We report the sequence, the catalytic properties, and a phylogenetic-comparative structural analysis of the RNase P RNA from Mycoplasma fermentans, at 276 nt the smallest known RNase P RNA. This RNA is noteworthy in that it lacks a stem-loop structure (helix P12) that was thought previously to be universally present in bacterial RNase P RNAs. This finding suggests that helix P12 is not required for catalytic activity in vivo. In order to test this possibility in vitro, the kinetic properties of M. fermentans RNase P RNA and a mutant Escherichia coli RNase P RNA that was engineered to lack helix P12 were determined. These RNase P RNAs are catalytically active with efficiencies (Kcat/Km) comparable to that of native E. coli RNase P RNA. These results show that helix P12 is dispensable in vivo in some organisms, and therefore is unlikely to be essential for the mechanism of RNase P action. The notion that all phylogenetically volatile structures in RNase P RNA are dispensable for the catalytic mechanism was tested. A synthetic RNA representing the phylogenetic minimum RNase P RNA was constructed by deleting all evolutionarily variable structures from the M. fermentans RNA. This simplified RNA (Micro P RNA) was catalytically active in vitro with approximately 600-fold decrease in catalytic efficiency relative to the native RNA.     

Characterization of the RNA processing enzyme RNase III from wild type and overexpressing Escherichia coli cells in processing natural RNA substrates.

1. A precursor to small stable RNA, 10Sa RNA, accumulates in large amounts in a temperature sensitive RNase E mutant at non-permissive temperatures, and somewhat in an rnc (RNase III-) mutant, but not in an RNase P- mutant (rnp) or wild type E. coli cells. 2. Since p10Sa RNA was not processed by purified RNase E and III in customary assay conditions, we purified p10Sa RNA processing activity about 700-fold from wild type E. coli cells. 3. Processing of p10Sa RNA by this enzyme shows an absolute requirement for a divalent cation with a strong preference for Mn2+ over Mg2+. Other divalent cations could not replace Mn2+. 4. Monovalent cations (NH+4, Na+, K+) at a concentration of 20 mM stimulated the processing of p10Sa RNA and a temperature of 37 degrees C and pH range of 6.8-8.2 were found to be optimal. 5. The enzyme retained half of its p10Sa RNA processing activity after 30 min incubation at 50 degrees C. 6. Further characterization of this activity indicated that it is RNase III. 7. To further confirm that the p10Sa RNA processing activity is RNase III, we overexpressed the RNase III gene in an E. coli cells that lacks RNase III activity (rnc mutant) and RNase III was purified using one affinity column, agarose.poly(I).poly(C). 8. This RNase III preparation processed p10Sa RNA in a similar way as observed using the p10Sa RNA processing activity purified from wild type E. coli cells, confirming that the first step of p10Sa RNA processing is carried out by RNase III.     

A hybrid ribonuclease H. A novel RNA cleaving enzyme with sequence-specific recognition.

A hybrid enzyme which site-specifically hydrolyzes RNA was created by covalently linking an oligodeoxyribonucleotide to Escherichia coli ribonuclease HI, an enzyme which specifically cleaves RNA moiety of DNA/RNA hybrids. A cysteine residue was substituted for Glu135 by site-directed mutagenesis in the mutant enzyme, in which all 3 free cysteine residues were replaced by alanine (Kanaya, S., Kimura, S., Katsuda, C., and Ikehara, M. (1990) Biochem. J. 271, 59-66), and coupled with a maleimide group, which is attached to the 5' terminus of the nonadeoxyribonucleotide (5'-GTCATCTCC-3') with a flexible tether. The resulting hybrid enzyme, d9-C135/RNase H, cleaved the phosphodiester bond between the fifth and sixth residues of the complementary nonaribonucleotide, without addition of the oligodeoxyribonucleotide. The nonaribonucleotide is cleaved by the wild-type or unmodified mutant enzyme only when the complementary oligodeoxyribonucleotide is present. When the kinetic parameters of the hybrid enzyme for the hydrolysis of the nonaribonucleotide were compared with those of the unmodified mutant enzyme for the hydrolysis of the nonanucleotide duplex, the hybrid enzyme exhibited a 7- and 4-fold decreases in the Km and kcat values, respectively, indicating that it performs multiple turnovers and has a sufficiently high hydrolytic activity. Hybrid ribonucleases H with various oligodeoxyribonucleotides in size and sequence, therefore, might be used as excellent tools for structural and functional studies of RNA.     

Contribution of Tyr712 and Phe716 to the activity of human RNase L.

Ribonuclease L (RNase L) is a key enzyme in the 2-5A host defense system, and its activity is strictly regulated by an unusual 2',5'-linked oligoadenylate (2-5A). A bipartite model, in which the N-terminal half of RNase L is responsible for the 2-5A binding and the C-terminal half alone is able to hydrolyse the substrate RNA, has been proposed on the basis of the results of deletion mutant analyses [Dong, B. & Silverman, R.H. (1997) J. Biol. Chem.272, 22236-22242]. Above all, the region between Glu711 and His720 was revealed to be essential for RNA binding and/or hydrolysis. To dissect the function of the region, we performed scanning mutagenesis over the 10 residues of glutathione S-transferase (GST)-fusion RNase L. Among the single amino acid mutants examined, Y712A and F716A resulted in a significant decrease of RNase activity with a reduced RNA binding acitivity. The losses of the RNase activity were not restored by its conservative mutation, whereas the RNA binding activity was enhanced in the case of Y712F. These results indicate that both Tyr712 and Phe716 provide the enzyme with a RNA binding activity and catalytic environment.