Gel retardation analysis of E. coli M1 RNA-tRNA complexes.

We have analyzed complexes between tRNA and E. coli M1 RNA by electrophoresis in non-denaturing polyacrylamide gels. The RNA subunit of E. coli RNase P formed a specific complex with mature tRNA molecules. A derivative of the tRNA(Gly), endowed with the intron of yeast tRNA(ile) (60 nt), was employed to improve separation of complexed and unbound M1 RNA. Binding assays with tRNA(Gly) and intron-tRNA(Gly) as well as analysis of intron-tRNA/M1 RNA complexes on denaturing gels showed that one tRNA is bound per molecule of M1 RNA. A tRNA carrying a truncation as small as the 5'-nucleotide had a strongly reduced affinity to M1 RNA and was also a weak competitor in the cleavage reaction, suggesting that nucleotide +1 is a major determinant of tRNA recognition and that the thermodynamically stable tRNA-M1 RNA complex is relevant for enzyme function. Binding was shown to be dependent on the M1 RNA concentration in a cooperative fashion. Only a fraction of M1 RNAs (50-60%) readily formed a complex with intron-tRNA(Gly), indicating that distinct conformational subpopulations of M1 RNA may exist. Formation of the M1 RNA-tRNA(Gly), complex was very similar at 100 mM Mg++ and Ca++, corroborating earlier data that Ca++ is competent in promoting M1 RNA folding and tRNA binding. Determination of apparent equilibrium constants (app Kd) for tRNA(Gly) as a function of the Mg++ concentration supports an uptake of at least two additional Mg++ ions upon complex formation. At 20-30 mM Mg++, highest cleavage rates but strongly reduced complex formation were observed. This indicates that tight binding of the tRNA to the catalytic RNA at higher magnesium concentrations retards product release and therefore substrate turnover.     

Self-cleavage of p2Sp1 RNA with Mg2+ and non-ionic detergent (Brij 58)

The precursor of an RNA molecule from T4-infected E. coli cells (p2Sp1 RNA) has the capacity to cleave itself at specific positions [(UpA (139-140) and CpA (170-171)], within a putative loop and stem structure. This sequence-specific cleavage requires at least a monovalent cation and non-ionic detergents. We studied the self-cleavage reaction of an RNA fragment (GUUUCGUACAAAC) (R1) with the sequence corresponding to the p2Sp1 RNA in the presence of Mg2+ and non-ionic detergents. It requires Mg2+ and is aided by a non-ionic detergent, Brij 58. The cleavage reaction is time, temperature, and pH-dependent. The cleavage occurs at the phosphodiester bond between UpA and CpA on the RNA fragment (GUUUCGUACAAAC) (R1). Furthermore, the maximum of cleavage of R1 occurs at a very low Mg2+ concentration (< or = 5 mM).     

Dependence of M1 RNA substrate specificity on magnesium ion concentration.

We have constructed a plasmid expressing E. coli M1 RNA, the catalytic RNA subunit of ribonuclease P, under the control of a phage T7 promoter. The active M1 RNA species synthesized in vitro by T7 RNA polymerase from this vector was reacted with the tRNA(Gln) - tRNA(Leu) precursor RNA (Band K) encoded by phage T4. Only the tRNA(Leu) moiety of this dimeric precursor RNA contains the 3' terminal C-C-A sequence common to all tRNAs. We observed that protein-free M1 RNA was capable of processing the precursor RNA at the 5' ends of both tRNA tRNA sequences. The rate of cleavage of the tRNA(Gln) sequence was more strongly dependent on [Mg2+] than that of tRNA(Leu), increasing severalfold between 100 and 500 mM Mg2+, conditions under which the rate of cleavage at the tRNA(Leu) sequence was constant.     

Artificial self-cleaving molecules consisting of a tRNA precursor and the catalytic RNA of RNase P.

We synthesized two types of chimeric RNAs between the catalytic RNA subunit of RNase P from Escherichia coli (M1 RNA) and a tRNA precursor (pre-tRNA); one had pre-tRNA at the 3' side to the M1 RNA (M1 RNA-pre-tRNA). The second had pre-tRNA at the 5' side of the M1 RNA (pre-tRNA-M1 RNA). Both molecules were self-cleaving RNAs. The self-cleavage of M1 RNA-pre-tRNA occurred at the normal site (5'-end of mature tRNA sequence) and proceeded under the condition of 10 mM Mg2+ concentration. This reaction at 10 mM Mg2+ was an intramolecular reaction (cis-cleavage), while, at 40 mM and 80 mM Mg2+, trans-cleavage partially occurred. The self-cleavage rate was strictly affected by the distance between the M1 RNA and the pre-tRNA in the molecule. The self-cleavage of pre-tRNA-M1 RNA occurred mainly at three sites within the mature tRNA sequence. This cleavage did not occur at 10 mM Mg2+. Use of M1 RNA-pre-tRNA molecule for the in vitro evolution of M1 RNA is discussed.     

Iron(II)-ethylenediaminetetraacetic acid catalyzed cleavage of RNA and DNA oligonucleotides: similar reactivity toward single- and double-stranded forms

Fe(II)-EDTA catalyzes the cleavage of nucleic acids with little or no base-sequence specificity. We have now studied the preference of this reagent in catalyzing the cleavage of single- versus double-stranded nucleic acid structures. Three RNA and two DNA molecules, each expected to contain both single- and double-stranded regions, were synthesized and their structures characterized by enzymatic digestion using secondary structure specific nucleases. Fe(II)-EDTA catalyzed nearly uniform strand scission along the entire length of each molecule; no correlation with secondary structure was observed. The homopolymer sequence dA30:dT30, embedded in a mixed-sequence context to promote exact register of the homopolymer tract, was cleaved to an extent similar to that of flanking sequences. The reactions were relatively insensitive to K+, Na+, and Mg2+ in the range 10-100 mM and were quenched by Tris-HCl buffer. We conclude that the Fe(II)-EDTA-catalyzed strand scission reaction does not discriminate between typical single- and double-stranded regions, which simplifies the interpretation of experiments in which the reaction is used to probe the tertiary structure of RNA molecules [Latham, J. A., & Cech, T. R. (1989) Science 245, 276-282].     

In vitro selection of RNAs that undergo autolytic cleavage with Pb2+.

An in vitro selection method has been developed to obtain RNA molecules that specifically undergo autolytic cleavage reactions by Pb2+ ion. The method utilizes a circular RNA intermediate which is regenerated following the cleavage reaction to allow amplification and multiple cycles of selection. Pb2+ is known to catalyze a specific cleavage reaction between U17 and G18 of yeast tRNA(Phe). Starting from pools of RNA molecules which have a random distribution of sequences at nine or ten selected positions in the sequence of yeast tRNA(Phe), we have isolated many RNA molecules that undergo rapid and specific self-cleavage with Pb2+ at a variety of different sites. Terminal truncation experiments suggest that most of these self-cleaving RNA molecules do not fold like tRNA. However, two of the variants are cleaved rapidly with Pb2+ at U17 even though they lack the highly conserved nucleotides G18 and G19. Both specific mutations and terminal truncation experiments suggest that the D and T loops of these two variants interact in a manner similar to that of tRNA(Phe) despite the absence of the G18U55 and G19C56 tertiary interactions. A model for an alternate tertiary interaction involving a U17U55 pair is presented. This model may be relevant to the structure of about 100 mitochondrial tRNAs that also lack G18 and G19. The selection method presented here can be directly applied to isolate catalytic RNAs that undergo cleavage in the presence of other metal ions, modified nucleotides, or sequence-specific nucleases.     

Purification and characterization of RNase P from Clostridium sporogenes

RNase P is a multi-subunit enzyme responsible for the accurate processing of the 5' terminus of all tRNAs. The RNA subunit from Clostridium sporogenes has been partially purified and characterized. The RNA is approximately 400 nucleotides long and makes a precise endonucleolytic cleavage at the mature 5' terminus of tRNA. The RNA requires moderate concentrations of Mg2+ (20 mM) and relatively high concentrations of NH4Cl (800 mM) for optimal activity. Mn2+ effectively substitutes for Mg2+ at 2 mM. Zn2+, Ni2+, Ca2+, and Co2+ are ineffective at stimulating activity. Monovalent ions are, in general, more effective the greater the ionic radius (NH+4 greater than Cs greater than Rb greater than K greater than Na). In contrast to the activity of Bacillus subtilis, C. sporogenes RNase P RNA is significant more active in (NH4)2SO4 than in NH4Cl.     

Complexity in orchestration of chemical groups near different cleavage sites in RNase P RNA mediated cleavage

RNase P mediated cleavage of the tRNA(His) precursor does not rely on the formation of the "+73/294 interaction" to give the correct cleavage product, i.e. cleavage at -1, while other tRNA precursors that are cleaved at the canonical site +1 do. A previous model, here referred to as the "2'OH-model", predicts that the 2'OH at the canonical cleavage site would affect cleavage at -1. Here we used model RNA hairpin substrates mimicking the structural architecture of the tRNA(His) precursor cleavage site to investigate the role of 2'OH with respect to ground state binding and rate of cleavage in the presence and absence of the +73/294 interaction. Our data emphasize the importance of the 2'OH in the immediate vicinity of the scissile bond. Moreover, introduction of 2'H at the cleavage site did not affect cleavage at an alternative cleavage site to any significant extent. Our findings are therefore inconsistent with the 2'OH model. We favor a model where the 2'OH at the cleavage site influence Mg2+ binding in its vicinity, however we do not exclude the possibility that the 2'OH at the cleavage site interacts with RNase P RNA. Studying the importance of the 2'OH at different cleavage sites also indicated a higher dependence on the 2'OH at the cleavage site in the absence of the +73/294 interaction than in its presence. Finally, we provide data suggesting that N3 of U at position -1 in the substrate is most likely not involved in an interaction with RNase P RNA.     

Cleavage of DNA with methidiumpropyl-EDTA-iron(II): reaction conditions and product analyses

The synthesis of methidiumpropyl-EDTA (MPE) is described. The binding affinities of MPE, MPE.Ni(II), and MPE.Mg(II) to calf thymus DNA are 2.4 X 10(4) M-1, 1.5 X 10(5) M-1, and 1.2 X 10(5) M-1, respectively, in 50 mM NaCl, pH 7.4. The binding site size is two base pairs. MPE.Mg(II) unwinds PM2 DNA 11 +/- 3 degrees per bound molecule. MPE.Fe(II) in the presence of O2 efficiently cleaves DNA and with low sequence specificity. Reducing agents significantly enhance the efficiency of the cleavage reaction in the order sodium ascorbate greater than dithiothreitol greater than NADPH. At concentrations of 0.1-0.01 microM in MPE.Fe(II) and 10 microM in DNA base pairs, optimum ascorbate and dithiothreitol concentrations for DNA cleavage are 1-5 mM. Efficient cleavage of DNA (10 microM in base pairs) with MPE.Fe(II) (0.1-0.01 microM) occurs over a pH range of 7-10 with the optimum at 7.4 (Tris-HCl buffer). The optimum cleavage time is 3.5 h (22 degrees C). DNA cleavage is efficient in a Na+ ion concentration range of 5 mM to 1 M, with the optimum at 5 mM NaCl. The number of single-strand scissions on supercoiled DNA per MPE.Fe(II) under optimum conditions is 1.4. Metals such as Co(II), Mg(II), Ni(II), and Zn(II) inhibit strand scission by MPE. The released products from DNA cleavage by MPE.Fe(II) are the four nucleotide bases. The DNA termini at the cleavage site are 5'-phosphate and roughly equal proportions of 3'-phosphate and 3'-(phosphoglycolic acid). The products are consistent with the oxidative degradation of the deoxyribose ring of the DNA backbone, most likely by hydroxy radical.     

Cooperativity in low-affinity Mg2+ binding to tRNA

The Pb2+-catalyzed cleavage of tRNAPhe has been used to probe the effect of Na+ and Mg2+ binding to tRNA. Na+ is a noncompetitive inhibitor of the Pb2+-catalyzed cleavage. Millimolar Mg2+ is also a noncompetitive inhibitor. Analysis of the Mg2+ data show that at least two sites are involved in binding and that there is an interaction between the sites (cooperativity). Low-affinity Mg2+ binding is thus different from "weak" and "strong" Mg2+ binding to tRNA characterized previously. We postulate that the alterations induced by low-affinity Mg2+ binding in tRNA mimic to some extent those brought about in RNA by the interaction with a protein factor and that at appropriate [Mg2+] the whole structure of tRNA is able to respond in a concerted way to a signal from the environment such as aminoacylation or codon binding.     

Stimulatory effect of ascorbate on iron transfer from bleomycin to apotransferrin

The chemotherapeutic agent, bleomycin, forms a 1:1complex with both Fe(III) and Fe(II). The rate offerric ion transfer from bleomycin toapotransferrin is rather slow. However, when ascorbate was added toFe(III)-bleomycin priorto exposure to apotransferrin, the transfer rate was markedly increased. Ascorbatereadilyreduces Fe(III)-bleomycin to Fe(II)-bleomycin. A second order rate constant of 2.4 mM min wasestimated for this reaction. Fe(II)-bleomycinimmediately combines with O 2 , generating the so-called'acti-vatedbleomycin' complex. The data suggest that a reduced form of iron-bleomycin more readilydonatesits iron ion to apotransferrin. Reoxidation of ferrous ions, andFe(III)-transferrin formation occur rapidly.     

Cleavage properties of an oligonucleotide containing a bridged internucleotide 5'-phosphorothioate RNA linkage.

An oligonucleotide has been synthesized that contains a single bridging 5'-phosphorothioate at an RNA linkage (5'-ApCpGpGpTpCpTprCpsApCpGpApGpC-3'). This new phosphodiester linkage is found to be particularly susceptible to cleavage when compared with the corresponding oxo, deoxy and thiodeoxy derivatives. Divalent metal cations were observed to dramatically increase the cleavage rate. The products of the cleavage under a variety of conditions are a 5'-thiol-containing fragment (6mer) and a 2',3'-cyclic phosphate-containing fragment (8mer). The pseudo-first order rate constant, kobs, for cleavage at pH 7.5 (50 mM Tris-HCI) in the presence of 5 mM EDTA is 1.5 x 10(-4)/min. In the presence of 5 mM metal dichloride and 50 mM Tris-HCI, pH 7.5, the relative cleavage rate enhancements are 10, 24, 71, 98, 370 and 3400 for Mg2+, Ca2+, Mn2+, Co2+, Zn2+ and Cd2+ respectively. The rate enhancements correlate well with Pearson's HSAB principle, suggesting that cleavage is mediated in part by coordination of the metal to the 5'-mercapto leaving group. RNA linkages containing bridging 5'-phosphorothioates should prove valuable for studying the mechanistic details of a variety of RNA cleaving agents, such as ribozymes.     

P-3A and (−)-desacetamido P-3A: demonstration and Study of their effective functional cleavage of duplex DNA

A study of the Fe(II) complexes of P-3A (1) and (−)-desacetamido P-3A (2) abilities to cleave duplex DNA was conducted through examination of single-strand and double-strand cleavage of supercoiled φX174 RFI DNA (Form I) in the presence of O₂ to produce relaxed (Form II) and linear (Form III) DNA, respectively. Like Fe(II)-bleomycin A₂ and deglycobleomycin A₂, Fe(II)-1 and 2 effectively produced both single- and double-strand cleavage of supercoiled φX174 DNA. Unlike Fe(II)-bleomycin A₂ or deglycobleomycin A₂, Fe(II)-1 and 2 were found to cleave duplex w794 DNA with no discemible sequence selectively suggesting that the polynucleotide recognition of the C-terminus tetrapeptide S subunit of the bleomycins including the bithiazole may dominate the bleomycin A₂ DNA cleavage selectivity.     

Several regions of a tRNA precursor determine the Escherichia coli RNase P cleavage site.

The RNase P cleavage reaction was studied as a function of the number of base-pairs in the acceptor-stem and/or T-stem of a natural tRNA precursor, the tRNA(Tyr)Su3 precursor. Our data suggest that the location of the Escherichia coli RNase P cleavage site does not depend merely on the lengths of the acceptor-stem and T-stem as previously suggested. Surprisingly, we find that precursors with only four base-pairs in the acceptor-stem are cleaved by M1 RNA and by holoenzyme. Furthermore, we show that both disruption of base-pairing, and alteration of the nucleotide sequence (without disruption of base-pairing) proximal to the cleavage site result in aberrant cleavage. Thus, the identity of the nucleotides near the cleavage site is important for recognition of the cleavage site rather than base-pairing. The important nucleotides are those at positions -2, -1, +1, +72, +73 and +74. We propose that the nucleotide at position +1 functions as a guiding nucleotide. These results raise the possibility that Mg2+ binding near the cleavage site is dependent on the identity of the nucleotides at these positions. In addition, we show that disruption of base-pairing in the acceptor-stem affects both Michaelis-Menten constants, Km and kcat.     

tRNA(Phe) binds aminoglycoside antibiotics.

Aminoglycoside antibiotics have recently been found to bind to a variety of unrelated RNA molecules, including sequences that are important for retroviral replication. We report the binding of neomycin B, kanamycin A, and Neo-Neo (a synthetic neomycin-neomycin dimer) to tRNA(Phe). Using thermal denaturation studies, fluorescence spectroscopy, Pb2+-mediated tRNA(Phe) cleavage, and gel mobility shift assays, we have established that aminoglycosides interact with yeast tRNA(Phe) and are likely to induce a conformational change. Thermal denaturation studies revealed that aminoglycosides have a substantial stabilizing effect on tRNA(Phe) secondary and tertiary structures, much greater than the stabilization effect of spermine, an unstructured polyamine. Aminoglycoside-induced inhibition of Pb2+-mediated tRNA(Phe) cleavage yielded IC50 values of: 5 microM for Neo-Neo, 100 microM for neomycin B, > 1 mM for kanamycin A, and > 10 mM for spermine. Enzymatic and chemical footprinting indicate that the anticodon stem as well as the junction of the TpsiC and D loops are preferred aminoglycoside binding sites.     

Iron(III)-mediated photolysis of outer arm dynein ATPase from sea urchin sperm flagella

Irradiation of outer arm dynein ATPase from sea urchin sperm tail flagella at 365-410 nm in the presence of Fe(III)-gluconate complex and ATP produces photolytic cleavage at two distinct sites on the beta heavy chain, located approximately 250 and approximately 230 kDa from its amino terminus. The former cut is close to or identical with the V1 site of the vanadate-mediated photocleavage (Gibbons, I.R., Lee-Eiford, A., Mocz, G., Phillipson, C. A., Tang, W.-J.Y., and Gibbons, B.H. (1987) J. Biol. Chem. 262, 2780-2786. The rate of photolysis shows a hyperbolic dependence on Fe(III)-gluconate concentration with half-maximal rate occurring at 23 microM at pH 6.3. In the presence of 0.1-0.5 mM Fe(III)-gluconate-ATP, approximately 58% of the beta chain becomes cleaved with a half-time of about 34 s; the remainder of the beta chain and almost all of the alpha chain are resistant to cleavage. This photolytic cleavage of the beta chain is accompanied by an approximately parallel loss of the dynein latent ATPase activity, whereas the Triton-activated ATPase is lost to a somewhat greater extent. Mg2+ concentrations above approximately 3 mM inhibit photolysis. Substitution of ADP for ATP changes the pattern of cleavage so that both the alpha and beta heavy chain undergo scission but at the 250-kDa site only. AMP, adenyl-5'-yl imidodiphosphate and Fe(II) do not support cleavage at either site. Trivalent rhodium-ATP complexes, as models of MgATP, can also catalyze photolysis of the beta chain at the 250-kDa site. These results suggest that photolysis results from the activation of an Fe(III)-ATP complex bound to the hydrolytic ATP binding site of the beta chain and that both Fe(III) cleavage sites are located close to the nucleotide binding site in the tertiary folding of the beta heavy chain. The cleavage reaction possibly involves initial photoreduction of Fe(III) bound at the Mg2+ binding site in the dynein.Fe.ATP complex, followed by covalent modification of an amino acid side chain that leads to eventual peptide scission.     

Bleomycin may be activated for DNA cleavage by NADPH-cytochrome P-450 reductase

In the presence of NADPH and O2, NADPH-cytochrome P-450 reductase was found to activate Fe(III)-bleomycin A2 for DNA strand scission. Consistent with observations made previously when cccDNA was incubated in the presence of bleomycin and Fe(II) + O2 or Fe(III) + C6H5IO, degradation of DNA by NADPH-cytochrome P-450 reductase activated Fe(III)-bleomycin A2 produced both single- and double-strand nicks with concomitant formation of malondialdehyde (precursors). Cu(II)-bleomycin A2 also produced nicks in SV40 DNA following activation with NADPH-cytochrome P-450 reductase, but these were not accompanied by the formation of malondialdehyde (precursors). These findings confirm the activity of copper bleomycin in DNA strand scission and indicate that it degrades DNA in a fashion that differs mechanistically from that of iron bleomycin. The present findings also-establish the most facile pathways for enzymatic activation of Fe(III)-bleomycin and Cu(II)-bleomycin, provide data concerning the nature of the activated metallobleomycins, and extend the analogy between the chemistry of cytochrome P-450 and bleomycin.     

Altering the specificity of restriction endonuclease: effect of replacing Mg2+ with Mn2+.

In the presence of 100 mM Tris buffer (pH 7.5) and 1-10 mM Mg2+ EcoRI endonuclease cleaves DNA at a specific nucleotide sequence and in a characteristic way: -GAATTC-. But if Mg2+ is replaced by Mn2+, the specificity of the cleavage is relaxed and cleavages occur at many other sites; moreover, there appears to be a hierarchy of cleavage rates at the pseudo-EcoRI restriction sites. For example, SV40 DNA is cleaved only once in the usual digestion conditions, but with Mn2+ more than ten cleavages are made; the five most rapidly cleaved SV40 DNA map locations are 0/1.0 larger than 0.93 larger than 0.33 approximately equal to 0.42 larger than 0.29 approximately equal to 0.40 larger than 0.25. Mn2+ also alters the restriction specificity of HindIII but not HpaII endonuclease.