Recognition elements for 5' exon substrate binding to the Candida albicans group I intron

A group I intron precursor and ribozyme were cloned from the large subunit rRNA of the human pathogen Candida albicans. Both the precursor and ribozyme are functional as determined from in vitro assays. Comparisons of dissociation constants for oligonucleotide binding to the ribozyme and to a hexanucleotide mimic of its internal guide sequence lead to a model for recognition of the 5' exon substrate by this intron. In particular, tertiary contacts with the P1 helix that help align the splice site include three 2'-hydroxyl groups, a G.U pair that occurs at the intron's splice junction, and a G.A pair. The free energy contribution that each interaction contributes to tertiary binding is determined. When the G.A pair is replaced with a G-C pair, tertiary interactions to 5' exon mimic 2'-hydroxyl groups are significantly weakened. When the G.A pair is replaced with a G.U pair, tertiary interactions are retained and binding is 10-fold tighter. These results expand our knowledge of substrate recognition by group I introns, and also provide a basis for rational design of oligonucleotide-based therapeutics for targeting group I introns by binding enhancement by tertiary interactions and suicide inhibition strategies.     

Contributions of individual nucleotides to tertiary binding of substrate by a Pneumocystis carinii group I intron

Pneumocystis carinii is a mammalian pathogen that infects and kills immunocompromised hosts such as cancer and AIDS patients. The LSU rRNA precursor of P. carinii contains a conserved group I intron that is an attractive drug target because humans do not contain group I introns. The oligonucleotide r(AUGACU), whose sequence mimics the 3'-end of the 5'-exon, binds to a ribozyme derived from the intron with a K(d) of 5.2 nM, which is 61000-fold tighter than expected from base-pairing alone [Testa, S. M., Haidaris, G. C., Gigliotti, F., and Turner, D. H. (1997) Biochemistry 36, 9379-9385]. Thus, oligonucleotide binding is enhanced by tertiary interactions. To localize interactions that give rise to this tertiary stability, binding to the ribozyme has been measured as a function of oligonucleotide length and sequence. The results indicate that 4.3 kcal/mol of tertiary stability is due to a G.U pair that forms at the intron's splice junction. Eliminating nucleotides at the 5'-end of r(AUGACU) does not affect intron binding more than expected from differences in base-pairing until r((___)ACU), which binds much more tightly than expected. Adding a C at the 5'- or 3'-end that can potentially form a C-G pair with the target has little effect on binding affinity. Truncated oligonucleotides were tested for their ability to inhibit intron self-splicing via a suicide inhibition mechanism. The tetramer, r((__)GACU), retains similar binding affinity and reactivity as the hexamer, r(AUGACU). Thus oligonucleotides as short as tetramers might serve as therapeutics that can use a suicide inhibition mechanism to inhibit self-splicing. Results with a phosphoramidate tetramer and thiophosphoramidate hexamer indicate that oligonucleotides with backbones stable to nuclease digestion retain favorable binding and reactivity properties.     

Divalent metal ion binding to a conserved wobble pair defining the upstream site of cleavage of group I self-splicing introns.

The upstream site of cleavage of all group I self-splicing introns is identified by an absolutely conserved U.G base pair. Although a wobble C.A pair can substitute the U.G pair, all other combinations of nucleotides at this position abolish splicing, suggesting that it is an unusual RNA structure, rather than sequence, that is recognized by the catalytic intron core. RNA enzymes are metalloenzymes, and divalent metal ion binding may be an important requirement for splice site recognition and catalysis. The paramagnetic broadening of NMR resonances upon manganese binding at specific sites was used to probe the interaction between divalent metal ions and an oligonucleotide model of a group I intron ribozyme substrate. Unlike previous studies in which only imino proton resonances were monitored, we have used isotopically labelled RNA and a set of complete spectral assignments to identify the location of the divalent metal binding site with much greater detail than previously possible. Two independent metal binding sites were identified for this oligonucleotide. A first metal binding site is located in the major groove of the three consecutive G.C base pairs at the end of double helical stem. A second site is found in the major groove of the RNA double helix in the vicinity of the U.G base pair. These results suggest that metal ion coordination (or a metal bridge) and tertiary interactions identified biochemically, may be used by group I intron ribozymes for substrate recognition.     

Use of duplex rigidity for stability and specificity in RNA tertiary structure

The Tetrahymena group I ribozyme's oligonucleotide substrate, CCCUCUA(5), forms six base pairs with the ribozyme's internal guide sequence (IGS, 5'GGAGGG) to give the P1 duplex, and this duplex then docks into the active site via tertiary interactions. Shortening the substrate by three residues to give UCUA(5) reduces the equilibrium constant for P1 docking by approximately 200-fold even though UCUA(5) retains all the functional groups known to be involved in tertiary interactions [Narlikar, G. J., Bartley, L. E., Khosla, M., and Herschlag, D. (1999) Biochemistry 38, 14192-14204]. Here we show that the P1 duplex formed with UCUA(5) engages in all of the major tertiary interactions made by the standard P1 duplex. This suggests that the destabilization is not due to disruption of specific tertiary interactions. It therefore appears that the weaker docking of UCUA(5) arises from the increased conformational freedom of the undocked P1 duplex, which has three unpaired IGS residues and thus a larger entropic cost for docking. Further, a 2'-methoxy substitution at an IGS residue that is base-paired in the standard P1 duplex with CCCUCUA(5) but unpaired in the P1 duplex with UCUA(5) destabilizes docking of the standard P1 duplex approximately 300-fold more than it destabilizes docking of the P1 duplex formed with UCUA(5). These results suggest that fixation of groups in the context of a rigid duplex may be a general strategy used by RNA to substantially increase interaction specificity, both by aiding binding of the desired functional groups and by increasing the energetic cost of forming alternative interactions.     

Dynamics and hydration of the alpha-helices of apolipophorin III

Apolipophorin III (apoLp-III) is an exchangeable apolipoprotein whose structure is represented as a bundle of five amphipathic alpha-helices. In order to study the properties of the helical domains of apolipophorin III, we designed and obtained five single-tryptophan mutants of Locusta migratoria apoLp-III. The proteins were studied by UV absorption spectroscopy, time-resolved and steady-state fluorescence spectroscopy, and circular dichroism. Fluorescence anisotropy, near-UV CD and solute fluorescence quenching studies indicate that the Trp residues in helices 1 (N-terminal) and 5 (C-terminal) have the highest conformational flexibility. These two residues also showed the highest degree of hydration. Trp residues in helices 3 and 4 display the lowest mobility, as assessed by fluorescence anisotropy and near UV CD. The Trp residue in helix 2 is protected from the solvent but shows high mobility. As inferred from the properties of the Trp residues, helices 1 and 5 appear to have the highest conformational flexibility. Helix 2 has an intermediate mobility, whereas helices 3 and 4 appear to constitute a highly ordered domain. From the configuration of the helices in the tertiary structure of the protein, we estimated the relative strength of the five interhelical interactions of apoLp-III. These interactions can be ordered according to their apparent stabilizing strengths as: helix 3-helix 4 > helix 2-helix 3 > helix 4-helix 1 approximately helix 2-helix 5 > helix 1-helix 5. A new model for the conformational change that is expected to occur upon binding of the apolipoprotein to lipid is proposed. This model is significantly different from the currently accepted model (Breiter, D. R., Kanost, M. R., Benning, M. M., Wesemberg, G., Law, J. H., Wells, M. A., Rayment, I., and Holden, M. (1991) Biochemistry 30, 603-608). The model presented here predicts that the relaxation of the tertiary structure and the concomitant exposure of the hydrophobic core take place through the disruption of the weak interhelical contacts between helices 1 and 5. To some extent, the weakness of the helix 1-helix 5 interaction would be due to the parallel arrangement of these helices.     

Structural features and thermodynamics of the J4/5 loop from the Candida albicans and Candida dubliniensis group I introns

The J4/5 loop of group I introns has tertiary interactions with the P1 helix that position the P1 substrate for the self-splicing reaction. The J4/5 loop of Candida albicans and Candida dubliniensis, 5'GAAGG3'/3'UAAUU5', potentially contains two A.A pairs flanked by one G.U pair on one side and two G.U pairs on the other side. Results from optical melting, nuclear magnetic resonance spectroscopy, and functional group substitution experiments with a mimic of the C. albicans and C. dubliniensis J4/5 loop are consistent with the adenosines forming tandem sheared A.A pairs with a cross-strand stack and only the G.U pair not adjacent to an A.A pair forming a static wobble G.U pair. The two G.U pairs adjacent to the tandem A.A pairs are likely in a dynamic equilibrium between multiple conformations. Although Co(NH(3))(6)(3+) stabilizes the loop by several kilocalories per mole at 37 degrees C, addition of Mg(2+) or Co(NH(3))(6)(3+) has no effect on the structure of the loop. The tandem G.U pairs provide a pocket of negative charge for Co(NH(3))(6)(3+) to bind. The results contribute to understanding the structure and dynamics of purine-rich internal loops and potential G.U pairs adjacent to internal loops.     

NMR analysis of helix I from the 5S RNA of Escherichia coli.

The structure of helix I of the 5S rRNA from Escherichia coli has been determined using a nucleolytic digest fragment of the intact molecule. The fragment analyzed, which corresponds to bases (-1)-11 and 108-120 of intact 5S rRNA, contains a G-U pair and has unpaired bases at its termini. Its proton resonances were assigned by two-dimensional NMR methods, and both NOE distance and coupling constant information have been used to calculate structural models for it using the full relaxation matrix algorithm of the molecular dynamics program XPLOR. Helix I has A-type helical geometry, as expected. Its most striking departure from regular helical geometry occurs at its G-U, which stacks on the base pair to the 5' side of its G but not on the base pair to its 3' side. This stacking pattern maximizes interstrand guanine-guanine interactions and explains why the G-U in question fails to give imino proton NOE's to the base pair to 5' side of its G. These results are consistent with the crystal structures that have been obtained for wobble base pairs in tRNAPhe [Mizuno, H., & Sundaralingam, M. (1978) Nucleic Acids Res. 5, 4451-4461] and A-form DNA [Rabbinovich, D., Haran, T., Eisenstein, M., & Shakked, Z. (1988) J. Mol. Biol. 200, 151-161]. The conformations of the terminal residues of helix I, which corresponds to bases (-1)-11 and 108-120 of native 5S RNA, are less well-determined, and their sugar puckers are intermediate between C2' and C3'-endo, on average.     

Stability of XGCGCp, GCGCYp, and XGCGCYp helixes: an empirical estimate of the energetics of hydrogen bonds in nucleic acids

The stabilizing effects of dangling ends and terminal base pairs on the core helix GCGC are reported. Enthalpy and entropy changes of helix formation were measured spectrophotometrically for AGCGCU, UGCGCA, GGCGCCp, CGCGCGp, and the corresponding pentamers XGCGCp and GCGCYp containing the GCGC core plus a dangling end. Each 5' dangling end increases helix stability at 37 degrees C roughly 0.2 kcal/mol and each 3' end from 0.8 to 1.7 kcal/mol. The free energy increments for dangling ends on GCGC are similar to the corresponding increments reported for the GGCC core [Freier, S. M., Alkema, D., Sinclair, A., Neilson, T., & Turner, D. H. (1985) Biochemistry 24, 4533-4539], indicating a nearest-neighbor model is adequate for prediction of stabilization due to dangling ends. Nearest-neighbor parameters for prediction of the free energy effects of adding dangling ends and terminal base pairs next to G.C pairs are presented. Comparison of these free energy changes is used to partition the free energy of base pair formation into contributions of "stacking" and "pairing". If pairing contributions are due to hydrogen bonding, the results suggest stacking and hydrogen bonding make roughly comparable favorable contributions to the stability of a terminal base pair. The free energy increment associated with forming a hydrogen bond is estimated to be -1 kcal/mol of hydrogen bond.     

Binding enhancement by tertiary interactions and suicide inhibition of a Candida albicans group I intron by phosphoramidate and 2'-O-methyl hexanucleotides

Candida albicans is one of many infectious pathogens that are evolving resistance to current treatments. RNAs provide a large class of targets for new therapeutics for fighting these organisms. One strategy for targeting RNAs uses short oligonucleotides that exhibit binding enhancement by tertiary interactions in addition to Watson-Crick pairing. A potential RNA target in C. albicans is the self-splicing group I intron in the LSU rRNA precursor. The recognition elements that align the 5' exon splice site for a ribozyme derived from this precursor are complex [Disney, M. D., Haidaris, C. G., and Turner, D. H. (2001) Biochemistry 40, 6507-6519]. These recognition elements have been used to guide design of hexanucleotide mimics of the 5' exon that have backbones modified for nuclease stability. These hexanucleotides bind as much as 100000-fold more tightly to a ribozyme derived from the intron than to a hexanucleotide mimic of the intron's internal guide sequence, r(GGAGGC). Several of these oligonucleotides inhibit precursor self-splicing via a suicide inhibition mechanism. The most promising suicide inhibitor is the ribophosphoramidate rn(GCCUC)rU, which forms more trans-spliced than cis-spliced product at oligonucleotide concentrations of >100 nM at 1 mM Mg(2+). The results indicate that short oligonucleotides modified for nuclease stability can target catalytic RNAs when the elements of tertiary interactions are complex.     

Evidence for processivity and two-step binding of the RNA substrate from studies of J1/2 mutants of the Tetrahymena ribozyme.

J1/2 of the Tetrahymena ribozyme, a sequence of three A residues, connects the RNA-binding site to the catalytic core. Addition or deletion of bases from J1/2 improves turnover and substrate specificity in the site-specific endonuclease reaction catalyzed by this ribozyme: G2CCCUCUA5 (S) + G in-equilibrium G2CCCUCU (P) + GA5. These paradoxical enhancements are caused by decreased affinity of the ribozyme for S and P [Young, B., Herschlag, D., & Cech, T.R. (1991) Cell 67, 1007]. An additional property of these mutant ribozymes, decreased fidelity of RNA cleavage, is now analyzed. (Fidelity is the ability to cleave at the correct phosphodiester bond within a particular RNA substrate.) Introduction of deoxy residues to give "chimeric" ribo/deoxyribooligonucleotides changes the positions of incorrect cleavage. Previous work indicated that S is bound to the ribozyme by both base pairing and teritary interactions involving 2'-hydroxyl groups of S. The data herein strongly suggest that the P1 duplex, which consists of S base-paired with the 5' exon binding site of the ribozyme, can dock into tertiary interactions in different registers; different 2'-hydroxyl groups of S plug into tertiary contacts with the ribozyme in the different registers. It is concluded that the mutations decrease fidelity by increasing the probability of docking out of register relative to docking in the normal register, thereby giving cleavage at different positions along S. These data also show that the contribution of J1/2 to the teritiary interactions is indirect, not direct. Thus, a structural role of the nonconserved J1/2 is indicated: this sequence positions S to optimize tertiary binding interactions and to ensure cleavage at the phosphodiester bond corresponding to the 5' splice site. Substitution of sulfur for the nonbridging pro-RP oxygen atom at the normal cleavage site has no effect on (kcat/Km)S but decreases the fraction of cleavage at the normal site in reactions catalyzed by the -3A mutant ribozyme, which has all three A residues of J1/2 removed. Thus, the ribozyme chooses where to cleave S after rate-limiting binding of S, indicating that docking can change after binding and suggesting that the ribozyme could act processively. Indeed, it is shown that the +2A ribozyme cleaves at one position along an RNA substrate and then, before releasing that RNA product, cleaves it again.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structural determinants of salmon calcitonin bioactivity: the role of the Leu-based amphipathic alpha-helix

Salmon calcitonin (sCT) forms an amphipathic helix in the region 9-19, with the C-terminal decapeptide interacting with the helix (Amodeo, P., Motta, A., Strazzullo, G., Castiglione Morelli, M. A. (1999) J. Biomol. NMR 13, 161-174). To uncover the structural requirements for the hormone bioactivity, we investigated several sCT analogs. They were designed so as to alter the length of the central helix by removal and/or replacement of flanking residues and by selectively mutating or deleting residues inside the helix. The helix content was assessed by circular dichroism and NMR spectroscopies; the receptor binding affinity in human breast cancer cell line T 47D and the in vivo hypocalcemic activity were also evaluated. In particular, by NMR spectroscopy and molecular dynamics calculations we studied Leu(23),Ala(24)-sCT in which Pro(23) and Arg(24) were replaced by helix inducing residues. Compared with sCT, it assumes a longer amphipathic alpha-helix, with decreased binding affinity and one-fifth of the hypocalcemic activity, therefore supporting the idea of a relationship between a definite helix length and bioactivity. From the analysis of other sCT mutants, we inferred that the correct helix length is located in the 9-19 region and requires long range interactions and the presence of specific regions of residues within the sequence for high binding affinity and hypocalcemic activity. Taken together, the structural and biological data identify well defined structural parameters of the helix for sCT bioactivity.     

Thermodynamics of RNA-RNA duplexes with 2- or 4-thiouridines: implications for antisense design and targeting a group I intron

Antisense compounds are designed to optimize selective hybridization of an exogenous oligonucleotide to a cellular target. Typically, Watson-Crick base pairing between the antisense compound and target provides the key recognition element. Uridine (U), however, not only stably base pairs with adenosine (A) but also with guanosine (G), thus reducing specificity. Studies of duplex formation by oligonucleotides with either an internal or a terminal 2- or 4-thiouridine (s(2)U or s(4)U) show that s(2)U can increase the stability of base pairing with A more than with G, while s(4)U can increase the stability of base pairing with G more than with A. The latter may be useful when binding can be enhanced by tertiary interactions with a s(4)U-G pair. To test the effects of s(2)U and s(4)U substitutions on tertiary interactions, binding to a group I intron ribozyme from mouse-derived Pneumocystis carinii was measured for the hexamers, r(AUGACU), r(AUGACs(2)U), and r(AUGACs(4)U), which mimic the 3' end of the 5' exon. The results suggest that at least one of the carbonyl groups of the 3' terminal U of r(AUGACU) is involved in tertiary interactions with the catalytic core of the ribozyme and/or thio groups change the orientation of a terminal U-G base pair. Thus thio substitutions may affect tertiary interactions. Studies of trans-splicing of 5' exon mimics to a truncated rRNA precursor, however, indicate that thio substitutions have negligible effects on overall reactivity. Therefore, modified bases can enhance the specificity of base pairing while retaining other activities and, thus, increase the specificity of antisense compounds targeting cellular RNA.     

Sequence dependence for the energetics of dangling ends and terminal base pairs in ribonucleic acid

Stability increments of terminal unpaired nucleotides (dangling ends) and terminal base pairs on the core helixes AUGCAU and UGCGCA are reported. Enthalpy, entropy, and free energy changes of helix formation were measured spectrophotometrically for 18 oligoribonucleotides containing the core sequences. The results indicate 3' dangling purines add more stability than 3' dangling pyrimidines. In most cases, the additional stability from a 3' dangling end on an AU base pair is less than that on a GC base pair [Freier, S.M., Burger, B.J., Alkema, D., Neilson, T., & Turner, D.H. (1985) Biochemistry 22, 6198-6206]. The sequence dependence provides a test for the importance of dangling ends for various RNA interactions. Correlations are suggested with codon context effects and with the three-dimensional structure of yeast phenylalanine transfer RNA. In the latter case, all terminal unpaired nucleotides having stability increments more favorable than -1 kcal/mol are stacked on the adjacent base pair. All terminal unpaired nucleotides having stability increments less favorable than -0.3 kcal/mol are not stacked on the adjacent base pair. In several cases, this lack of stacking is associated with a turn in the sugar-phosphate backbone. This suggests stability increments measured on oligoribonucleotides may be useful for predicting tertiary structure in large RNA molecules. Comparison of the stability increments for terminal dangling ends and base pairs, and of terminal GC and AU base pairs, indicates the free energy increment associated with forming a hydrogen bond can be about -1 kcal/mol of hydrogen bond.     

Substitutions at histidine 74 and aspartate 278 alter ligand binding and allostery in lactose repressor protein

In the inducer-bound structure of the lac repressor protein, the side chains of H74 and D278 are positioned to form an ion pair between monomers that appears to be disrupted upon operator binding (Lewis, M., Chang, G., Horton, N. C., Kercher, M. A., Pace, H. C., Schumacher, M. A., Brennan, R. G., and Lu, P. (1996) Science 271, 1247-1254). A series of single substitutions at H74 and D278 and a double mutant, H74D-D278H, were generated to determine the influence of this interaction on ligand binding and allostery in lac repressor. Introduction of apolar amino acids at H74 resulted in distinct effects on ligand binding. Alanine and leucine substitutions decreased operator binding, while tryptophan and phenylalanine increased affinity for operator DNA. Introduction of a negatively charged residue at position 74 in H74D had minimal effects, and "inverting" the side chains in H74D/D278H did not significantly alter inducer or operator binding at neutral pH. In contrast, all substitutions of D278 increased affinity for operator DNA and diminished inducer binding. These observations can be interpreted in the context of the Monod-Wyman-Changeux model. If a salt bridge were essential for stabilizing or destabilizing the inducer-bound conformation, a mutation at either residue that interrupts this interaction should have a similar effect on allostery. Because the type and degree of alteration in ligand binding properties depended on the nature of the substitution at these residues, the individual roles played by H74 and D278 in lac repressor allostery appear more important than their direct contact across the monomer-monomer interface.     

Contributions of dangling end stacking and terminal base-pair formation to the stabilities of XGGCCp, XCCGGp, XGGCCYp, and XCCGGYp helixes

The role of stacking in terminal base-pair formation was studied by comparison of the stability increments for dangling ends to those for fully formed base pairs. Thermodynamic parameters were measured spectrophotometrically for helix formation of the hexanucleotides AGGCCUp, UGGCCAp, CGGCCGp, GCCGGCp, and UCCGGAp and for the corresponding pentanucleotides containing a 5'-dangling end on the GGCCp or CCGGp core helix. In 1 M NaCl at 1 X 10(-4) M strands, a 5'-dangling nucleotide in this series increases the duplex melting temperature (Tm) only 0-4 degrees C, about the same as adding a 5'-phosphate. In contrast, a 3'-dangling nucleotide increases the Tm at 1 X 10(-4) M strands 7-23 degrees C, depending on the sequence [Freier, S. M., Burger, B. J., Alkema, D., Neilson, T., & Turner, D. H. (1983) Biochemistry 22, 6198-6206]. These results are consistent with stacking patterns observed in A-form RNA. The stability increments from terminal A.U, C.G, or U.A base pairs on GGCC or a terminal U.A pair on CCGG are nearly equal to the sums of the stability increments from the corresponding dangling ends. This suggests stacking plays a large role in nucleic acid stability. The stability increment from the terminal base pairs in GCCGGCp, however, is about 5 times the sum of the corresponding dangling ends, suggesting hydrogen bonding can also make important contributions.     

Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 2. Kinetic description of the reaction of an RNA substrate that forms a mismatch at the active site

The site-specific endonuclease reaction catalyzed by the ribozyme from the Tetrahymena pre-rRNA intervening sequence has been characterized with a substrate that forms a "matched" duplex with the 5' exon binding site of the ribozyme [G2CCCUCUA5 + G in equilibrium with G2CCCUCU + GA5 (G = guanosine); Herschlag, D., & Cech, T.R. (1990) Biochemistry (preceding paper in this issue)]. The rate-limiting step with saturating substrate is dissociation of the product G2CCCUCU. Here we show that the reaction of the substrate G2CCCGCUA5, which forms a "mismatched" duplex with the 5' exon binding site at position -3 from the cleavage site, has a value of kcat that is approximately 10(2)-fold greater than kcat for the matched substrate (50 degrees C, 10 mM MgCl2, pH 7). This is explained by the faster dissociation of the mismatched product, G2CCCGCU, than the matched product. With subsaturating oligonucleotide substrate and saturating G, the binding of the oligonucleotide substrate and the chemical step are each partially rate-limiting. The rate constant for the chemical step of the endonuclease reaction and the rate constant for the site-specific hydrolysis reaction, in which solvent replaces G, are each within approximately 2-fold with the matched and mismatched substrates, despite the approximately 10(3)-fold weaker binding of the mismatched substrate. This can be described as "uniform binding" of the base at position -3 in the ground state and transition state [Albery, W.J., & Knowles, J. R. (1976) Biochemistry 15, 5631-5640]. Thus, the matched substrate does not use its extra binding energy to preferentially stabilize the transition state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of a polycyclic aromatic hydrocarbon bay region ring in modulating DNA adduct structure: the non-bay region (8S,9R,10S, 11R)-N(6)-[11-(8,9,10,11-tetrahydro-8,9, 10-trihydroxybenz[a]anthracenyl)]-2'-deoxyadenosyl adduct in codon 61 of the human N-ras protooncogene.

The structure of the non-bay region (8S,9R,10S,11R)-N(6)-[11-(8,9,10, 11-tetrahydro-8,9,10-trihydroxybenz[a]anthracenyl)]-2'-de oxyadenosyl adduct at X(6) of 5'-d(CGGACXAGAAG)-3'.5'-d(CTTCTTGTCCG)-3', incorporating codons 60, 61 (underlined), and 62 of the human N-ras protooncogene, was determined. Molecular dynamics simulations were restrained by 475 NOEs from (1)H NMR. The benz[a]anthracene moiety intercalated above the 5'-face of the modified base pair and from the major groove. The duplex suffered distortion at and immediately adjacent to the adduct site. This was evidenced by the disruption of the Watson-Crick base pairing for X(6) x T(17) and A(7) x T(16) and the increased rise of 7.7 A between base pairs C(5) x G(18) and X(6) x T(17). Increased disorder was observed as excess line width of proton resonances near the lesion site. Comparison with the bay region benzo[a]pyrene [Zegar, I. S., Kim, S. J., Johansen, T. N., Horton, P. J., Harris, C. M., Harris, T. M., and Stone, M. P. (1996) Biochemistry 35, 6212-6224] and bay region benz[a]anthracene [Li, Z., Mao, H., Kim, H.-Y., Tamura, P. J., Harris, C. M., Harris, T. M., and Stone, M. P. (1999) Biochemistry 38, 2969-2981] adducts with the corresponding stereochemistry and at the same site shows that this non-bay region benz[a]anthracene lesion assumes different base pair geometry, in addition to exhibiting greater disorder. These differences are attributed to the loss of the bay region ring. The results suggest the bay region ring contributes to base stacking interactions at the lesion site. These structural differences between the non-bay and bay region lesions are correlated with site-specific mutagenesis data. The bay region benzo[a]pyrene and bay region benz[a]anthracene adducts were poorly replicated in vivo, and induced A --> G mutations. In contrast, the non-bay region benz[a]anthracene adduct was easily bypassed in vivo and was nonmutagenic.     

Topological comparison of two helix destabilizing proteins: ribonuclease A and the gene 5 DNA binding protein

A topological comparison of the two helix destabilizing proteins, pancreatic ribonuclease A and the gene 5 DNA binding protein of bacteriophage fd has been completed utilizing the available high resolution tertiary structures of each protein. The results indicate these two proteins are structurally if not also evolutionarily related. Regions of closet topological equivalence occur between beta loops directly involved in nucleotide binding or are required for the maintenance of their respective oligonucleotide binding channels. In addition, there is a similar placement of critical amino acid side chains about the binding site. Further evidence for this structural relationship is obtained by comparison of structural data for the mode of complexation of polynucleotides to each protein. The results of topological comparison suggest the essential property shared by helix destabilizing proteins, whether specialized DNA binding proteins such as G5BP or proteins with other primary functional roles, like ribonuclease A, is the presence of an elongated oligonucleotide binding channel. Although ribonuclease A and G5BP are structurally related, it seems likely any protein with this structural feature will exhibit a helix destabilizing capacity. This conclusion is supported by the diversity of molecular characteristics shown by other proteins having this activity.