The DNase activity of RNase T and its application to DNA cloning.

RNase T is one of eight distinct 3'-->5' exoribonucleases present in Escherichia coli. The enzyme plays an important role in stable RNA metabolism, including tRNA end turnover and 3' maturation of most stable RNAs because it is the only RNase that can efficiently remove residues near a double-stranded (ds) stem. In the course of study of its specificity and mechanism, we found that RNase T also has single-strand-specific DNase activity. Purified RNase T degrades both single-stranded (ss)RNA and ssDNA in a non-processive manner. However, in contrast to its action on RNA, RNase T binds ssDNA much more tightly and shows less sequence specificity. As with RNA, DNA secondary structure strongly affects its degradation by RNase T. Thus, RNase T action on a dsDNA with a single-stranded 3'-extension efficiently generates blunt-ended DNA. This property of RNase T suggested that it might be a useful enzyme for blunt-ended DNA cloning. We show here that RNase T provides much higher cloning efficiency than the currently used mung bean nuclease.     

Substrate recognition and catalysis by the exoribonuclease RNase R

RNase R is a processive, 3' to 5' hydrolytic exoribonuclease that together with polynucleotide phosphorylase plays an important role in the degradation of structured RNAs. However, RNase R differs from other exoribonucleases in that it can by itself degrade RNAs with extensive secondary structure provided that a single-stranded 3' overhang is present. Using a variety of specifically designed substrates, we show here that a 3' overhang of at least 7 nucleotides is required for tight binding and activity, whereas optimum binding and activity are achieved when the overhang is 10 or more nucleotides in length. In contrast, duplex RNAs with no overhang or with a 4-nucleotide overhang bind extremely poorly to RNase R and are inactive as substrates. A duplex RNA with a 10-nucleotide 5' overhang also is not a substrate. Interestingly, this molecule is bound only weakly, indicating that RNase R does not simply recognize single-stranded RNA, but the RNA must thread into the enzyme with 3' to 5' polarity. We also show that ribose moieties are required for recognition of the substrate as a whole since RNase R is unable to bind or degrade single-stranded DNA. However, RNA molecules with deoxyribose or dideoxyribose residues at their 3' termini can be bound and degraded. Based on these data and a homology model of RNase R, derived from the structure of the closely related enzyme, RNase II, we present a model for how RNase R interacts with its substrates and degrades RNA.     

Purification and characterization of the Escherichia coli exoribonuclease RNase R. Comparison with RNase II

Escherichia coli RNase R, a 3' --> 5' exoribonuclease homologous to RNase II, was overexpressed and purified to near homogeneity in its native untagged form by a rapid procedure. The purified enzyme was free of nucleic acid. It migrated upon gel filtration chromatography as a monomer with an apparent molecular mass of approximately 95 kDa, in close agreement with its expected size based on the sequence of the rnr gene. RNase R was most active at pH 7.5-9.5 in the presence of 0.1-0.5 mm Mg(2+) and 50-500 mm KCl. The enzyme shares many catalytic properties with RNase II. Both enzymes are nonspecific processive ribonucleases that release 5'-nucleotide monophosphates and leave a short undigested oligonucleotide core. However, whereas RNase R shortens RNA processively to di- and trinucleotides, RNase II becomes more distributive when the length of the substrate reaches approximately 10 nucleotides, and it leaves an undigested core of 3-5 nucleotides. Both enzymes work on substrates with a 3'-phosphate group. RNase R and RNase II are most active on synthetic homopolymers such as poly(A), but their substrate specificities differ. RNase II is more active on poly(A), whereas RNase R is much more active on rRNAs. Neither RNase R nor RNase II can degrade a complete RNA-RNA or DNA-RNA hybrid or one with a 4-nucleotide 3'-RNA overhang. RNase R differs from RNase II in that it cannot digest DNA oligomers and is not inhibited by such molecules, suggesting that it does not bind DNA. Although the in vivo function of RNase R is not known, its ability to digest certain natural RNAs may explain why it is maintained in E. coli together with RNase II.     

Cleavage of T4 species I ribonucleic acid by Escherichia coli ribonuclease III.

T4 Species I RNA, a molecule 140 nucleotides in length with some structural features very much like a tRNA, is specifically cleaved by an enzymatic activity in Escherichia coli extracts to give three segments with 19, 48 and 73 nucleotides. We report the purification and characterization of the E. coli RNase which cleaves two 3' phosphodiester bonds of T4 Species I RNA. This reaction has many properties in common with those catalyzed by E. coli RNase III, although the optimal salt conditions for T4 Species I RNA cleavage differ significantly from those for other RNase III-catalyzed reactions. The reaction is not catalyzed by extracts from an E. coli strain lacking RNase III activity. Furthermore, T4 Species I RNA is cleaved by highly purified E. coli RNase III to yield the same three specific fragments. We conclude that this specific cleavage is due to the action of RNase III, and that the requirement for lower ionic strength may reveal further important properties about this RNA processing enzyme.     

Mechanism of action of Moloney murine leukemia virus RNase H III.

The mechanism of action of Moloney murine leukemia virus RNase H III was studied, utilizing the model substrate (A)n. (dT)n and polyacrylamide gel electrophoresis to assay enzyme activity. Examination by electrophoresis on 15% polyacrylamide gels in 7 M urea and on DEAE-cellulose paper in 7 M urea revealed that, early in a reaction with [3H](A)n. (dT)n as substrate, RNase H III generated products ranging in length from 80 to 90 nucleotides to less than 10 nucleotides and that after extended incubation the limit digest products generated were 3 to 15 nucleotides long. Product oligomers were of the following configuration: [5'-P, 3'-OH](A)n. RNase H III was shown to be an exonuclease requiring free ends in its substrate for activity by the inability to degrade RNA inserted in Escherichia coli ColE1 plasmid DNA. The enzyme was capable of attacking RNA in RNA-DNA hybrids in the 5' to 3' and 3' to 5' directions as demonstrated by the use of [3H, 5'-32P](A)600. (dT)n and cellulose-[3H](A)n. (dT)n. Rnase H III was random in its mode of action because addition of excess unlabeled (A)n. (dT)n to an ongoing reaction with [3H](A)n. (dT)n as substrate resulted in immediate inhibition of enzyme activity.     

Purification and characterization of the tRNA-processing enzyme RNase BN

RNase BN, a tRNA-processing enzyme previously shown to be required for the 3'-maturation of certain bacteriophage T4-encoded tRNAs, was overexpressed and purified to near homogeneity from Escherichia coli. The purified enzyme, which is free of nucleic acid, is an alpha(2)-dimer with a molecular mass of approximately 65 kDa. RNase BN displays a number of unusual catalytic properties compared with the other exoribonucleases of E. coli. The enzyme is most active at pH 6.5 in the presence of Co(2+) and high concentrations of monovalent salts. It is highly specific for tRNA substrates containing an incorrect residue within the universal 3'-CCA sequence. Thus, tRNA-CU and tRNA-CA are effective substrates, whereas intact tRNA-CCA, elongated tRNA-CCA-Cn, phosphodiesterase-treated tRNA, and the closely related tRNA-CC are essentially inactive as substrates. RNA or DNA oligonucleotides also are not substrates. These data indicate that RNase BN has an extremely narrow substrate specificity. However, since tRNA molecules with incorrect residues within the -CCA sequence are not normally produced in E. coli, the role of RNase BN in uninfected cells remains to be determined.     

Interplay among processing and degradative enzymes and a precursor ribonucleic acid in the selective maturation and maintenance of ribonucleic acid molecules

In order to understand why the first tRNA (tRNAGln) in the T4 tRNA gene cluster is not produced when T4 infects an RNase III- mutant of Escherichia coli, RNA metabolism was analyzed in RNase III- RNase P- (rnc, rnp) cells infected with bacteriophage T4. After such an infection a new dimeric precursor RNA molecule of tRNAGln and tRNALeu has been identified and analyzed. This molecule is structurally very similar to K band RNA that accumulates in rnc+ rnp strains. It is four nucleotides shorter than K RNA at the 5' end. This molecule like K RNA contains two RNase P processing sites at the 5' ends of each tRNA. Both sites are accessible to RNase P. However, while in the K RNA the site at the 5' end of tRNALeu (the site in the middle of the substrate) is more efficiently cleaved than the other site, this differential is even increased in the Ks (K like) molecule. This difference is sufficiently large that in vivo in the RNase III- strain the smaller precursor of tRNAGln is degraded rather than being matured to tRNAGln by RNase P. This information contributes to the elucidation of the key role of RNase III in the processing of T4 tRNA. It shows the dependence of RNase P activity at the 5' end of tRNAGln on a correct and specific cleavage by RNase III at a position six nucleotides proximal to the RNase P site, and it explains why in the absence of RNase III the first tRNA in the T4 tRNA cluster, tRNAGln, does not accumulate.     

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.     

Crystal structure of RNase T, an exoribonuclease involved in tRNA maturation and end turnover

The 3' processing of most bacterial precursor tRNAs involves exonucleolytic trimming to yield a mature CCA end. This step is carried out by RNase T, a member of the large DEDD family of exonucleases. We report the crystal structures of RNase T from Escherichia coli and Pseudomonas aeruginosa, which show that this enzyme adopts an opposing dimeric arrangement, with the catalytic DEDD residues from one monomer closely juxtaposed with a large basic patch on the other monomer. This arrangement suggests that RNase T has to be dimeric for substrate specificity, and agrees very well with prior site-directed mutagenesis studies. The dimeric architecture of RNase T is very similar to the arrangement seen in oligoribonuclease, another bacterial DEDD family exoribonuclease. The catalytic residues in these two enzymes are organized very similarly to the catalytic domain of the third DEDD family exoribonuclease in E. coli, RNase D, which is monomeric.     

Substrate shape specificity of E coli RNase P ribozyme is dependent on the concentration of magnesium ion

The bacterial RNase P ribozyme can accept a hairpin RNA with CCA-3' tag sequence as well as a cloverleaf pre-tRNA as substrate in vitro, but the details are not known. By switching tRNA structure using an antisense guide DNA technique, we examined the Escherichia coli RNase P ribozyme specificity for substrate RNA of a given shape. Analysis of the RNase P reaction with various concentrations of magnesium ion revealed that the ribozyme cleaved only the cloverleaf RNA at below 10 mM magnesium ion. At 10 mM magnesium ion or more, the ribozyme also cleaved a hairpin RNA with a CCA-3' tag sequence. At above 20 mM magnesium ion, cleavage site wobbling by the enzyme in tRNA-derived hairpin occurred, and the substrate specificity of the enzyme became broader. Additional studies using another hairpin substrate demonstrated the same tendency. Our data strongly suggest that raising the concentration of metal ion induces a conformational change in the RNA enzyme.     

RNase PH catalyzes a synthetic reaction, the addition of nucleotides to the 3' end of RNA.

Escherichia coli RNase PH is a phosphate-dependent exoribonuclease that has been implicated in the 3' processing of tRNA precursors. It degrades RNA chains in a phosphorolytic manner releasing nucleoside diphosphates as products. Here we show that RNase PH also catalyzes a synthetic reaction, the addition of nucleotides to the 3' termini of RNA molecules. The synthetic activity co-purifies with RNase PH throughout an extensive enrichment indicating that it is due to the same enzyme. The synthetic activity can incorporate all nucleoside diphosphates, but not triphosphates, and is strongly inhibited by Pi, but not PPi. Various RNA molecules stimulate nucleotide incorporation, and with tRNA the 3' end of the molecule serves a primer function. RNA chains as long as 40 residues can be synthesized in this system. As with polynucleotide phosphorylase, the synthetic activity of RNase PH apparently represents the reversal of the degradative reaction.     

Recognition of the 5' leader of pre-tRNA substrates by the active site of ribonuclease P

The bacterial tRNA processing enzyme ribonuclease P (RNase P) is a ribonucleoprotein composed of a approximately 400 nucleotide RNA and a smaller protein subunit. It has been established that RNase P RNA contacts the mature tRNA portion of pre-tRNA substrates, whereas RNase P protein interacts with the 5' leader sequence. However, specific interactions with substrate nucleotides flanking the cleavage site have not previously been defined. Here we provide evidence for an interaction between a conserved adenosine, A248 in the Escherichia coli ribozyme, and N(-1), the substrate nucleotide immediately 5' of the cleavage site. Specifically, mutations at A248 result in miscleavage of substrates containing a 2' deoxy modification at N(-1). Compensatory mutations at N(-1) restore correct cleavage in both the RNA-alone and holoenzyme reactions, and also rescue defects in binding thermodynamics caused by A248 mutation. Analysis of pre-tRNA leader sequences in Bacteria and Archaea reveals a conserved preference for U at N(-1), suggesting that an interaction between A248 and N(-1) is common among RNase P enzymes. These results provide the first direct evidence for RNase P RNA interactions with the substrate cleavage site, and show that RNA and protein cooperate in leader sequence recognition.     

Important 2'-hydroxyl groups in model substrates for M1 RNA, the catalytic RNA subunit of RNase P from Escherichia coli.

The role of 2'-hydroxyl groups in a model substrate for RNase P from Escherichia coli was studied using mixed DNA/RNA derivatives of such a substrate. The presence of the 2'-hydroxyl groups of nucleotides at positions -1 and -2 in the leader sequence and at position 1, as well as at the first C in the 3'-terminal CCA sequence, are important but not absolutely essential for efficient cleavage of the substrate by RNase P or its catalytic RNA subunit, M1 RNA. The 2'-hydroxyl groups in the substrate that are important for efficient cleavage also participate in the binding of Mg2+. An all-DNA external guide sequence (EGS) can efficiently render a potential substrate, derived from the model substrate, susceptible to cleavage by the enzyme or its catalytic RNA subunit. Furthermore, both DNA and RNA EGSs turn over during the reaction with RNase P in vitro. The identity of the nucleotide at position 1 in the substrate, the adjacent Mg(2+)-binding site in the leader sequence, and the junction of the single and double-stranded regions are the important elements in the recognition of model substrates, as well as in the identification of the sites of cleavage in those model substrates.     

Comparative Surface Structure of 16S Ribosomal Ribonucleic Acid of 30S Ribosomes of Procaryotic Cells

Ribonuclease T(1) treatment of 30S ribosomes of Escherichia coli converts a large region at the 3' OH end of 16S ribosomal ribonucleic acid (rRNA) to low-molecular-weight RNA. The final 25 nucleotides at the 3' terminus of the molecule emerge relatively intact, whereas most of the region "upstream," for about 150 nucleotides, is converted to oligonucleotides. Identical enzyme treatment generates a fragment of about 60 nucleotides from the middle of 16S rRNA (section D'). To determine whether there are similar sequences in other bacteria, which occupy similar accessible surface locations, we treated 30S ribosomes from Azotobacter vinelandii and Bacillus stearothermophilus with RNase T(1). In each case, a fragment of RNA about 25 nucleotides in length containing the 3' OH end of 16S rRNA and a fragment of about 60 nucleotides in length similar, but not identical, in oligonucleotide composition to section D' of E. coli 16S rRNA were obtained from nuclease-treated 30S ribosomes. These data indicate that, although the primary structure at the 3' end and the middle (section D') of the various 16S rRNA's is not completely conserved, their respective conformations are conserved. A number of identical oligonucleotides were found in the low-molecular-weight fraction obtained from RNase T(1)-treated E. coli, A. vinelandii, and B. stearothermophilus 30S ribosomes. These results show that identical RNase T(1)-sensitive sequences are present in all three bacteria. Hydrolysis of these regions leads to the production of the fragments 25 and 60 nucleotides in length.     

The reaction mechanism of ribonuclease II and its interaction with nucleic acid secondary structures.

Ribonuclease II is a processive 3'- to 5'-exoribonuclease in Escherichia coli with two binding sites: a catalytic site associated with the first few 3'-nucleotides and an anchor site binding nucleotides approximately 15 to 25 from the 3'-end. When RNase II degrades single-stranded helical poly(C), the enzyme-substrate complex dissociates at discrete intervals of 12 nucleotides. RNase II stalled at the last rC of single-stranded 3'-(rC)(n)(dC)(m) oligonucleotides. The more residues released, the faster the stalled complex dissociated and the less it inhibited RNase II activity, i.e. the enzyme-substrate association weakened progressively. Using phosphodiesterase I (PDE I) as a probe, a method was developed to identify cytidine residues in (32)P-oligonucleotides interacting with a protein. PAGE bands corresponding to nucleotides 1-6 from the 3'-end were consistent with interaction at the catalytic site, and following a gap, bands approximately 15 to 25 from the 3'-end, with anchor site association. Both 3' and 5' binding were necessary to maintain the complex. Of most significance, the original anchor site nucleotides remained fixed at the anchor site while the 3'-end was pulled, or threaded, through the catalytic site, i.e. the substrate did not 'slide' through the enzyme. DNA oligonucleotides with double-stranded stem-loops were good competitive inhibitors of RNase II. A 3'-single-stranded arm was essential, while optimal binding required both 5'- and 3'-arms. PDE I probing indicated that the nucleotides at the anchor site were specified by the spatial distance from the catalytic site, and on only one of the duplex strands. When degradation of a structured RNA paused or stopped, the RNase II-product commenced cycles of dissociation-reassociation. Duplex strand binding by RNase II made complex DNA or RNA structures accessible to degradation by other nucleases and further verified the PDE I footprinting method.     

Defining the enzyme binding domain of a ribonuclease III processing signal. Ethylation interference and hydroxyl radical footprinting using catalytically inactive RNase III mutants.

Ethylation interference and hydroxyl radical footprinting were used to identify substrate ribose-phosphate backbone sites that interact with the Escherichia coli RNA processing enzyme, ribonuclease III. Two RNase III mutants were employed, which bind substrate in vitro similarly as wild-type enzyme, but lack detectable phosphodiesterase activity. Specifically, altering glutamic acid at position 117 to lysine or alanine uncouples substrate binding from cleavage. The two substrates examined are based on the bacteriophage T7 R1.1 RNase III processing signal. One substrate, R1.1 RNA, undergoes accurate single cleavage at the canonical site, while a close variant, R1.1[WC-L] RNA, undergoes coordinate double cleavage. The interference and footprinting patterns for each substrate (i) overlap, (ii) exhibit symmetry and (iii) extend approximately one helical turn in each direction from the RNase III cleavage sites. Divalent metal ions (Mg2+, Ca2+) significantly enhance substrate binding, and confer stronger protection from hydroxyl radicals, but do not significantly affect the interference pattern. The footprinting and interference patterns indicate that (i) RNase III contacts the sugar-phosphate backbone; (ii) the RNase III-substrate interaction spans two turns of the A-form helix; and (iii) divalent metal ion does not play an essential role in binding specificity. These results rationalize the conserved two-turn helix motif seen in most RNase III processing signals, and which is necessary for optimal processing reactivity. In addition, the specific differences in the footprint and interference patterns of the two substrates suggest why RNase III catalyzes the coordinate double cleavage of R1.1[WC-L] RNA, and dsRNA in general, while catalyzing only single cleavage of R1.1 RNA and related substrates in which the scissle bond is within an asymmetric internal loop.     

Regions of RNase E important for 5'-end-dependent RNA cleavage and autoregulated synthesis

RNase E is an important regulatory enzyme that plays a key role in RNA processing and degradation in Escherichia coli. Internal cleavage by this endonuclease is accelerated by the presence of a monophosphate at the RNA 5' end. Here we show that the preference of E. coli RNase E for 5'-monophosphorylated substrates is an intrinsic property of the catalytically active amino-terminal half of the enzyme and does not require the carboxy-terminal region. This property is shared by the related E. coli ribonuclease CafA (RNase G) and by a cyanobacterial RNase E homolog derived from Synechocystis, indicating that the 5'-end dependence of RNase E is a general characteristic of members of this ribonuclease family, including those from evolutionarily distant species. Although it is dispensable for 5'-end-dependent RNA cleavage, the carboxy-terminal half of RNase E significantly enhances the ability of this ribonuclease to autoregulate its synthesis in E. coli. Despite similarities in amino acid sequence and substrate specificity, CafA is unable to replace RNase E in sustaining E. coli cell growth or in regulating RNase E production, even when overproduced sixfold relative to wild-type RNase E levels.     

Specificity of cleavage by ribonuclease III

The specificity of RNase III for various synthetic homopolymeric doublestranded RNA substrates have been examined. Although RNase III appears to cleave all homopolymeric RNA duplex structures, with Poly (U)·Poly (A) as the substrate, the enzyme cleaves the Poly (U) strand much faster than it cleaves the Poly (A) strand. Under conditions where the Poly (U) strand is quantitatively cleaved into acid-soluble fragments ranging in size between 5–8 nucleotides in length, the poly (A) strand is cleaved into large fragments 40–60 nucleotides in length. These results indicate that RNase III recognizes duplex RNA structures for binding, and makes single-stranded scissions and suggests that the enzyme has a preference for cleaving adjacent to UMP residues over AMP residues in polynucleotide chains.