An affinity adsorbent, 5'-adenylate-aminohexyl-sepharose. II. Purification and characterization of multi-forms of RNase T2

RNase T2 bound to an affinity adsorbent, 5'-adenylate-aminohexyl-Sepharose 4B, specifically at pH 4.5. The colorless enzyme was eluted only by the simultaneous addition of 2'(3')-AMP (1 mM) and NaCl (greater than 1 M) at pH 4.5. By applying this affinity chromatography to the purification of RNase T2, pure enzyme with a specific activity of 60 was obtained in only four steps and the yield was about 10 times higher than that of the previous purification method. This enzyme preparation was found to be heterogeneous in molecular weight and was separated into two fractions on Sephadex G-75 gel filtration. As the smaller enzyme with a molecular weight of 36,000 was identical with RNase T2 in every property examined, we tentatively designated the larger one with an apparent molecular weight of 80,000 as high molecular weight RNase T2 (RNase T2-L). RNase T2-L was still heterogeneous and was separated into five fractions, RNases T2-L 1-5, by repeated Sephadex G-150 gel filtration. The amino acid and carbohydrate analyses revealed that each of these fractions has a protein moiety in common with RNase T2 and the heterogeneities were due to the carbohydrate content, mainly galactose content.     

A thermoresistant mutant of ribonuclease T1 having three disulfide bonds

Molecular-dynamic calculations predict that, if Tyr24 and Asn84 are each replaced by a Cys residue, it should be possible to form a third disulfide bond in ribonuclease T1 (RNase T1) between these residues, with only minimal conformational changes at the catalytic site. The gene encoding such a mutant variant of RNase T1 (Tyr24----Cys24, Asn84----Cys84) was constructed by the cassette mutagenesis method using a chemically synthesized gene. In order to reduce the toxic effect of the mutant enzyme (RNase T1S) on an Escherichia coli host, we arranged for the protein to be secreted into the periplasmic space by using a vector that harbors a gene for an alkaline phosphatase signal peptide under the control of the trp promoter. The nucleolytic activity of RNase T1S toward pGpC was approximately the same as that of RNase T1 at 37 degrees C (pH 7.5). Moreover, at 55 degrees C, RNase T1S retained nearly 70% of its activity while the activity of the wild-type enzyme was reduced to less than 10%. RNase T1S was also more resistant to denaturation by urea than the wild-type enzyme. However, unlike RNase T1, RNase T1S was irreversibly and almost totally inactivated by boiling at 100 degrees C for 15 min.     

Increase in nucleolytic activity of ribonuclease T1 by substitution of tryptophan 45 for tyrosine 45

The preparation and analysis of a mutant ribonuclease (RNase) T1 which possesses higher nucleolytic activity than the wild-type enzyme are described. The gene for the mutant RNase T1 (Tyr45----Trp45), in which a single amino acid at the binding site of the guanine base has been changed, was constructed by the cassette mutangenesis method using a chemically synthesized gene [Ikehara, M. et al. (1986) Proc. Natl Acad. Sci. USA 83, 4695-4699]. In order to reduce the nucleolytic activity of the enzyme in vivo, this gene was expressed in Escherichia coli as a fused protein connected through methionine residues to other proteins at both the N- and C-termini. After liberation from the fused protein by cleavage with cyanogen bromide at the methionine junctions, the mutant RNase T1 was purified by column chromatography. The nucleolytic activity toward pGpC increased to 120% of that of wild-type RNase T1. The kinetic parameters of the mutant enzyme demonstrate that this higher nucleolytic activity is due to a higher affinity for the substrate, probably because of an increased stacking effect in the binding pocket for the guanine base. This mutant enzyme also possessed a higher nucleolytic activity against pApC than wild-type RNase T1.     

Conformational stability and activity of ribonuclease T1 and mutants. Gln25----Lys, Glu58----Ala, and the double mutant

Ribonuclease T1 (RNase T1) and mutants Gln25----Lys, Glu58----Ala, and the double mutant were prepared from a chemically synthesized gene, cloned and expressed in Escherichia coli. The wild-type RNase T1 prepared from the cloned gene was identical in every functional and physical property examined to RNase T1 prepared from Aspergillus oryzae. Urea and thermal unfolding experiments show that Gln25----Lys is 0.9 kcal/mol more stable and Glu58----Ala is 0.8 kcal/mol less stable than wild-type RNase T1. In the double mutant, these contributions cancel and the stability does not differ significantly from that of wild-type RNase T1. For the double mutant, the dependence of delta G on urea concentration is significantly greater than for wild-type RNase T1 or the single mutants. This suggests that the double mutant unfolds more completely in urea than the other proteins. The activity of Gln25----Lys is identical with that of wild-type RNase T1. The activities of Glu58----Ala and the double mutant are 7% of wild-type when GpC hydrolysis is measured (due to a 35-fold decrease in kcat), and 37% of wild-type when RNA hydrolysis is measured. Thus, Glu58 is important, but not essential to the activity of RNase T1.     

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.     

Ribonuclease F,a putative processing endoribonuclease from Escherichia coli

A new endoribonuclease activity, RNase F, was partially purified from $\text{Escherichia coli}$ cells. This activity can cleave a precursor RNA molecule (of Species 1), isolated from T4 infected cells, in a specific site. This activity is different from the other three know processing endoribonucleases of $\text{E. coli}$ RNase III, RNase E and RNase P.     

Tertiary structure of RNase Pch1 predicted from the model structure of RNase Ms and the crystal structure of RNase T1

In this paper we predict the structure of RNase Pch1 as modelled from the previously predicted structure of RNase Ms and the crystal structure of RNase T1 in the complex with 2GMP. The predicted structures and their initial energy minimized structural RNase T1 template are compared. The predicted structures of RNase Pch1 show, independent of their prediction form RNase Ms or T1, a higher structural similarity to RNase T1 than to RNase Ms, in agreement with higher sequence similarity and specificity — RNaes T1 and Pchl are specific for guanine whereas RNase Ms is base-unspecific with preference for guanine. Offprint requests to: W Saenger     

Purification and characterization of a non-S-RNase and S-RNases from styles of Japanese pear (Pyrus pyrifolia)

In this study we biochemically characterized stylar ribonucleases (RNases) of Japanese pear (Pyrus pyrifolia), which exhibits S-RNase-based gametophytic self-incompatibility. We separated the RNase fractions NS-1, NS-2, and NS-3 from stylar extracts of the cultivar Nijisseiki (S(2)S(4)). The RNase in each fraction was purified to homogeneity through a series of chromatographic steps. Chemical analysis of the proteins revealed that the basic RNases in the NS-2 and NS-3 fractions were the S(4)- and S(2)-RNases, respectively. Five additional S-RNases were purified from other cultivars. An acidic RNase in the NS-1 fraction was also purified from other cultivars, and identified as a non-S-allele-associated RNase (non-S-RNase). The non-S-RNase is composed of 203 amino acids, is non-glycosylated and is a N-terminal-pyroglutamylated enzyme of the RNase T(2) family. The substrate specificities and optimum pH levels of the non-S-RNase and S-RNases were similar. Interestingly, the specific activity of the non-S-RNase was 7.5-221-fold higher than those of the S-RNases when tolura yeast RNA was used as the substrate. The specific activity of the S(2)-RNase was 8.8-28.6-fold lower than those of the other S-RNases. These differences in specific activities among the stylar RNases are discussed.     

The Inhibition of Human Tumor Cell Proliferation by RNase Pol,a Member of the RNase T1 Family,from Pleurotus ostreatus

RNase Po1 is a guanylic acid-specific ribonuclease (a RNase T1 family RNase) from Pleurotus ostreatus. We determined the cDNA sequence encoding RNase Po1 and expressed RNase Po1 in Escherichia coli. A comparison of the enzymatic properties of RNase Po1 and RNase T1 indicated that the optimum temperature for RNase Po1 activity was 20 °C higher than that for RNase T1. An MTT assay indicated that RNase Po1 inhibits the proliferation of human neuroblastoma cells (IMR-32 and SK-N-SH) and human leukemia cells (Jurkat and HL-60). Furthermore, Hoechst 33342 staining showed morphological changes in HL-60 cells due to RNase Po1, and flow cytometry indicated the appearance of a sub-G1 cell population. The extent of these changes was dependent on the concentration of RNase Pol. We suggest that RNase Po1 induces apoptosis in tumor cells.     

Isolation, characterization, and primary structure of a base non-specific and adenylic acid preferential ribonuclease with higher specific activity from Trichoderma viride.

In order to elucidate the structure-function relationship of RNases belonging to the RNase T2 family (base non-specific and adenylic acid-preferential RNase), an RNase of this family was purified from Trichoderma viride (RNase Trv) to give three closely adjacent bands with RNase activity on slab-gel electrophoresis in a yield of 20%. The three RNases gave single band with the same mobility on slab-gel electrophoresis after endoglycosidase F digestion. The enzymatic properties including base specificity of RNase Trv were very similar to those of typical T2-family RNases such as RNase T2 from Aspergillus oryzae and RNase M from A. saitoi. The specific activity of RNase Trv towards yeast RNA was about 13-fold higher than that of RNase M. The complete primary structure of RNase Trv was determined by analyses of the peptides generated by digestion of reduced and carboxymethylated RNase Trv with Staphylococcus aureus V8 protease, lysylendopeptidase and alpha-chymotrypsin. The molecular weight of the protein moiety deduced from the sequence was 25,883. The locations of 10 half-cystine residues were almost superimposable upon those of other RNases of this family. The homologies between RNase Trv and RNase T2, RNase M, and RNase Rh (Rhizopus niveus) were 124, 132, and 92 residues, respectively. The sequences around three histidine residues, His52, His109, and His114, were highly conserved in these 4 RNases.     

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.     

T cells contain an RNase-insensitive inhibitor of APOBEC3G deaminase activity

The deoxycytidine deaminase APOBEC3G (A3G) is expressed in human T cells and inhibits HIV-1 replication. When transfected into A3G-deficient epithelial cell lines, A3G induces catastrophic hypermutation by deaminating the HIV-1 genome. Interestingly, studies suggest that endogenous A3G in T cells induces less hypermutation than would be expected. However, to date, the specific deaminase activity of endogenous A3G in human CD4+ T cells has not been examined directly. Here, we compared deaminase activity of endogenous and exogenous A3G in various human cell lines using a standard assay and a novel, quantitative, high-throughput assay. Exogenous A3G in epithelial cell lysates displayed deaminase activity only following RNase treatment, as expected given that A3G is known to form an enzymatically inactive RNA-containing complex. Surprisingly, comparable amounts of endogenous A3G from T cell lines or from resting or activated primary CD4+ T cells exhibited minimal deaminase activity, despite RNase treatment. Specific deaminase activity of endogenous A3G in H9, CEM, and other T cell lines was up to 36-fold lower than specific activity of exogenous A3G in epithelial-derived cell lines. Furthermore, RNase-treated T cell lysates conferred a dose-dependent inhibition to epithelial cell lysates expressing enzymatically active A3G. These studies suggest that T cells, unlike epithelial-derived cell lines, express an unidentified RNase-resistant factor that inhibits A3G deaminase activity. This factor could be responsible for reduced levels of hypermutation in T cells, and its identification and blockade could offer a means for increasing antiretroviral intrinsic immunity of T cells.     

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.     

Escherichia coli rnlA and rnlB compose a novel toxin-antitoxin system

RNase LS was originally identified as a potential antagonist of bacteriophage T4 infection. When T4 dmd is defective, RNase LS activity rapidly increases after T4 infection and cleaves T4 mRNAs to antagonize T4 reproduction. Here we show that rnlA, a structural gene of RNase LS, encodes a novel toxin, and that rnlB (formally yfjO), located immediately downstream of rnlA, encodes an antitoxin against RnlA. Ectopic expression of RnlA caused inhibition of cell growth and rapid degradation of mRNAs in ΔrnlAB cells. On the other hand, RnlB neutralized these RnlA effects. Furthermore, overexpression of RnlB in wild-type cells could completely suppress the growth defect of a T4 dmd mutant, that is, excess RnlB inhibited RNase LS activity. Pull-down analysis showed a specific interaction between RnlA and RnlB. Compared to RnlA, RnlB was extremely unstable, being degraded by ClpXP and Lon proteases, and this instability may increase RNase LS activity after T4 infection. All of these results suggested that rnlA-rnlB define a new toxin-antitoxin (TA) system.     

The subsite structures of guanine-specific ribonucleases and a guanine-preferential ribonuclease. Cleavage of oligoinosinic acids and poly I

In order to estimate the size of the active site of guanylic acid specific RNases (RNase T1 from Aspergillus oryzae and RNase St from Streptomyces erythreus) and guanine-preferential RNase (RNase Ms from A. saitoi), the depolymerization reaction of oligoinosinic acid, (Ip)nI greater than p, having various chain lengths was studied. The kinetic parameters for depolymerization of oligoinosinic acids, (pKm, log V and log V/Km) by the three RNases increased with increase of the chain length of the substrates, and became almost constant at n = 2 or more. Thus, the size of the active site of RNase T1, RNase St, and RNase Ms was estimated to be three nucleotides in length.     

Characterization of a unique nonsecretory ribonuclease from urine of pregnant women.

We have reported previously [Sakakibara, et al. (1991) Chem. Pharm. Bull. 39, 146-149] that a protein purified from a partially purified pharmaceutical preparation of human chorionic gonadotropin (a urinary protein preparation from pregnant women) is a unique nonsecretory ribonuclease (RNase)-like protein on the basis of its amino terminal sequence homology. We purified the protein further from the same materials by gel filtration and reversed-phase column chromatographies with RNase activity as an index. The purified protein was designated RNase UpI-2. The catalytic activity and its sensitivity to inhibition by divalent cations suggest that the protein is related to nonsecretory RNase. The estimated molecular weight of RNase UpI-2 (38 kDa) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was significantly higher than that of urinary nonsecretory RNases (13 to 19 kDa) reported so far. After trifluoromethanesulfonic acid treatment, the molecular weight of RNase UpI-2 was reduced and approached that of nonsecretory RNase, which indicated that the protein contains a significant amount of carbohydrate (approximately 50%). RNase UpI-2 was immunoreactive with antibodies to a nonsecretory RNase, RNAase 1 [Yasuda et al. (1988) Biochim. Biophys. Acta 965, 185-194]. By immunoblot analysis of the protein freshly prepared from various urine samples, it was shown that a considerable amount of RNase UpI-2 is present in urine of pregnant women, but only a trace of RNase UpI-2, if any, was detected in urine of nonpregnant women and men. These results suggest the possibility that RNase UpI-2 may have been formed via a specific protein modification in pregnant women.     

Crystal structures of the ribonuclease MC1 mutants N71T and N71S in complex with 5'-GMP: structural basis for alterations in substrate specificity

Ribonuclease MC1 (RNase MC1), isolated from bitter gourd seeds, is a uridine specific RNase belonging to the RNase T2 family. Mutations of Asn71 in RNase MC1 to the amino acids Thr (N71T) and Ser (N71S) in guanosine preferential RNases altered the substrate specificity from uridine specific to guanosine specific, as shown by the transphosphorylation of diribonucleoside monophosphates [Numata, T., et al. (2001) Biochemistry 40, 524-530]. To elucidate the structural basis for the alteration of substrate specificity, crystal structures of the RNase MC1 mutants N71T and N71S, free or complexed with 5'-GMP, were determined at resolutions higher than 2 A. In the N71T-5'-GMP and N71S-5'-GMP complexes, the guanine moiety was, as in the case of the uracil moiety bound to wild-type RNase MC1, firmly stabilized in the B2 site by an extensive network of hydrogen bonds and hydrophobic interactions. Structure comparisons showed that mutations of Asn71 to Thr or Ser cause an enlargement of the B2 site, which then make it feasible to insert a guanine base into the B2 site of mutants N71T and N71S. This binding further allows for hydrogen bonding interaction of the side chain hydroxyl groups of Thr71 or Ser71 with the N7 atom of the guanine base. The mode of guanine binding of mutants N71T and N71S was found to be essentially identical to that of a guanosine preferential RNase NW from Nicotiana glutinosa. In particular, hydrogen bonds between the N7 atom of the guanine base and the hydroxyl groups of the amino acids at position 71 (RNase MC1 numbering) were completely conserved in three guanosine preferential enzymes, thereby indicating that the hydrogen bond may play an essential role in guanine binding in guanosine preferential RNases in the RNase T2 family. Consequently, it can be concluded that amino acids at position 71 (RNase MC1 numbering) serve as one of the determinants for substrate specificity (or preference) in the RNase T2 fimily by changing the size and shape of the B2 site.     

An affinity adsorbent, 5'-adenylate-aminohexyl-sepharose. I. Purification and properties of two forms of RNase U2

An affinity adsorbent, 5'-adenylate-aminohexyl-Sepharose 4B, was prepared by the periodate oxidation of AMp followed by coupling and condensation with amino-hexyl-Sepharose 4B. RNase U2, a purine-specific RNase, was specifically bound to this adsorbent at pH 4.5 and eluted critically at pH 5.9 in the presence of 1 M NaCl, corresponding to the pH dependence of the binding of 2'-AMP to RNase U2. By using this affinity chromatography as a main tool, a simplified and effective purification method for RNase U2 was established with a high yield of 58%. Another form of RNase U2 with low specific activity, named RNase U2-B, was eluted at a slightly higher pH from this adsorbent. RNase U2-B was indistinguishable from the original enzyme (RNase U2-A) in base specificity, affinity for ApA, molecular weight and amino acid composition, but was clearly different in specific activity, molecular activity for ApA, isoelectric point and conformation of molecule. This affinity adsorbent is also effective for the detection or isolation of small amounts of base-specific RNases in crude cell extract.