RNA ligase in bacteria: formation of a 2',5' linkage by an E. coli extract

Ligase activity was detected in extracts of Escherichia coli, Clostridium tartarivorum, Rhodospirillum salexigens, Chromatium gracile, and Chlorobium limicola. Ligase was measured by joining of tRNA halves produced from yeast IVS-containing tRNA precursors by a yeast endonuclease. The structure of tRNATyr halves joined by an E. coli extract was examined. The ligated junction is resistant to nuclease P1 and RNAase T2 but sensitive to venom phosphodiesterase and alkaline hydrolysis, consistent with a 2',5' linkage. The nuclease-resistant junction dinucleotide comigrates with authentic (2',5') APA marker in thin-layer chromatography. The phosphate in the newly formed phosphodiester bond is derived from the pre-tRNA substrate. The widespread existence of a bacterial ligase raises the possibility of a novel class of RNA processing reactions.     

Residues 36-42 of liver RNase PL3 contribute to its uridine-preferring substrate specificity. Cloning of the cDNA and site-directed mutagenesis studies.

Within the superfamily of homologous mammalian ribonucleases (RNases) 4 distinct families can be recognized. Previously, representative members of three of these have been cloned and studied in detail. Here we report on the cloning of a cDNA encoding a member of the fourth family, RNase PL3 from porcine liver. The deduced amino acid sequence showed the presence of a signal peptide, confirming the notion that RNase PL3 is a secreted RNase. Expression of the cDNA in Escherichia coli yielded 1.5 mg of purified protein/liter of culture. The recombinant enzyme was indistinguishable from the enzyme isolated from porcine liver based on the following criteria: amino acid analysis, N-terminal amino acid sequence, molecular weight, specific activity toward yeast RNA, and kinetic parameters for the hydrolysis of uridylyl(3',5')adenosine and cytidylyl(3',5')adenosine. Interestingly, the kinetic data showed that RNase PL3 has a very low activity toward yeast RNA, i.e., 2.5% compared to pancreatic RNase A. Moreover, using the dinucleotide substrates and homopolymers it was found that RNase PL3, in contrast to most members of the RNase superfamily, strongly prefers uridine over cytidine on the 5' side of the scissile bond. Replacement, by site-directed mutagenesis, of residues 36-42 of RNase PL3 by the corresponding ones from bovine pancreatic RNase A resulted in a large preferential increase in the catalytic efficiency for cytidine-containing substrates. This suggests that this region of the molecule contains some of the elements that determine substrate specificity.     

Characterization of Escherichia coli RNase PH.

We have previously shown that the orfE gene of Escherichia coli encodes RNase PH. Here we show that the OrfE protein (purified as described in the accompanying paper) (Jensen, K. F., Andersen, J. T., and Poulsen, P. (1992) J. Biol. Chem. 267, 17147-17152) has both the degradative and synthetic activities of RNase PH. This highly purified protein was used to characterize the enzymatic and structural properties of RNase PH. The enzyme requires a divalent cation and phosphate for activity, the latter property indicating that RNase PH is exclusively a phosphorolytic enzyme. Among tRNA-type substrates, the enzyme is most active against synthetic tRNA precursors containing extra residues following the -CCA sequence, and it can act on these molecules to generate mature tRNA with amino acid acceptor activity; 3'-phosphoryl-terminated molecules are not active as substrates. The equilibrium constant for RNase PH is near unity, suggesting that at the phosphate concentration present in vivo, the enzyme would participate in RNA degradation. The synthetic reaction of RNase PH displays a nonlinear response to increasing enzyme concentrations, and this may be due to self-aggregation of the protein. Higher order multimers of RNase PH could be detected by gel filtration at higher protein concentrations and by protein cross-linking. The possible role of RNase PH in tRNA processing is discussed.     

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.     

Identification of a cytidine-specific ribonuclease from chicken liver

Rapid RNA sequencing technology was used to determine if the base specificities of an RNase recently purified from chicken liver would prove useful for RNA sequence analysis. Escherichia coli 5 S [5'-32P]rRNA or yeast 5.8 S [5'-32P]rRNA was digested with the enzyme and this digest, along with digests derived from RNases of known specificity (U2, T1, T2) were subjected to electrophoresis through denaturing polyacrylamide slab gels. Following autoradiography, the banding patterns arising from the activity of each enzyme were compared, and the base specificity of the unknown RNase was established. The chicken liver RNase was found to have a marked preference for phosphodiester bonds containing cytidylic acid residues, a property which should make the enzyme useful for distinguishing between pyrimidines in RNA sequencing.     

Effects of C5 protein on Escherichia coli RNase P catalysis with a precursor tRNA(Phe) bearing a single mismatch in the acceptor stem

Escherichia coli RNase P, an RNA-processing enzyme that cleaves precursor tRNAs to generate the mature 5'-end, is composed of a catalytic component (M1 RNA) and a protein cofactor (C5 protein). In this study, effects of C5 protein on the RNase P catalysis with a precursor E. coli tRNA(Phe) having a single mismatch in the acceptor stem were examined. This mutant precursor unexpectedly generated upstream cleavage products at the -8 position as well as normal cleavage products at the +1 position. The cleavage at the -8 position was essentially effective only in the presence of C5 protein. Possible secondary structures for cleavage at the -8 position deviate significantly from the structures of the known RNase P substrates, implying that C5 protein can allow the enzyme to broaden the substrate specificity more than previously appreciated.     

Chloroplast ribonuclease P does not utilize the ribozyme-type pre-tRNA cleavage mechanism

The transfer RNA 5' maturation enzyme RNase P has been characterized in Bacteria, Archaea, and Eukarya. The purified enzyme from all three kingdoms is a ribonucleoprotein containing an essential RNA subunit; indeed, the RNA subunit of bacterial RNase P RNA is the sole catalytic component. In contrast, the RNase P activity isolated from spinach chloroplasts lacks an RNA component and appears to function as a catalytic protein. Nonetheless, the chloroplast enzyme recognizes a pre-tRNA substrate for E. coli RNase P and cleaves it as efficiently and precisely as does the bacterial enzyme. To ascertain whether there are differences in catalytic mechanism between an all-RNA and an all-protein RNase P, we took advantage of the fact that phosphodiester bond selection and hydrolysis by the E. coli RNase P ribozyme is directed by a Mg2+ ion coordinated to the nonbridging pro-Rp oxygen of the scissile bond, and is blocked by sulfur replacement of this oxygen. We therefore tested the ability of the chloroplast enzyme to process a precursor tRNA containing this sulfur substitution. Partially purified RNase P from spinach chloroplasts can accurately and efficiently process phosphorothioate-substituted pre-tRNAs; cleavage occurs exclusively at the thio-containing scissile bond. The enzymatic throughput is fivefold slower, consistent with a general chemical effect of the phosphorothioate substitution rather than with a metal coordination deficiency. The chloroplast RNase P reaction mechanism therefore does not involve a catalytic Mg2+ bonded to the pro-Rp phosphate oxygen, and hence is distinct from the mechanism of the bacterial ribozyme RNase P.     

Identification of a ribonuclease P-like activity from human KB cells.

An endoribonuclease which cleaves tRNA precursor molecules has been partially purified from human KB tissue culture cells. This activity is found in cytoplasmic fractions but is not detectable in the nucleoplasm. tRNA precursor molecules from both E. coli and KB cells are cleaved by this novel activity to produce 5' phosphate-terminated oligonucleotides. E coli RNAase P and the KB cell nuclease both make a single endonucleolytic scission in E. coli tRNATyr precursor, thereby separating the 41 extra nucleotides on the 5' end of the precursor molecule from the 5' terminal sequence of the mature tRNATyr molecule. The cleavage products generated from other E. coli tRNA precursors by the KB cell activity are identical in size to those produced by RNAase P. The KB cell endoribonuclease requires Mg2+ and a monovalent cation (Na+, K+, or NH4+) for function. The enzymatic activity has a broad pH optimum, centered near pH 8.0, and the activity is inhibited by tRNA. Several KB cell RNAs with long half-lives in vivo, including 5S and bulk 4S RNA, are not cleaved by this nuclease. The KB cell endoribonuclease resembles E. coli RNAase P in its substrate specificity, pH optimum, ion requirements, and sensitivity to tRNA. These properties and the cytoplasmic localization of the novel endoribonuclease indicate its involvement in the biosynthesis of KB cell tRNA.     

Separation and characterization of 5'- and 3'-tRNA processing nucleases from rat liver mitochondria

The 5'- and 3'-tRNA processing nucleases have been isolated from rat liver mitochondria. The two activities co-purified through heparin-agarose and phenyl-Sepharose columns and then efficiently separated on a DEAE-cellulose column. The 5' processing nuclease was found in the flow-through fraction, and the 3' processing activity eluted with 0.5 M KCl. Both enzymes were greater than 500-fold purified over the high speed supernatant of a mitoplast extract. The 159-base pre-tRNATyr used as a substrate in this study was synthesized in vitro and contained the Escherichia coli suppressor III tRNATyr plus a 49-base leader sequence and a 25-base trailing sequence. The 5' processing nuclease converted the pre-tRNATyr into two discrete RNA species, identified as the 5'-processed intermediate and the 5' flanking fragment, by endonucleolytic cleavage at the 5' end of the mature tRNATyr sequence. The 3' processing nuclease was inactive with the intact pre-tRNATyr as substrate but efficiently converted the 5'-processed intermediate to the mature tRNATyr, indicating an obligatory order of processing in which 5' maturation was necessary before cleavage by the 3' processing nuclease could occur. The mitochondrial enzymes exhibited optimal activity in the presence of about 2 mM Mg2+, but both enzymes were nearly fully active without addition of exogenous Mg2+ to the reaction mixtures. In contrast, a partially purified 5' processing endonuclease present in the postmitochondrial cytosolic fraction required higher [Mg2+] for activity, thus providing a means for differentiating between these similar enzyme activities obtained from the cytosolic and mitochondrial fractions.     

The influence of elongation-factor-Tu . GTP and anticodon-anticodon interactions on the anticodon loop conformation of yeast tRNATyr

The interactions of yeast tRNATyr, spin-labelled at position i6A-37 next to the anticodon, with EF-Tu . GTP and with Escherichia coli tRNAVal (which has a complementary anticodon) have been studied. The immobilization of the spin label upon ternary complex formation shows a conformational change of the anticodon region, although this part of tRNATyr is not in direct contact with the protein, as indicated by RNase T1 digestion. Upon anticodon-anticodon interaction, no conformational change of the anticodon loop of tRNATyr was observed.     

Heat labile ribonuclease HI from a psychrotrophic bacterium: gene cloning, characterization and site-directed mutagenesis.

The rnhA gene encoding RNase HI from a psychrotrophic bacterium, Shewanella sp. SIB1, was cloned, sequenced and overexpressed in an rnh mutant strain of Escherichia coli. SIB1 RNase HI is composed of 157 amino acid residues and shows 63% amino acid sequence identity to E.coli RNase HI. Upon induction, the recombinant protein accumulated in the cells in an insoluble form. This protein was solubilized and purified in the presence of 7 M urea and refolded by removing urea. Determination of the enzymatic activity using M13 DNA-RNA hybrid as a substrate revealed that the enzymatic properties of SIB1 RNase HI, such as divalent cation requirement, pH optimum and cleavage mode of a substrate, are similar to those of E.coli RNase HI. However, SIB1 RNase HI was much less stable than E.coli RNase HI and the temperature (T(1/2)) at which the enzyme loses half of its activity upon incubation for 10 min was approximately 25 degrees C for SIB1 RNase HI and approximately 60 degrees C for E.coli RNase HI. The optimum temperature for the SIB1 RNase HI activity was also shifted downward by 20 degrees C compared with that of E.coli RNase HI. Nevertheless, SIB1 RNase HI was less active than E.coli RNase HI even at low temperatures. The specific activity determined at 10 degrees C was 0.29 units/mg for SIB1 RNase HI and 1.3 units/mg for E.coli RNase HI. Site-directed mutagenesis studies suggest that the amino acid substitution in the middle of the alphaI-helix (Pro52 for SIB1 RNase HI and Ala52 for E.coli RNase HI) partly accounts for the difference in the stability and activity between SIB1 and E.coli RNases HI.     

Sequence changes in both flanking sequences of a pre-tRNA influence the cleavage specificity of RNase P.

The cleavage specificities of the RNase P holoenzymes from Escherichia coli and the yeast Schizosaccharomyces pombe and of the catalytic M1 RNA from E. coli were analyzed in 5'-processing experiments using a yeast serine pre-tRNA with mutations in both flanking sequences. The template DNAs were obtained by enzymatic reactions in vitro and transcribed with phage SP6 or T7 RNA polymerase. The various mutations did not alter the cleavage specificity of the yeast RNase P holoenzyme; cleavage always occurred predominantly at position G + 1, generating the typical seven base-pair acceptor stem. In contrast, the specificity of the prokaryotic RNase P activities, i.e. the catalytic M1 RNA and the RNase P holoenzyme from E. coli, was influenced by some of the mutated pre-tRNA substrates, which resulted in an unusual cleavage pattern, generating extended acceptor stems. The bases G - 1 and C + 73, forming the eighth base pair in these extended acceptor stems, were an important motif in promoting the unusual cleavage pattern. It was found only in some natural pre-tRNAs, including tRNA(SeCys) from E. coli, and tRNAs(His) from bacteria and chloroplasts. Also, the corresponding mature tRNAs in vivo contain an eight base pair acceptor stem. The presence of the CCA sequence at the 3' end of the tRNA moiety is known to enhance the cleavage efficiency with the catalytic M1 RNA. Surprisingly, the presence or absence of this sequence in two of our substrate mutants drastically altered the cleavage specificity of M1 RNA and of the E. coli holoenzyme, respectively. Possible reasons for the different cleavage specificities of the enzymes, the influence of sequence alterations and the importance of stacking forces in the acceptor stems are discussed.     

Directly photocrosslinked nucleotides joining transfer RNA to aminoacyl-tRNA synthetase in methionine and tyrosine systems

We have used ultraviolet photocrosslinking and 32P post-labeling to help define the contact surface between transfer RNAs and aminoacyl-tRNA synthetases for the methionine and tyrosine systems. Photocrosslinking between tRNAs and synthetases is shown to occur only in cognate complexes. The increased sensitivity of our procedures reduces the amounts of interacting macromolecules and permits lower ultraviolet light doses, thereby minimizing radiation damage. These procedures have detected crosslinks only within the 3'-terminal RNase T1 fragments in yeast tRNAMeti and Escherichia coli tRNATyr2; and although the photoadducts were unstable, we have identified the crosslinked nucleotides. These crosslinks occur at positions C74 and A76 in yeast tRNAMeti and position U64 in E. coli tRNATyr1&2 (conventional tRNA numbering system of Gauss & Sprinzl, 1981). This work demonstrates that even labile photocrosslinks can be exploited for mapping crosslinked nucleotides.     

Purification and characterization of the Pac1 ribonuclease of Schizosaccharomyces pombe.

The pac1+ gene of the fission yeast Schizosaccharomyces pombe is essential for viability and its overexpression induces sterility and suppresses mutations in the pat1+ and snm1+ genes. The pac1+ gene encodes a protein that is structurally similar to RNase III from Escherichia coli, but its normal function is unknown. We report here the purification and characterization of the Pac1 protein after overexpression in E. coli. The purified protein is a highly active, double-strand-specific endoribonuclease that converts long double-stranded RNAs into short oligonucleotides and also cleaves a small hairpin RNA substrate. The Pac1 RNase is inhibited by a variety of double- and single-stranded polynucleotides, but polycytidylic acid greatly enhances activity and also promotes cleavage specificity. The Pac1 RNase produces 5'-phosphate termini and requires Mg2+; Mn2+ supports activity but causes a loss of cleavage specificity. Optimal activity was obtained at pH 8.5, at low ionic strength, in the presence of a reducing agent. The enzyme is relatively insensitive to N-ethylmaleimide but is strongly inhibited by ethidium bromide and vanadyl ribonucleoside complexes. The properties of the Pac1 RNase support the hypothesis that it is a eukaryotic homolog of RNase III.     

Partial characterization of ribonuclease (RNase) activity from the isolated and solubilized brush border of Hymenolepis diminuta

The isolated brush border membrane of Hymenolepis diminuta contained ribonuclease (RNase) activity which was demonstrable using yeast RNA or synthetic homopolymers of adenylic, cytidylic, inosinic, or uridylic acids as substrates. Polyguanylic acid was not hydrolyzed by worm RNase. RNase activity was inhibited by EDTA and divalent cations as well as sulfhydryl blocking and reducing agents. Polyguanylic acid and DNA were also inhibitors of RNase activity; these compounds were not hydrolyzed, but inhibited the hydrolysis of other substrates, possibly by nonproductive substrate binding. Data suggested that RNase (endonuclease) was probably the major enzyme activity in the degradation of long chain polyribonucleotides at the work's surface, while phosphodiesterase (exonuclease) activity did not contribute significantly to the hydrolysis of these compounds.     

Catalysis by the RNA subunit of RNase P--a minireview

RNase P, an enzyme that contains both RNA and protein components, cleaves tRNA precursors to generate mature 5' termini. The catalytic activity of RNase P resides in the RNA component, with the protein cofactor affecting the rate of the cleavage reaction. The reaction is also influenced by the nature of the tRNA substrate.     

The pH-dependence of the Escherichia coli RNase HII-catalysed reaction suggests that an active site carboxylate group participates directly in catalysis

RNase HII specifically catalyses the hydrolysis of phosphate diester linkages contained within the RNA portion of DNA/RNA hybrids. The catalytic parameters of the enzyme derived from Escherichia coli BL21 have been measured using 5'-fluorescent oligodeoxynucleotide substrates containing embedded ribonucleotides. The products of the reaction and the chemistry of phosphate diester hydrolysis were assigned unequivocally using mass spectrometry. The pH-dependence of the catalytic parameters was measured under conditions of optimal magnesium ion concentration. The logarithm of the turnover number of the enzyme increases steeply with pH until a pH-independent region is reached close to neutrality. The slope of the pH-dependent region is 2, indicating that the catalytically proficient form of RNase HII is di-anionic. The pH-dependence of log 1/K(M) is a sigmoidal curve reaching a maximal value at higher pH, suggesting deprotonation of a residue stabilises substrate binding. Possible mechanisms for the RNase HII-catalysed reaction consistent with the pH-dependent behaviour of the enzyme are discussed. The active sites of RNase H enzymes contain a cluster of four strictly conserved carboxylate groups. Together, the data suggest a requirement for ionisation of an active site carboxylic acid for metal ion binding or correct positioning of metal ion(s) in the enzyme-substrate complex and a role for a second active site carboxylate in general base catalysis.     

The protein component of bacterial ribonuclease P flickers the metal ion response to the substrate shape preference of the ribozyme

The substrate shape specificity of the Escherichia coli ribonuclease P (RNase P) ribozyme depends on the concentration of magnesium ion. At 10 mM or more, it can cleave a hairpin substrate as well as a cloverleaf pre-transfer RNA (tRNA). The results showed, however, that the holo enzyme cleaved the hairpin substrate at low concentrations of magnesium ion. Considering that the homologous E. coli tRNAs are resistant to internal cleavage by the RNase P, the phenomena suggest that this catalytic activity might take part in the removing the mis-folded RNAs in the cell.