The SCO2299 gene from Streptomyces coelicolor A3(2) encodes a bifunctional enzyme consisting of an RNase H domain and an acid phosphatase domain

The SCO2299 gene from Streptomyces coelicolor encodes a single peptide consisting of 497 amino acid residues. Its N-terminal region shows high amino acid sequence similarity to RNase HI, whereas its C-terminal region bears similarity to the CobC protein, which is involved in the synthesis of cobalamin. The SCO2299 gene suppressed a temperature-sensitive growth defect of an Escherichia coli RNase H-deficient strain, and the recombinant SCO2299 protein cleaved an RNA strand of RNA.DNA hybrid in vitro. The N-terminal domain of the SCO2299 protein, when overproduced independently, exhibited RNase H activity at a similar level to the full length protein. On the other hand, the C-terminal domain showed no CobC-like activity but an acid phosphatase activity. The full length protein also exhibited acid phosphatase activity at almost the same level as the C-terminal domain alone. These results indicate that RNase H and acid phosphatase activities of the full length SCO2299 protein depend on its N-terminal and C-terminal domains, respectively. The physiological functions of the SCO2299 gene and the relation between RNase H and acid phosphatase remain to be determined. However, the bifunctional enzyme examined here is a novel style in the Type 1 RNase H family. Additionally, S. coelicolor is the first example of an organism whose genome contains three active RNase H genes.     

Crystal structure of type 1 ribonuclease H from hyperthermophilic archaeon Sulfolobus tokodaii: role of arginine 118 and C-terminal anchoring

The crystal structure of ribonuclease HI from the hyperthermophilic archaeon Sulfolobus tokodaii (Sto-RNase HI) was determined at 1.6 A resolution. Sto-RNase HI exhibits not only RNase H activity but also double-stranded RNA-dependent ribonuclease (dsRNase) activity. The main-chain fold and steric configurations of the four acidic active-site residues of Sto-RNase HI are very similar to those of other type 1 RNases H. However, Arg118 of Sto-RNase HI is located at the position in which His124 of E. coli RNase HI, His539 of HIV-1 RNase H, and Glu188 of Bacillus halodurans RNase H are located. The mutation of this residue to Ala considerably reduced both the RNase H and dsRNase activities without seriously affecting substrate binding, suggesting that Arg118 is involved in catalytic function. This residue may promote product release by perturbing the coordination of the metal ion A as proposed for Glu188 of B. halodurans RNase H. In addition, the extreme C-terminal region of Sto-RNase HI is anchored to its core region by one disulfide bond and several hydrogen bonds. Differential scanning calorimetry measurements indicated that Sto-RNase HI is a hyperstable protein with a melting temperature of 102 degrees C. The mutations of the cysteine residues forming disulfide bond or elimination of the extreme C-terminal region greatly destabilized the protein, indicating that anchoring of the C-terminal tail is responsible for hyperstabilization of Sto-RNase HI.     

Identification of the gene encoding a type 1 RNase H with an N-terminal double-stranded RNA binding domain from a psychrotrophic bacterium

The gene encoding a bacterial type 1 RNase H, termed RBD-RNase HI, was cloned from the psychrotrophic bacterium Shewanella sp. SIB1, overproduced in Escherichia coli, and the recombinant protein was purified and biochemically characterized. SIB1 RBD-RNase HI consists of 262 amino acid residues and shows amino acid sequence identities of 26% to SIB1 RNase HI, 17% to E. coli RNase HI, and 32% to human RNase H1. SIB1 RBD-RNase HI has a double-stranded RNA binding domain (RBD) at the N-terminus, which is commonly present at the N-termini of eukaryotic type 1 RNases H. Gel mobility shift assay indicated that this domain binds to an RNA/DNA hybrid in an isolated form, suggesting that this domain is involved in substrate binding. SIB1 RBD-RNase HI exhibited the enzymatic activity both in vitro and in vivo. Its optimum pH and metal ion requirement were similar to those of SIB1 RNase HI, E. coli RNase HI, and human RNase H1. The specific activity of SIB1 RBD-RNase HI was comparable to that of E. coli RNase HI and was much higher than those of SIB1 RNase HI and human RNase H1. SIB1 RBD-RNase HI showed poor cleavage-site specificity for oligomeric substrates. SIB1 RBD-RNase HI was less stable than E. coli RNase HI but was as stable as human RNase H1. Database searches indicate that several bacteria and archaea contain an RBD-RNase HI. This is the first report on the biochemical characterization of RBD-RNase HI.     

A common 40 amino acid motif in eukaryotic RNases H1 and caulimovirus ORF VI proteins binds to duplex RNAs.

Eukaryotic RNases H from Saccharomyces cerevisiae , Schizosaccharomyces pombe and Crithidia fasciculata , unlike the related Escherichia coli RNase HI, contain a non-RNase H domain with a common motif. Previously we showed that S.cerevisiae RNase H1 binds to duplex RNAs (either RNA-DNA hybrids or double-stranded RNA) through a region related to the double-stranded RNA binding motif. A very similar amino acid sequence is present in caulimovirus ORF VI proteins. The hallmark of the RNase H/caulimovirus nucleic acid binding motif is a stretch of 40 amino acids with 11 highly conserved residues, seven of which are aromatic. Point mutations, insertions and deletions indicated that integrity of the motif is important for binding. However, additional amino acids are required because a minimal peptide containing the motif was disordered in solution and failed to bind to duplex RNAs, whereas a longer protein bound well. Schizosaccharomyces pombe RNase H1 also bound to duplex RNAs, as did proteins in which the S.cerevisiae RNase H1 binding motif was replaced by either the C.fasciculata or by the cauliflower mosaic virus ORF VI sequence. The similarity between the RNase H and the caulimovirus domain suggest a common interaction with duplex RNAs of these two different groups of proteins.     

Different cleavage specificities of RNases III from Rhodobacter capsulatus and Escherichia coli.

23S rRNA in Rhodobacter capsulatus shows endoribonuclease III (RNase III)-dependent fragmentation in vivo at a unique extra stem-loop extending from position 1271 to 1331. RNase III is a double strand (ds)-specific endoribonuclease. This substrate preference is mediated by a double-stranded RNA binding domain (dsRBD) within the protein. Although a certain degree of double strandedness is a prerequisite, the question arises what structural features exactly make this extra stem-loop an RNase III cleavage site, distinguishing it from the plethora of stem-loops in 23S rRNA? We used RNase III purified from R.capsulatus and Escherichia coli, respectively, together with well known substrates for E.coli RNase III and RNA substrates derived from the special cleavage site in R.capsulatus 23S rRNA to study the interaction between the Rhodobacter enzyme and the fragmentation site. Although both enzymes are very similar in their amino acid sequence, they exhibit significant differences in binding and cleavage of these in vitro substrates.     

Eukaryotic RNases H1 act processively by interactions through the duplex RNA-binding domain

Ribonucleases H have mostly been implicated in eliminating short RNA primers used for initiation of lagging strand DNA synthesis. Escherichia coli RNase HI cleaves these RNA-DNA hybrids in a distributive manner. We report here that eukaryotic RNases H1 have evolved to be processive enzymes by attaching a duplex RNA-binding domain to the RNase H region. Highly conserved amino acids of the duplex RNA-binding domain are required for processivity and nucleic acid binding, which leads to dimerization of the protein. The need for a processive enzyme underscores the importance in eukaryotic cells of processing long hybrids, most of which remain to be identified. However, long RNA-DNA hybrids formed during immunoglobulin class-switch recombination are potential targets for RNase H1 in the nucleus. In mitochondria, where RNase H1 is essential for DNA formation during embryogenesis, long hybrids may be involved in DNA replication.     

Functional analysis of the domain organization of Trypanosoma brucei RNase HI

The structure-function relationship of Trypanosoma brucei RNase HI was investigated by evaluating the abilities of truncated forms of the enzyme to convert RNase H substrate to product. Our studies identify a 42-amino-acid noncanonical RNase HI spacer domain essential for function. We also show that the enzyme's nuclear localization domain is not required for RNase H activity but functions as an RNA binding domain which modulates the enzyme's Mn(2+)-dependent activity. These findings show that the enzyme's RNA binding/nuclear targeting and RNase H activities are organized into discrete N- and C-terminal domains with boundaries established by its spacer domain. This is the first report of the unusual structure to function relationship of a protozoal RNase H. This relationship may be conserved in other eukaryotic RNases H suggesting that criteria preserving their structure and function may be important to their roles in nucleic acid metabolism.     

NMR structure of the N-terminal domain of Saccharomyces cerevisiae RNase HI reveals a fold with a strong resemblance to the N-terminal domain of ribosomal protein L9.

In addition to the conserved and well-defined RNase H domain, eukaryotic RNases HI possess either one or two copies of a small N-terminal domain. The solution structure of one of the N-terminal domains from Saccharomyces cerevisiae RNase HI, determined using NMR spectroscopy, is presented. The 46 residue motif comprises a three-stranded antiparallel beta-sheet and two short alpha-helices which pack onto opposite faces of the beta-sheet. Conserved residues involved in packing the alpha-helices onto the beta-sheet form the hydrophobic core of the domain. Three highly conserved and solvent exposed residues are implicated in RNA binding, W22, K38 and K39. The beta-beta-alpha-beta-alpha topology of the domain differs from the structures of known RNA binding domains such as the double-stranded RNA binding domain (dsRBD), the hnRNP K homology (KH) domain and the RNP motif. However, structural similarities exist between this domain and the N-terminal domain of ribosomal protein L9 which binds to 23 S ribosomal RNA.     

E. coli RNase I exhibits a strong Ca2+-dependent inherent double-stranded RNase activity

Since its initial characterization, Escherichia coli RNase I has been described as a single-strand specific RNA endonuclease that cleaves its substrate in a largely sequence independent manner. Here, we describe a strong calcium (Ca²⁺)-dependent activity of RNase I on double-stranded RNA (dsRNA), and a Ca²⁺-dependent novel hybridase activity, digesting the RNA strand in a DNA:RNA hybrid. Surprisingly, Ca²⁺ does not affect the activity of RNase I on single stranded RNA (ssRNA), suggesting a specific role for Ca²⁺ in the modulation of RNase I activity. Mutation of a previously overlooked Ca²⁺ binding site on RNase I resulted in a gain-of-function enzyme that is highly active on dsRNA and could no longer be stimulated by the metal. In summary, our data imply that native RNase I contains a bound Ca²⁺, allowing it to target both single- and double-stranded RNAs, thus having a broader substrate specificity than originally proposed for this traditional enzyme. In addition, the finding that the dsRNase activity, and not the ssRNase activity, is associated with the Ca²⁺-dependency of RNase I may be useful as a tool in applied molecular biology.