Initiator-Elongator Discrimination in Vertebrate tRNAs for Protein Synthesis

Initiator tRNAs are used exclusively for initiation of protein synthesis and not for the elongation step. We show, in vivo and in vitro, that the primary sequence feature that prevents the human initiator tRNA from acting in the elongation step is the nature of base pairs 50:64 and 51:63 in the TΨC stem of the initiator tRNA. Various considerations suggest that this is due to sequence-dependent perturbation of the sugar phosphate backbone in the TΨC stem of initiator tRNA, which most likely blocks binding of the elongation factor to the tRNA. Because the sequences of all vertebrate initiator tRNAs are identical, our findings with the human initiator tRNA are likely to be valid for all vertebrate systems. We have developed reporter systems that can be used to monitor, in mammalian cells, the activity in elongation of mutant human initiator tRNAs carrying anticodon sequence mutations from CAU to CCU (the C35 mutant) or to CUA (the U35A36 mutant). Combination of the anticodon sequence mutation with mutations in base pairs 50:64 and 51:63 yielded tRNAs that act as elongators in mammalian cells. Further mutation of the A1:U72 base pair, which is conserved in virtually all eukaryotic initiator tRNAs, to G1:C72 in the C35 mutant background yielded tRNAs that were even more active in elongation. In addition, in a rabbit reticulocyte in vitro protein-synthesizing system, a tRNA carrying the TΨC stem and the A1:U72-to-G1:C72 mutations was almost as active in elongation as the elongator methionine tRNA. The combination of mutant initiator tRNA with the CCU anticodon and the reporter system developed here provides the first example of missense suppression in mammalian cells.     

Specific type of interactions in the quaternary system of Cu(II), adenosine 5'-triphosphate, 1,11-diamino-4,8-diazaundecane and uridine

Complexation reactions in the quaternary system Cu/ATP/3,3,3-tet/Urd have been studied. The stability constants of the complexes of the Cu(ATP)(3,3,3-tet)H(x)(Urd) type have been determined by computer analysis of the potentiometric titration. On the basis of the results of spectroscopic as well as equilibrium studies, the mode of interactions has been proposed. Metal ions coordinate phosphate groups of ATP and nitrogen atoms of polyamine. It has been established that in the conditions of the complex Cu(ATP)(3,3,3-tet) formation, uridine introduced into the Cu(II)/ATP/3,3,3-tet ternary system is involved in hydrogen bonding with the endocyclic nitrogen atoms N(1) and N(7) of the ATP purine ring and formation of the adduct Cu(ATP)(3,3,3-tet)H(Urd) is observed. Introduction of metal ions into the system changes substantially the mode of interactions between complementary base pairs relative to that proposed in the Watson and Crick model.     

Suppression of amber codons in vivo as evidence that mutants derived from Escherichia coli initiator tRNA can act at the step of elongation in protein synthesis

The absence of a Watson-Crick base pair at the end of the amino acid acceptor stem is one of the features which distinguishes prokaryotic initiator tRNAs as a class from all other tRNAs. We show that this structural feature prevents Escherichia coli initiator tRNA from acting as an elongator in protein synthesis in vivo. We generated a mutant of E. coli initiator tRNA in which the anticodon sequence is changed from CAU to CUA (the T35A36 mutant). This mutant tRNA has the potential to read the amber termination codon UAG. We then coupled this mutation to others which change the C1.A72 mismatch at the end of the acceptor stem to either a U1:A72 base pair (T1 mutant) or a C1:G72 base pair (G72 mutant). Transformation of E. coli CA274 (HfrC Su- lacZ125am trpEam) with multicopy plasmids carrying the mutant initiator tRNA genes show that mutant tRNAs carrying changes in both the anticodon sequence and the acceptor stem suppress amber codons in vivo, whereas mutant tRNA with changes in the anticodon sequence alone does not. Mutant tRNAs with the above anticodon sequence change are aminoacylated with glutamine in vitro. Measurement of kinetic parameters for aminoacylation by E. coli glutaminyl-tRNA synthetase show that both the nature of the base pair at the end of the acceptor stem and the presence or absence of a base pair at this position can affect aminoacylation kinetics. We discuss the implications of this result on recognition of tRNAs by E. coli glutaminyl-tRNA synthetase.     

Structure of transfer RNA molecules containing the long variable loop

A structure is proposed for the type II tRNA molecules containing the long variable loop and the tertiary base interactions here are compared with type I tRNAs having the short variable loop. The type II tRNAs are similar to the type I tRNAs in their tertiary base pairing interactions but differ from them generally by not having the tertiary base triples. The long variable loop, which is comprised of a helical stem and a loop at the end of it, emerges from the deep groove side of the dihydrouridine helix, and is tilted roughly 30° to the plane formed by the amino acid-pseudouridine and anticodon-dihydrouridine helices found in yeast tRNA^Phe. The fact that many of the type I tRNAs also lack the full compliment of base triples suggests that the tertiary base pairs may alone suffice to sustain the tRNA fold required for its biological function. The base triples and the variable loop appear to have little functional significance. The base type at position 9 is correlated with the number of base triples and G-C base pairs in the dihydrouridine stem.     

The effect of chemical modification of 3-(3-amino-3-carboxypropyl)uridine on tRNA function.

The minor base 3-(3-amino-3-carboxypropyl)uridine (acp3U) in Escherichia coli tRNAPhe was acylated with the N-hydroxysuccinimide esters of acetic, phenoxy-acetic, and naphthoxyacetic acid, as well as the ester of 5-dimethylaminonaphthalene-1-sulfonyl (dansyl)-glycine. The derivatives of tRNAPhe formed were all capable of accepting phenylalanine. There were only minor effects on the kinetic parameters of these derivatives for E. coli phenylalanyl-tRNA synthetase. There was no effect on the ability of tRNAPhe to participate in poly(U)- or poly(ACU)-directed polypeptide synthesis or in the poly(U)-stimulated binding to E. coli ribosomes. The rate of photodynamic cross-linking of 4-Srd 8 to Cyd 13 was decreased in tRNAs containing the acetyl and dansyl-glycyl derivatives of acp3U, indicating that acylation of this base may perturb the tertiary structure of the tRNA. This base in tRNAPhe does not appear to play any role in the known biological functions of tRNAPhe.     

Identification of the tRNA anticodon recognition site of Escherichia coli methionyl-tRNA synthetase

We have previously shown that the anticodon of methionine tRNAs contains most, if not all, of the nucleotides required for specific recognition of tRNA substrates by Escherichia coli methionyl-tRNA synthetase [Schulman, L. H., & Pelka, H. (1988) Science 242, 765-768]. Previous cross-linking experiments have also identified a site in the synthetase that lies within 14 A of the anticodon binding domain [Leon, O., & Schulman, L. H. (1987) Biochemistry 26, 5416-5422]. In the present work, we have carried out site-directed mutagenesis of this domain, creating conservative amino acid changes at residues that contain side chains having potential hydrogen-bond donors or acceptors. Only one of these changes, converting Trp461----Phe, had a significant effect on aminoacylation. The mutant enzyme showed an approximately 60-100-fold increase in Km for methionine tRNAs, with little or no change in the Km for methionine or ATP or in the maximal velocity of the aminoacylation reaction. Conversion of the adjacent Pro460 to Leu resulted in a smaller increase in Km for tRNA(Mets), with no change in the other kinetic parameters. Examination of the interaction of the mutant enzymes with a series of tRNA(Met) derivatives containing base substitutions in the anticodon revealed sequence-specific interactions between the Phe461 mutant and different anticodons. Km values were highest for tRNA(mMet) derivatives containing the normal anticodon wobble base C. Base substitutions at this site decreased the Km for aminoacylation by the Phe461 mutant, while increasing the Km for the wild-type enzyme and for the Leu460 mutant to values greater than 100 microM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Covalent cross-linking of transfer ribonucleic acid to the ribosomal P site. Mechanism and site of reaction in transfer ribonucleic acid.

The covalent cross-linking of unmodified Escherichia coli N-acetylvalyl-tRNA to the 16S RNA of Escherichia coli ribosomes upon near-UV irradiation previously reported by us [Schwartz, I., & Ofengand, J. (1978) Biochemistry 17, 2524--2530] has been studied further. Up to 70% of the unmodified tRNA, nonenzymatically bound to tight-couple ribosomes at 7 mM Mg2+, could be cross-linked by 310--335-nm light. Covalent attachment was solely to the 16S RNA. It was dependent upon both irradiation and the presence of mRNA but was unaffected by the presence or absence of 4-thiouridine in the tRNA. The kinetics of cross-linking showed single-hit behavior. Twofold more cross-linking was obtained w-th tight-couple ribosomes than with salt-washed particles. Puromycin treatment after irradiation released the bound N-acetyl[3H]valine, demonstrating that the tRNA was covalently bound at the P site and that irradiation and covalent linking did not affect the peptidyl transferase reaction. Cross-linking was unaffected by the presence of O2, argon, ascorbate (1 mM), or mercaptoethanol (10 mM). Prephotolysis of a mixture of tRNA and ribosomes in the absence of puly(U2,G) did not block subsequent cross-linking in its presence nor did it generate any long-lived chemically reactive species. There was a strong tRNA specificity. E. coli tRNA1Val and tRNA1Ser and Bacillus subtilis tRNAVal and tRNAThr could be cross-linked, but E. coli tRNA2Val, 5-fluorouracil-substituted tRNA1Val, tRNAPhe, or tRNAFMet could not. By sequence comparison of the reactive and nonreactive tRNAs, the site of attachment in the tRNA was deduced to be the 5'-anticodon base, cmo5U, or ,o5U in all of the reactive tRNAs. The attachment site in 16S RNA is described in the accompanying paper [Zimmerman, R. A., Gates, S. M., Schwartz, I., & Ofengand, J. (1979) Biochemistry (following paper in this issue)]. The link between tRNA and 16S RNA is either direct or involves mRNA bases at most two nucleotides apart since use of the trinucleotide GpUpU in place of poly(U2,G) to direct the binding and cross-linking of N-acetylvalyl-tRNA to the P site did not affect either the rate or yield of cross-linking. Both B. subtilis tRNAVal (mo5U) and E. coli tRNA1Val (cmo5U) gave the same rate and yield of cross-linking when directed by the trinucleotide GpUpU. Therefore, the presence of the charged carboxyl group in the cmo5U-containing tRNA apparently does not markedly perturb the orientation of this base with respect to its reaction partner in the 16S RNA. The cross-linking of AcVal-tRNA only takes place from the P site. At 75 mM KCl and 75 mM NH4Cl, less than 0.4% cross-linking was found at the A site, while 55.5% was obtained at the P site. However, when the salt concentration was lowered to 50 mM NH4Cl, 5% cross-linking to the A site was detected, compared to 49% at the P site. Thus, a simple change in the ionic strength of the incubation mixture was able to alter the affinity labeling pattern of the ribosome.     

Insertional Editing of Mitochondrial tRNAs of Physarum polycephalum and Didymium nigripes

tRNAs encoded on the mitochondrial DNA of Physarum polycephalum and Didymium nigripes require insertional editing for their maturation. Editing consists of the specific insertion of a single cytidine or uridine relative to the mitochondrial DNA sequence encoding the tRNA. Editing sites are at 14 different locations in nine tRNAs. Cytidine insertion sites can be located in any of the four stems of the tRNA cloverleaf and usually create a G·C base pair. Uridine insertions have been identified in the T loop of tRNA^Lys from Didymium and tRNA^Glu from Physarum. In both tRNAs, the insertion creates the GUUC sequence, which is converted to GTΨC (Ψ = pseudouridine) in most tRNAs. This type of tRNA editing is different from other, previously described types of tRNA editing and resembles the mRNA and rRNA editing in Physarum and Didymium. Analogous tRNAs in Physarum and Didymium have editing sites at different locations, indicating that editing sites have been lost, gained, or both since the divergence of Physarum and Didymium. Although cDNAs derived from single tRNAs are generally fully edited, cDNAs derived from unprocessed polycistronic tRNA precursors often lack some of the editing site insertions. This enrichment of partially edited sequences in unprocessed tRNAs may indicate that editing is required for tRNA processing or at least that RNA editing occurs as an early event in tRNA synthesis.     

Divergence of tRNA genes in chloroplast DNA of higher plants

The saturation hybridization between spinach chloroplast (ct) DNA and spinach ¹²⁵I-labelled chloroplast tRNA has shown that about 1.1% of the spinach ctDNA codes for tRNAs. The observed hybridization is a result of specific base-pairing as shown by competition hybridization experiments and thermal stability of the ctDNA-tRNA hybrids. The amount of hybridization shows that spinach ctDNA contains about 40 tRNA genes. Similar hybridization studies have shown that corn ctDNA contains about 28 tRNA genes. The cross-hybridizations between ctDNA and tRNAs of corn, spinach and pea have shown that tRNAs in chloroplasts of higher plants have undergone significant divergence. The pea and spinach tRNAs have been found to have 50% of the base sequences in common. The corn tRNAs have been found to have only about 30% of the base sequences in common with pea and spinach. These data have been confirmed by extensive heterologous competition experiments and thermal stability of the heterologous DNA-tRNA hybrids. The experiments have also shown that the base sequences of tRNAs common in all three plants are the same.     

Ribosomal proteins S7 and L1 are located close to the decoding site of E. coli ribosome--affinity labeling studies with modified tRNAs carrying photoreactive probes attached adjacent to the 3''-end of the anticodon.

Two photoreactive azidonitrophenyl probes have been attached to Yeast methionine elongator tRNA by chemical modification of the N6-(threoninocarbonyl)adenosine located next to the 3'-end of the anticodon. The maximum distance between the purine ring and the azido group estimated for the two probes is 16-17 and 23-24A, respectively. Binding and cross-linking of the uncharged, modified tRNAs to E. coli ribosomes have been studied with and without poly(A,U,G) as a message, under conditions directing uncharged tRNAs preferentially to the P-site. The modified tRNAs retain their binding activity and upon irradiation bind covalently to the ribosome with very high yields. Protein S7 is the major cross-linking target for both modified tRNAs, in the presence or absence of poly(A,U,G). Protein L1 and to a lesser extent proteins L33 and L27 have been found to be cross-linked with the short probe. Cross-linking to 168 rRNA reaches significant levels only in the absence of the message.     

Relationship between the structure and function of Escherichia coli initiator tRNA

Through functional studies of mutant tRNAs, we have identified sequence and/or structural features important for specifying the many distinctive properties of E coli initiator tRNA. Many of the mutant tRNAs contain an anticodon sequence change from CAU→CUA and are now substrates for E coli glutaminyl-tRNA synthetase (GlnRS). We describe here the effect of further mutating the discriminator base 73 and nucleotide 72 at the end of the acceptor stem on: i) recognition of the mutant tRNAs by E coli GlnRS; ii) recognition by E coli methionyl-tRNA transformylase; and iii) activity of the mutant tRNAs in initiation in E coli. For GlnRS recognition, our results are, in general, consistent with interactions found in the crystal structure of the E coli GlnRS-glutamine tRNA complex. The results also support our previous conclusion that formylation of initiator tRNA is important for its function in initiation.     

Missense and nonsense suppressors can correct frameshift mutations

Missense and nonsense suppressor tRNAs, selected for their ability to read a new triplet codon, were observed to suppress one or more frameshift mutations in trpA of Escherichia coli. Two of the suppressible frameshift mutants, trpA8 and trpA46AspPR3, were cloned, sequenced, and found to be of the +1 type, resulting from the insertion of four nucleotides and one nucleotide, respectively. Twenty-two suppressor tRNAs were examined, 20 derived from one of the 3 glycine isoacceptor species, one from lysT, and one from trpT. The sequences of all but four of the mutant tRNAs are known, and two of those four were converted to suppressor tRNAs that were subsequently sequenced. Consideration of the coding specificities and anticodon sequences of the suppressor tRNAs does not suggest a unitary mechanism of frameshift suppression. Rather, the results indicate that different suppressors may shift frame according to different mechanisms. Examination of the suppression windows of the suppressible frameshift mutations indicates that some of the suppressors may work at cognate codons, either in the 0 frame or in the +1 frame, and others may act at noncognate codons (in either frame) by some as-yet-unspecified mechanism. Whatever the mechanisms, it is clear that some +1 frameshifting can occur at non-monotonous sequences. A striking example of a frameshifting missense suppressor is a mutant lysine tRNA that differs from wild-type lysine tRNA by only a single base in the amino acid acceptor stem, a C to U70 transition that results in a G.U base pair. It is suggested that when this mutant lysine tRNA reads its cognate codon, AAA, the presence of the G.U base pair sometimes leads either to a conformational change in the tRNA or to an altered interaction with some component of the translation machinery involved in translocation, resulting in a shift of reading frame. In general, the results indicate that translocation is not simply a function of anticodon loop size, that different frameshifting mechanisms may operate with different tRNAs, and that conformational features, some far removed from the anticodon region, are involved in maintaining fidelity in translocation.     

Uridine phosphorylase from Schistosoma mansoni

Uridine phosphorylase is the only pyrimidine nucleoside cleaving activity that can be detected in extracts of Schistosoma mansoni. The enzyme is distinct from the two purine nucleoside phosphorylases contained in this parasite. Although Urd is the preferred substrate, uridine phosphorylase can also catalyze the reversible phosphorolysis of dUrd and dThd, but not Cyd, dCyd, or orotidine. The enzyme was purified 170-fold to a specific activity of 2.76 nmol/min/mg of protein with a 16% yield. It has a Mr of 56,000 as determined by molecular sieving on Sephadex G-100. The mechanism of uridine phosphorylase is sequential. When Urd was the substrate, the KUrd = 13 microM and the KPi = 533 +/- 78 microM. When dThd was used as a substrate, the KdThd = 54 microM and the KPi = 762 +/- 297 microM. The Vmax with dThd was 53 +/- 9.8% that of Urd. dThd was a competitive inhibitor when Urd was used as a substrate. The enzyme showed substrate inhibition by Urd, dThd (greater than 0.125 mM) and phosphate (greater than 10 mM). 5-(Benzyloxybenzyloxybenzyl)acyclouridine was identified as a potent and specific inhibitor of parasite (Ki = 0.98 microM) but not host uridine phosphorylase. Structure-activity relationship studies suggest that uridine phosphorylase from S. mansoni has a hydrophobic pocket adjacent to the 5-position of the pyrimidine ring and indicate differences between the binding sites of the mammalian and parasite enzymes. These differences may be useful in designing specific inhibitors for schistosomal uridine phosphorylase which will interfere selectively with nucleic acids synthesis in this parasite.     

Photocross-linking analysis of the contact surface of tRNA Met in complexes with Escherichia coli methionine:tRNA ligase.

Photoinduced covalent cross-linking has been used to identify a common surface of four methionine-accepting tRNAs which interact specifically with the Escherichia coli methionine:tRNA ligase (EC 6.1.1.10). tRNA--ligase mixtures were irradiated, and the covalently linked complexes were isolated and digested with T1 RNase (Schimmel & Budzik, 1977). The fragments lost from the elution profile of the T1 RNase digest were considered to have been cross-linked to the protein and therefore in intimate contact with the enzyme. Only specific cognate tRNA--ligase pairs produce covalently linked complexes. The four substrate tRNAs used in this study have substantially different sequences, but all showed a common cross-linking pattern, supporting the view that the sites cross-linked to the enzyme reflect the functionally common contact surface rather than particularly photoreactivity regions of tRNA. The cross-linked contact surface is comprised of three regions: (1) the narrow groove of the anticodon stem and its extension into the anticodon loop; (2) the 3' terminal residues; and (3) the 3' side of the "T arm". Unlike previous studies with other tRNAs, the D arm is not involved and significant radiation damage is suffered by the tRNA which must be taken into account in the analysis. The results are consistent with and complement chemical modification studies [Schulman, L. H., & Pelka, H. (1977) Biochemistry 16, 4256].     

Effect of zinc ions on tRNA structure: imino proton NMR spectroscopy

The structure of tRNA in solution was explored by NMR spectroscopy to evaluate the effect of divalent cations, especially zinc, which has a profound effect on the chromatographic behaviour of tRNAs in certain systems. The divalent ions Mg2+ and Zn2+ have specific effects on the imino proton region of the 1H NMR spectrum of valine transfer RNA (tRNA(Val] of Escherichia coli and of phenylalanine transfer RNA (tRNA(Phe] of yeast. The dependence of the imino proton spectra of the two tRNAs was examined as a function of Zn2+ concentration. In both tRNAs the tertiary base pair (G-15).(C-48) was markedly affected by Zn2+ (shifted downfield possibly by as much as 0.4 ppm); this is the terminal base pair in the augmented dihydrouridine helix (D-helix). Base pair (U-8).(A-14) in yeast tRNA(Phe) or (s4U-8).(A-14) in tRNA1(Val), which are stacked on (G-15).(C-48), was not affected by Zn2+, except when 1-2 Mg2+ ions per tRNA were also present. Another imino proton that may be affected by Zn2+ in both tRNAs is that of the tertiary base pair (G-19).(C-46). The assignment of this resonance in yeast tRNA(Phe) is tentative since it is located in the region of highly overlapping resonances between 12.6 and 12.3 ppm. This base pair helps to anchor the D-loop to the T psi C loop.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of modified nucleosides in intact transfer ribonucleic acid by pyrolysis-electron impact-collisional activation mass spectrometry.

A novel mass spectrometric method has been developed for the detection and identification of dihydrouridine, ribothymidine, 4-thiouridine, and 7-methylguanosine in Escherichia coli tRNAs. The method utilizes (a) Pyrolysis-Electron Impact-Mass Spectrometry (PYEIMS), a procedure which releases the purine and pyrimidine bases from the intact, underivatized tRNA molecule. The mass spectrum exhibits intense peaks for the bases deriving from the common nucleosides in tRNA as well as peaks of much lower intensity at mass values expected for the bases from modified components known to be present in the tRNA; and, (b) Collisional Activation Mass Spectrometry (CAMS), a technique which permits the isolation of a single ion species from a complex mass spectrum. Subsequent fragmentation of that species yields a characteristic collisional activation spectrum. Such analyses of the ion species that were presumed to originate from H2Urd, rThd, 4SUrd, and 7MeGuo in the tRNA were used to define the structure and, thus, the identity of each component. Attributes of the PYEICAMS technique are that (a) precise structural elucidation of minor nucleosides present in tRNAs at the 1 - 4% level is obtained; (b) the high order of sensitivity allows the analysis to be done on microgram amounts of tRNA; and (c) there is no requirement for enzymatic or chemical hydrolysis of the tRNA or for subsequent chromatographic separation methods.     

15N-labeled tRNA. Identification of 4-thiouridine in Escherichia coli tRNASer1 and tRNATyr2 by 1H-15N two-dimensional NMR spectroscopy

Uridine is uniquely conserved at position 8 in elongator tRNAs and binds to A14 to form a reversed Hoogsteen base pair which folds the dihydrouridine loop back into the core of the L-shaped molecule. On the basis of 1H NMR studies, Hurd and co-workers (Hurd, R. E., Robillard, G. T., and Reid, B. R. (1977) Biochemistry 16, 2095-2100) concluded that the interaction between positions 8 and 14 is absent in Escherichia coli tRNAs with only 3 base pairs in the dihydrouridine stem. We have taken advantage of the unique 15N chemical shift of N3 in thiouridine to identify 1H and 15N resonances for the imino units of S4U8 and s4U9 in E. coli tRNASer1 and tRNATyr2. Model studies with chloroform-soluble derivatives of uridine and 4-thiouridine show that the chemical shifts of the protons in the imino moieties move downfield from 7.9 to 14.4 ppm and from 9.1 to 15.7 ppm, respectively; whereas, the corresponding 15N chemical shifts move downfield from 157.5 to 162.5 ppm and from 175.5 to 180.1 ppm upon hydrogen bonding to 5'-O-acetyl-2',3'-isopropylidene adenosine. The large difference in 15N chemical shifts for U and s4U allows one to unambiguously identify s4U imino resonances by 15N NMR spectroscopy. E. coli tRNASer1 and tRNATyr2 were selectively enriched with 15N at N3 of all uridines and modified uridines. Two-dimensional 1H-15N chemical shift correlation NMR spectroscopy revealed that both tRNAs have resonances with 1H and 15N chemical shifts characteristic of s4UA pairs. The 1H shift is approximately 1 ppm upfield from the typical s4U8 resonance at 14.8 ppm, presumably as a result of local diamagnetic anisotropies. An additional s4U resonance with 1H and 15N shifts typical of interaction of a bound water or a sugar hydroxyl group with s4U9 was discovered in the spectrum of tRNATyr2. Our NMR results for tRNAs with 3-base pair dihydrouridine stems suggest that these molecules have an U8A14 tertiary interaction similar to that found in tRNAs with 4-base pair dihydrouridine stems.     

DNA-mediated charge transport as a probe of MutY/DNA interaction

MutY is an Escherichia coli DNA repair enzyme that binds to 8-oxo-G:A and G:A mismatches and catalyzes the deglycosylation of the mismatched 2'-deoxyadenosine. We have applied DNA-mediated charge transport to probe the interaction of MutY with its DNA substrate. Oligonucleotides synthesized with a tethered rhodium intercalator and guanine doublets placed before and after the MutY binding site are used to assay for base flipping activity by MutY. On the basis of this assay, we find no evidence that MutY uses progressive base flipping as a means to find its binding site; protein binding does not perturb long-range DNA charge transport. DNA-mediated charge transport can be utilized to promote protein-DNA cross-linking from a distance. Long-range oxidation of 8-oxo-G within the MutY binding site using tethered rhodium intercalators promoted cross-linking and yielded information on MutY side chains that interact with this base. On the basis of photooxidative cross-linking of the wild type but not K142A mutant, it is evident that, within the protein complex, lysine 142 makes important contacts with 8-oxo-G.     

On the mechanism of the acid-catalyzed hydrolysis of uridine to uracil. Evidence for 6-hydroxy-5,6-dihydrouridine intermediates

In acidic media, the 5,6-double bond of uridine is rapidly hydrated to give a small amount of 6-hydroxy-5,6-dihydrouridine (Urd-H2O), the mechanism of which is known from studies of the acid-catalyzed dehydration of Urd-H2O (Prior, J. J., Maley, J., and Santi, D. V. (1984) J. Biol. Chem. 258, 2422-2428). In addition to dehydration, Urd-H2O also undergoes direct hydrolysis of the N-glycosidic bond in acidic solution. The kinetics of the above reaction demonstrates that Urd-H2O, or an intermediate in the pathway leading from Urd to Urd-H2O, is kinetically competent to account for the hydrolysis of the N-glycosidic bond of Urd. The hydrolysis of (1'-2H)Urd proceeds with an alpha-secondary deuterium isotope effect of kH/kD of 1.11 at 25 degrees C. This isotope effect is sufficiently large to implicate carbonium ion character at the 1'-carbon during hydrolysis but, since it is not the maximal value expected, suggests that N-glycoside cleavage is rate-determining with a transition state intermediate between reactant and products. Importantly, the hydrolysis of [6-3H]Urd proceeds with a substantial inverse secondary isotope effect of kT/kH = 1.15 at 25 degrees C which indicates some degree of sp2 to sp3 rehybridization of C-6 of the pyrimidine moiety during hydrolysis. From the data available, it appears that an important pathway in the hydrolysis of the N-glycoside bond of Urd involves either spontaneous cleavage of Urd which is protonated at the 5-carbon or a protonated species of Urd-H2O. The studies described here, together with the known susceptibility of the 6-position of pyrimidine heterocycles toward nucleophiles, permits the proposal of chemically reasonable mechanisms for enzyme-catalyzed cleavage of N-glycosidic bonds of pyrimidines.