R Kierzek

Publications (52)278.16 Total impact

• Article: Substrate recognition by a yeast 2'-phosphotransferase involved in tRNA splicing and by its Escherichia coli homolog.

  M A Steiger, R Kierzek, D H Turner, E M Phizicky
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  ABSTRACT: The final step of tRNA splicing in Saccharomyces cerevisiae requires 2'-phosphotransferase (Tpt1) to transfer the 2'-phosphate from ligated tRNA to NAD, producing mature tRNA and ADP ribose-1' '-2' '-cyclic phosphate. To address how Tpt1 protein recognizes substrate RNAs, we measured the steady-state kinetic parameters of Tpt1 protein with 2'-phosphorylated ligated tRNA and a variety of related substrates. Tpt1 protein has a high apparent affinity for ligated tRNA (K(m,RNA), 0.35 nM) and a low turnover rate (k(cat), 0.3 min(-1)). Tpt1 protein recognizes both tRNA and the internal 2'-phosphate of RNAs. Steady-state kinetic analysis reveals that as RNAs lose structure and length, K(m,RNA) and k(cat) both increase commensurately. For a 2'-phosphorylated octadecamer derived from the anticodon stem-loop of ligated tRNA, K(m,RNA) and k(cat) are 5- and 8-fold higher, respectively, than for ligated tRNA, whereas for a simple substrate like pApA(p)pA, K(m,RNA) and k(cat) are 430- and 150-fold higher, respectively. Tpt1 is not detectably active on a trimer with a terminal 5'- or 3'-phosphate and is very inefficient at removal of a terminal 2'-phosphate unless there is an adjacent 3'-phosphate or phosphodiester. The K(m,NAD) for Tpt1 is substrate dependent: K(m,NAD) is 10 microM with ligated tRNA, 200 microM with pApA(p)pA, and 600 microM with pApApA(p). Preliminary analysis of KptA, a functional Tpt1 protein homologue from Escherichia coli, reveals that KptA protein is strikingly similar to yeast Tpt1 in its kinetic parameters, although E. coli is not known to have a 2'-phosphorylated RNA substrate.
  Biochemistry 12/2001; 40(46):14098-105. · 3.42 Impact Factor
• Article: Influence of N6-isopentenyladenosine (i(6)A) on thermal stability of RNA duplexes.

  E Kierzek, R Kierzek
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  ABSTRACT: The thermodynamic stability of self-complementary oligoribonucleotides containing N6-isopentenyladenosine (i(6)A) or N6-isopentanyladenosine (p(6)A) was determined. The base pairs i(6)A.U and p(6)A.U were placed in either an internal (separated and tandem) and a terminal position within the duplex, or unpaired i(6)A and p(6)A as a 3'-dangling ends. The thermal unfolding of the oligomers was determined by means of UV melting profiles and the thermodynamic parameters: enthalpy (DeltaH degrees ), entropy (DeltaS degrees) and free energy (DeltaG degrees (37)) as well as the melting temperature (T(m)) were calculated. Both modified nucleosides destabilized the duplexes, however, the effect depended on the position of the modified adenosine within the duplex. The similarity of the behavior of oligomers containing i(6)A and p(6)A suggests a negligible effect of the double bond on the thermal stability. The largest destabilization was observed when derivatives of adenosine were placed in an internal position. The effect of 3'-dangling ends suggests that the presence of the N6-isopentenyl- or N6-isopentanyl substitutent affects hydrogen bonding rather than stacking within duplex.
  Biophysical Chemistry 08/2001; 91(2):135-40. · 2.20 Impact Factor
• Article: Stability and structure of RNA duplexes containing isoguanosine and isocytidine.

  X Chen, R Kierzek, D H Turner
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  ABSTRACT: Isoguanosine (iG) and isocytidine (iC) differ from guanosine (G) and cytidine (C), respectively, in that the amino and carbonyl groups are transposed. The thermodynamic properties of a set of iG, iC containing RNA duplexes have been measured by UV optical melting. It is found that iG-iC replacements usually stabilize duplexes, and the stabilization per iG-iC pair is sequence-dependent. The sequence dependence can be fit to a nearest-neighbor model in which the stabilities of iG--iC pairs depend on the adjacent iG--iC or G--C pairs. For 5'-CG-3'/3'-GC-5' and 5'-GG-3'/3'-CC-5' nearest neighbors, the free energy differences upon iG-iC replacement are smaller than 0.2 kcal/mol at 37 degrees C, regardless of the number of replacements. For 5'-GC-3'/3'-CG-5', however, each iG--iC replacement adds 0.6 kcal/mol stabilizing free energy at 37 degrees C. Stacking propensities of iG and iC as unpaired nucleotides at the end of a duplex are similar to those of G and C. An NMR structure is reported for r(CiGCGiCG)(2) and found to belong to the A-form family. The structure has substantial deviations from standard A-form but is similar to published NMR and/or crystal structures for r(CGCGCG)(2) and 2'-O-methyl (CGCGCG)(2). These results provide benchmarks for theoretical calculations aimed at understanding the fundamental physical basis for the thermodynamic stabilities of nucleic acid duplexes.
  Journal of the American Chemical Society 03/2001; 123(7):1267-74. · 9.91 Impact Factor
• Article: Nonenzymatic cleavage of oligoribonucleotides.

  R Kierzek
  Methods in Enzymology 02/2001; 341:657-75. · 2.04 Impact Factor
• Article: Elements of thermodynamics in RNA evolution.

  E Kierzek, E Biała, R Kierzek
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  ABSTRACT: The paper presents some aspects correlating thermal stability of RNA folding and the occurrence of structural motifs in natural ribonucleic acids. Particularly, the thermodynamic stability of 2'-5' and 3'-5' linked RNA and the contribution of unpaired terminal nucleotides (dangling ends) in secondary (2D) and tertiary (3D) structures of RNA are discussed. Both examples suggest that during evolution nature selected sequences and structures of RNA which are the most thermally stable and efficient for their biological function.
  Acta biochimica Polonica 02/2001; 48(2):485-93. · 1.49 Impact Factor
• Source

  Available from: actabp.pl

  Article: How RNA viruses exchange their genetic material.

  M Alejska, A Kurzyńiska-Kokorniak, M Broda, R Kierzek, M Figlerowicz
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  ABSTRACT: One of the most unusual features of RNA viruses is their enormous genetic variability. Among the different processes contributing to the continuous generation of new viral variants RNA recombination is of special importance. This process has been observed for human, animal, plant and bacterial viruses. The collected data reveal a great susceptibility of RNA viruses to recombination. They also indicate that genetic RNA recombination (especially the nonhomologous one) is a major factor responsible for the emergence of new viral strains or species. Although the formation and accumulation of viral recombinants was observed in numerous RNA viruses, the molecular basis of this phenomenon was studied in only a few viral species. Among them, brome mosaic virus (BMV), a model (+)RNA virus offers the best opportunities to investigate various aspects of genetic RNA recombination in vivo. Unlike any other, the BMV-based system enables homologous and nonhomologous recombination studies at both the protein and RNA levels. As a consequence, BMV is the virus for which the structural requirements for genetic RNA recombination have been most precisely established. Nevertheless, the previously proposed model of genetic recombination in BMV still had one weakness: it could not really explain the role of RNA structure in nonhomologous recombination. Recent discoveries concerning the latter problem give us a chance to fill this gap. That is why in this review we present and thoroughly discuss all results concerning nonhomologous recombination in BMV that have been obtained until now.
  Acta biochimica Polonica 02/2001; 48(2):391-407. · 1.49 Impact Factor
• Article: Nuclear magnetic resonance spectroscopy and molecular modeling reveal that different hydrogen bonding patterns are possible for G.U pairs: one hydrogen bond for each G.U pair in r(GGCGUGCC)(2) and two for each G.U pair in r(GAGUGCUC)(2).

  X Chen, J A McDowell, R Kierzek, T R Krugh, D H Turner
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  ABSTRACT: G.U pairs occur frequently and have many important biological functions. The stability of symmetric tandem G.U motifs depends both on the adjacent Watson-Crick base pairs, e.g., 5'G > 5'C, and the sequence of the G.U pairs, i.e., 5'-UG-3' > 5'-GU-3', where an underline represents a nucleotide in a G.U pair [Wu, M., McDowell, J. A., and Turner, D. H. (1995) Biochemistry 34, 3204-3211]. In particular, at 37 degrees C, the motif 5'-CGUG-3' is less stable by approximately 3 kcal/mol compared with other symmetric tandem G.U motifs with G-C as adjacent pairs: 5'-GGUC-3', 5'-GUGC-3', and 5'-CUGG-3'. The solution structures of r(GAGUGCUC)(2) and r(GGCGUGCC)(2) duplexes have been determined by NMR and restrained simulated annealing. The global geometry of both duplexes is close to A-form, with some distortions localized in the tandem G.U pair region. The striking discovery is that in r(GGCGUGCC)(2) each G.U pair apparently has only one hydrogen bond instead of the two expected for a canonical wobble pair. In the one-hydrogen-bond model, the distance between GO6 and UH3 is too far to form a hydrogen bond. In addition, the temperature dependence of the imino proton resonances is also consistent with the different number of hydrogen bonds in the G.U pair. To test the NMR models, U or G in various G.U pairs were individually replaced by N3-methyluridine or isoguanosine, respectively, thus eliminating the possibility of hydrogen bonding between GO6 and UH3. The results of thermal melting studies on duplexes with these substitutions support the NMR models.
  Biochemistry 09/2000; 39(30):8970-82. · 3.42 Impact Factor
• Source

  Available from: uni-leipzig.de

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

  S M Testa, M D Disney, D H Turner, R Kierzek
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  ABSTRACT: 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.
  Biochemistry 01/2000; 38(50):16655-62. · 3.42 Impact Factor
• Article: The non-enzymatic hydrolysis of oligoribonucleotides VI. The role of biogenic polyamines.

  A Bibillo, M Figlerowicz, R Kierzek
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  ABSTRACT: Single-stranded oligoribonucleotides containing UA and CA phosphodiester bonds can be hydrolyzed specifically under non-enzymatic conditions in the presence of spermidine, a biogenic amine found in a wide variety of organisms. In the present study, the rate of oligonucleotide and tRNA(i)(Met)hydrolysis was measured in the presence of spermidine and other biogenic amines. It was found that spermine [H(3)N(+)(CH(2))(3)(+)NH(2)(CH(2))(4)(+)NH(2)(CH(2))(3)(+)NH(3)] and putrescine [H(3)N(+)(CH(2))(4)(+)NH(3)] can replace spermidine [H(3)N(+)-(CH(2))(4)(+)NH(2)(CH(2))(3)(+)NH(3)] to induce the hydrolysis. For all three polyamines, a bell-shaped cleavage rate versus concentration relationship was observed. The maximum rate of hydrolysis was achieved at 0.1, 1.0 and 10 mM spermine, spermidine and putrescine, respectively. Moreover, we found that the hydrolysis requires at least two linked amino groups since two aminoalcohols, 2-aminoethanol and 3-aminopropanol, were not able to induce the cleavage of the phospho-diester bond. The optimal cleavage rate of the oligo-ribonucleotides was observed when amino groups were separated by tri- or tetramethylene linkers. The methylation of the amino groups reduced the ability of diamines to induce oligoribonucleotide hydrolysis. Non-enzymatic cleavage of tRNA(i)(Met)from Lupinus luteus and tRNA(i)(Met)from Escherichia coli demonstrate that both RNAs hydrolyze as expected from principles derived from oligoribonucleotide models.
  Nucleic Acids Research 11/1999; 27(19):3931-7. · 8.03 Impact Factor
• Source

  Available from: uni-leipzig.de

  Article: Thermodynamics of single mismatches in RNA duplexes.

  R Kierzek, M E Burkard, D H Turner
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  ABSTRACT: The thermodynamic properties and structures of single mismatches in short RNA duplexes were studied in optical melting and imino proton NMR experiments. The free energy increments at 37 degrees C measured for non-GU single mismatches range from -2.6 to 1.7 kcal/mol. These increments depend on the identity of the mismatch, adjacent base pairs, and the position in the helix. UU and AA mismatches are more stable close to a helix end, but GG mismatch stability is essentially unaffected by the position in the helix. Approximations are suggested for predicting stabilities of single mismatches in short RNA duplexes.
  Biochemistry 11/1999; 38(43):14214-23. · 3.42 Impact Factor
• Article: Thermodynamics of unpaired terminal nucleotides on short RNA helixes correlates with stacking at helix termini in larger RNAs.

  M E Burkard, R Kierzek, D H Turner
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  ABSTRACT: Free energies for stacking of unpaired nucleotides (dangling ends) at the termini of oligoribonucleotide Watson-Crick helixes (DeltaG(0)37,stack) depend on sequence for 3' ends but are always small for 5' ends. Here, these free energies are correlated with stacking at helix termini in a database of 34 RNA structures determined by X-ray crystallography and NMR spectroscopy. Stacking involving GA pairs is considered separately. A base is categorized as stacked by its distance from (</=4.0 A), angle with (</=30 degrees), and overlap with the terminal helix base-pair. A base is unstacked if it does not satisfy one or more of these criteria. Of the 36 unpaired bases in sequences with DeltaG(0)37,stackmore favorable than -0.7 kcal/mol, 30 (83 %) are stacked on the adjacent base-pair, indicating a propensity for such sequences to stack in the 3D structure. Structures containing the strongly stacked sequence [sequence: see text] show that the amino group of C closely overlaps the carbonyl-4 of U. Thermodynamic measurement of U stacking on a 2-pyrimidinone-guanine base-pair, where the amino group of C is replaced by hydrogen, suggests that interactions with the cytosine amino group contribute approximately 0.5 kcal/mol to DeltaG(0)37,stack. For GA mismatches at helix termini, the nucleotide at the 3' helix end is always stacked, and the nucleotide at the 5' end is stacked in almost 90 % of occurrences. In available structures, non-Watson-Crick paired bases 3' to an imino-hydrogen bonded GA are also always stacked; the GA provides a large platform for favorable stacking. For the 56 sequences associated with DeltaG(0)37,stackless favorable than -0.4 kcal/mol, 19 (34 %) are stacked; these sequences have a propensity for not stacking on adjacent base-pairs. Phylogenetic conservation of weakly stacking sequences at 3' ends may be a predictor of a backbone turn.
  Journal of Molecular Biology 08/1999; 290(5):967-82. · 4.00 Impact Factor
• Article: [The secondary structural motifs of RNA].

  K Ziomek, R Kierzek
  Postepy biochemii 02/1999; 45(2):80-7.
• Article: [Predicting the secondary structures of the ribonucleic acids (RNA)].

  K Ziomek, R Kierzek
  Postepy biochemii 02/1999; 45(2):74-80.
• Source

  Available from: actabp.pl

  Article: Nonenzymatic hydrolysis of oligoribonucleotides. V. The elements affecting the process of self-hydrolysis.

  A Bibiłło, K Ziomek, M Figlerowicz, R Kierzek
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  ABSTRACT: Chemical instability of some of the phosphodiester bonds, often observed in large RNAs, visualizes the autocatalytic properties of this class of nucleic acids. Unexpectedly, selective hydrolysis occurs also in short oligoribonucleotides (as short as a tetramer or hexamer). Herein, we describe additional experiments which support the conclusion that the hydrolysis is not due to ribonuclease contamination but is of autocatalytic origin and is related to the sequence and structure of single-stranded oligomers. Moreover, we show that the presence in the reaction mixture of polyamines, such as spermidine, is essential for hydrolysis of oligoribonucleotides.
  Acta biochimica Polonica 02/1999; 46(1):145-53. · 1.49 Impact Factor
• Article: Transient ADP-ribosylation of a 2'-phosphate implicated in its removal from ligated tRNA during splicing in yeast.

  S L Spinelli, R Kierzek, D H Turner, E M Phizicky
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  ABSTRACT: The last step of tRNA splicing in yeast is catalyzed by Tpt1 protein, which transfers the 2'-phosphate from ligated tRNA to NAD to produce ADP-ribose 1"-2"-cyclic phosphate (Appr>p). Structural and functional TPT1 homologs are found widely in eukaryotes and, surprisingly, also in Escherichia coli, which does not have this class of tRNA splicing. To understand the possible roles of the Tpt1 enzymes as well as the unusual use of NAD, the reaction mechanism of the E. coli homolog KptA was investigated. We show here that KptA protein removes the 2'-phosphate from RNA via an intermediate in which the phosphate is ADP-ribosylated followed by a presumed transesterification to release the RNA and generate Appr>p. The intermediate was characterized by analysis of its components and their linkages, using various labeled substrates and cofactors. Because the yeast and mouse Tpt1 proteins, like KptA protein, can catalyze the conversion of the KptA-generated intermediate to both product and the original substrate, these enzymes likely use the same reaction mechanism. Step 1 of this reaction is strikingly similar to the ADP-ribosylation of proteins catalyzed by a number of bacterial toxins.
  Journal of Biological Chemistry 01/1999; 274(5):2637-44. · 4.77 Impact Factor
• Source

  Available from: uni-leipzig.de

  Article: Thermodynamic parameters for an expanded nearest-neighbor model for formation of RNA duplexes with Watson-Crick base pairs.

  T Xia, J SantaLucia, M E Burkard, R Kierzek, S J Schroeder, X Jiao, C Cox, D H Turner
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  ABSTRACT: Improved thermodynamic parameters for prediction of RNA duplex formation are derived from optical melting studies of 90 oligoribonucleotide duplexes containing only Watson-Crick base pairs. To test end or base composition effects, new sets of duplexes are included that have identical nearest neighbors, but different base compositions and therefore different ends. Duplexes with terminal GC pairs are more stable than duplexes with the same nearest neighbors but terminal AU pairs. Penalizing terminal AU base pairs by 0.45 kcal/mol relative to terminal GC base pairs significantly improves predictions of DeltaG degrees37 from a nearest-neighbor model. A physical model is suggested in which the differential treatment of AU and GC ends accounts for the dependence of the total number of Watson-Crick hydrogen bonds on the base composition of a duplex. On average, the new parameters predict DeltaG degrees37, DeltaH degrees, DeltaS degrees, and TM within 3.2%, 6.0%, 6.8%, and 1.3 degreesC, respectively. These predictions are within the limit of the model, based on experimental results for duplexes predicted to have identical thermodynamic parameters.
  Biochemistry 11/1998; 37(42):14719-35. · 3.42 Impact Factor
• Article: Guanosine binds to the Tetrahymena ribozyme in more than one step, and its 2'-OH and the nonbridging pro-Sp phosphoryl oxygen at the cleavage site are required for productive docking.

  L A Profenno, R Kierzek, S M Testa, D H Turner
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  ABSTRACT: The dynamics of binding of various guanosine, or G, substrates to the Tetrahymena thermophila L-21 ScaI ribozyme have been investigated by fluorescence-detected stopped-flow experiments. Upon rapid mixing of various G substrates with a preformed complex of the ribozyme and the fluorescent 5' splice site analogue CCUCUepsilonA, fluorescence transients that provide rates for binding of G substrates and the rate-limiting step for transesterification are observed. The measured apparent bimolecular rate constant for binding of pG is 10(3) M-1 s-1, much slower than expected for diffusion. pG appears to bind to the preformed complex of the ribozyme and CCUCUepsilonA in at least two steps, a bimolecular step followed by at least one conformational change. This two-step binding of pG, involving a rapid pre-equilibrium, leads to the slow apparent rate constant for binding of pG. Furthermore, the 2'-OH of pG and of the 3' terminal G of the G substrate GUCG and the nonbridging pro-Sp phosphoryl oxygen atom at the site of phosphoryl transfer on CCUCUepsilonA appear to mediate formation of a properly conformed docked ternary complex of the G substrate, 5' splice site, and ribozyme which may represent an intermediate required for initiation of transesterification. It is possible that the 2'-OH of pG and this nonbridging pro-Sp phosphoryl oxygen interact, directly or indirectly, with one another.
  Biochemistry 11/1997; 36(41):12477-85. · 3.42 Impact Factor
• Article: An NAD derivative produced during transfer RNA splicing: ADP-ribose 1"-2" cyclic phosphate.

  G M Culver, S M McCraith, M Zillmann, R Kierzek, N Michaud, R D LaReau, D H Turner, E M Phizicky
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  ABSTRACT: Transfer RNA (tRNA) splicing is essential in Saccharomyces cerevisiae as well as in humans, and many of its features are the same in both. In yeast, the final step of this process is removal of the 2' phosphate generated at the splice junction during ligation. A nicotinamide adenine dinucleotide (NAD)-dependent phosphotransferase catalyzes removal of the 2' phosphate and produces a small molecule. It is shown here that this small molecule is an NAD derivative: adenosine diphosphate (ADP)-ribose 1"-2" cyclic phosphate. Evidence is also presented that this molecule is produced in Xenopus laevis oocytes as a result of dephosphorylation of ligated tRNA.
  Science 08/1993; 261(5118):206-8. · 31.20 Impact Factor
• Article: Binding of guanosine and 3' splice site analogues to a group I ribozyme: interactions with functional groups of guanosine and with additional nucleotides.

  S Moran, R Kierzek, D H Turner
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  ABSTRACT: Dissociation constants, Kd, were measured by equilibrium dialysis at 5 degrees C for a series of substrates binding to the L-21 ScaI ribozyme derived from the Tetrahymena thermophila self-splicing large subunit (LSU) ribosomal RNA intron. These substrates are analogues for the 3' exon splice site, the cyclization site, and the exogenous G that initiates group I splicing. UCG has a Kd of 17 microM. Lengthening the substrate to GUCG and GGUCG enhances binding but by less than expected from potential base pairing. Functional groups on the 3'-terminal G of GUCG were replaced with H to test their effect on binding. GUC(2'dG) binds slightly tighter than the all-ribose molecule but shows no reactivity as a substrate. GUC(3'dG) binds weaker than GUCG. Inosine and 2-aminopurine ribonucleoside at the 3' position weaken binding by 16- and 26-fold, respectively, but both tetramers are reactive. Thus hydrogen bonds to Watson-Crick pairing positions of the 3'G of GUCG contribute 1-2 kcal/mol to the free energy change for binding. Similar results are found in comparisons of UCG with UC(2'dG), UC(3'dG), and UCI. The nonreactive substrate GUCdGA includes a phosphodiester bond 3' to the guanosine that is the site of chemistry for the all-ribose substrate GUCGA; GUCdGA binds 50 times more weakly than GUCdG. A similar result is obtained for GUCdGU. Competition experiments show that guanosine and guanosine 5'-monophosphate bind with dissociation constants of about 0.9 mM. The monomers 2'dG and 3'dG have Kd's of 0.5 and > or = 3 mM, respectively. This suggests that sugar pucker and/or interactions with hydroxyl groups affect binding. Implications for ribozyme catalysis, splicing, cyclization, and design of antisense oligomers are discussed.
  Biochemistry 05/1993; 32(19):5247-56. · 3.42 Impact Factor
• Source

  Available from: psu.edu

  Article: Dynamics of ribozyme binding of substrate revealed by fluorescence-detected stopped-flow methods.

  P C Bevilacqua, R Kierzek, K A Johnson, D H Turner
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  ABSTRACT: Fluorescence-detected stopped-flow and equilibrium methods have been used to study the mechanism for binding of pyrene (pyr)-labeled RNA oligomer substrates to the ribozyme (catalytic RNA) from Tetrahymena thermophila. The fluorescence of these substrates increases up to 25-fold on binding to the ribozyme. Stopped-flow experiments provide evidence that pyr experiences at least three different microenvironments during the binding process. A minimal mechanism is presented in which substrate initially base pairs to ribozyme and subsequently forms tertiary contacts in an RNA folding step. All four microscopic rate constants are measured for ribozyme binding of pyrCCUCU.
  Science 12/1992; 258(5086):1355-8. · 31.20 Impact Factor