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R Kierzek

Publications and source records attributed to R Kierzek.

At least 37 records · Page 2Linked to original sources
PubMed1993

Binding of guanosine and 3' splice site analogues to a group I ribozyme: interactions with functional groups of guanosine and with additional nucleotides.

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.

Animals↗
PubMed1992

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

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.

Animals↗
PubMed1992

Hydrolysis of oligoribonucleotides: influence of sequence and length.

The chemical stability of phosphodiester bonds of some oligoribonucleotides in the presence of a cofactor like polyvinylpyrolidine (PVP) is sequence dependent. It was found that pyrimidine-A (YA) and pyrimidine-C (YC) are especially susceptible to hydrolysis. The hydrolyzability of this same phosphodiester bond is dependent on its position in the oligomer. The presence of 3' and 5'-adjacent nucleotides enhances hydrolysis of the UA phosphodiester bond. The acceleration of the hydrolysis of UA by a 5'-adjacent nucleotide is not base dependent. However, a 3'-adjacent purine increases hydrolysis of a UA phosphodiester bond more than a 3'-pyrimidine. The presence of the exoamino group on the 3'-side base (on 6 and 4 position for adenosine and cytidine, respectively) of YA or YZ phosphodiester bond is required for hydrolysis.

Base Sequence↗
PubMed1992

Nonenzymatic hydrolysis of oligoribonucleotides.

Selective cleavage of phosphodiester bonds in RNA is important in the processing of large RNA molecules. This paper reports specific cleavage at UA sequences in single stranded oligoribonucleotides as short as hexamers. The hydrolysis between U and A leaves a 2',3'-cyclic phosphate on the 5'-side and a 5'-hydroxyl group on the 3' side of the cleavage. The hydrolysis is promoted by a wide range of cofactors, including polymeric organic compounds such as polyvinylpyrrolydone (PVP) and by proteins. A variety of experiments suggests the cleavage is not due to contamination by ribonuclease. The rate of cleavage is a function of oligoribonucleotide, PVP and spermidine concentrations. Mg2+ is not required. The phenomenon described here can potentially provide a relatively simple way of coding chemical stability into single stranded RNA based on its sequence and structure. This process seems to be similar to that involved in post-transcriptional degradation of mRNA.

Animals↗
PubMed1992

Association of 2'-5' oligoribonucleotides.

Oligoribonucleotides with 2'-5' linkages have been synthesized on solid support. UV melting and CD experiments indicate complementary strands associate to give complexes with melting temperatures 30 to 40 degrees C lower than for duplexes formed by 3'-5' oligoribonucleotides with the same sequence. UV melting and imino proton NMR spectra and NOEs for (2'-5') CGGCGCCG are consistent with formation of an antiparallel duplex. The results suggest greater duplex stability was one factor favoring 3'-5' over 2'-5' linkages in evolution.

Base Sequence↗
PubMed1992

Context dependence of hydrogen bond free energy revealed by substitutions in an RNA hairpin.

Prediction and modeling of RNA structure requires knowledge of the free energy contributions of various interactions. Many unusual hydrogen bonds were recently proposed in the structure of a GCAA hairpin determined from nuclear magnetic resonance. The contributions of these hydrogen bonds to the folding stability of the hairpin formed by rGGCGCAAGCC have now been investigated through the use of functional group substitutions. These and previous results suggest a strong context dependence for the free energy of hydrogen bond formation. The results also suggest that the phylogenetic preference for GNRA (where N = A, C, G, or U and R = A or G) tetraloops may have a functional rather than thermodynamic basis.

Base Sequence↗
PubMed1991

Nearest-neighbor parameters for G.U mismatches: [formula; see text] is destabilizing in the contexts [formula; see text] and [formula; see text] but stabilizing in [formula; see text].

Thermodynamic parameters derived from optical melting studies are reported for duplex formation by a series of oligoribonucleotides containing G.U mismatches. The results are used to determine nearest-neighbor parameters for helix propagation by G.U mismatches. Surprisingly, the [formula; see text] nearest-neighbor free energy increment in unfavorable in the contexts [formula; see text], and [formula; see text] but favorable in the context [formula; see text]. This is a non-nearest-neighbor effect. In contrast, the [formula; see text] free energy increment is favorable and independent of context. Circular dichroism and imino proton NMR spectra of several sequences do not reveal an obvious structural basis for this dichotomy. For example, all the G.U mismatches have two slowly exchanging imino protons. The imino resonances for the G.U mismatches in GGAGUUCC, GUCGUGAC, and CCUGUAGG, however, broaden at lower temperature than the imino resonances for the interior Watson-Crick base pairs. In contrast, the imino resonances for the G.U mismatches in GGAUGUCC remain sharp at high temperature. The improved parameters for G.U mismatches should improve predictions of RNA structure from sequence.

Base Composition↗
PubMed1991

Stabilities of consecutive A.C, C.C, G.G, U.C, and U.U mismatches in RNA internal loops: Evidence for stable hydrogen-bonded U.U and C.C.+ pairs.

The stability and structure of RNA duplexes with consecutive A.C, C.A, C.C, G.G, U.C, C.U, and U.U mismatches were studied by UV melting, CD, and NMR. The results are compared to previous results for GA and AA internal loops [SantaLucia, J., Kierzek, R., & Turner, D. H. (1990) Biochemistry 29, 8813-8819; Peritz, A., Kierzek, R., & Turner, D.H. (1991) Biochemistry 30, 6428-6436)]. The observed order for stability increments of internal loop formation at pH 7 is AG = GA approximately UU greater than GG greater than or equal to CA greater than or equal to AA = CU = UC greater than or equal to CC greater than or equal to AC. The results suggest two classes for internal loops with consecutive mismatches: (1) loops that stabilize duplexes and have strong hydrogen bonding and (2) loops that destabilize duplexes and may not have strong hydrogen bonding. Surprisingly, rCGCUUGCG forms a very stable duplex at pH 7 in 1 M NaCl with a TM of 44.8 degrees C at 1 x 10(-4) M and a delta G degrees 37 of -7.2 kcal/mol. NOE studies of the imino protons indicate hydrogen bonding within the U.U mismatches in a wobble-type structure. Resonances corresponding to the hydrogen-bonded uridines are located at 11.3 and 10.4 ppm. At neutral pH, rCGCCCGCG is one of the least stable duplexes with a TM of 33.2 degrees C and delta G degrees 37 of -5.1 kcal/mol. Upon lowering the pH to 5.5, however, the TM increases by 12 degrees C, and delta G degrees 37 becomes more favorable by 2.5 kcal/mol. The pH dependence of rCGCCCGCG may be due to protonation of the internal loop C's, since no changes in thermodynamic parameters are observed for rCGCUUGCG between pH 7 and 5.5. Furthermore, two broad imino proton resonances are observed at 10.85 and 10.05 ppm for rCGCCCGCG at pH 5.3, but not at pH 6.5. This is also consistent with C.C+ base pairs forming at pH 5.5. rCGCCAGCG and rGGCACGCC have a small pH dependence, with TM increases of 5 and 3 degrees C, respectively, upon lowering the pH from 7 to 5.5. rCGCCUGCG and rCGCUCGCG also show little pH dependence, with TM increases of 0.8 and 1.4 degrees C, respectively, upon lowering the pH to 5.5.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Composition↗
PubMed1991

Thermodynamic study of internal loops in oligoribonucleotides: symmetric loops are more stable than asymmetric loops.

Thermodynamic parameters for internal loops of unpaired adenosines in oligoribonucleotides have been measured by optical melting studies. Comparisons are made between helices containing symmetric and asymmetric loops. Asymmetric loops destabilize a helix more than symmetric loops. The differences in free energy between symmetric and asymmetric loops are roughly half the magnitude suggested from a study of parameters required to give accurate predictions of RNA secondary structure [Papanicolaou, C., Gouy, M., & Ninio, J. (1984) Nucleic Acids Res. 12, 31-44]. Circular dichroism spectra indicate no major structural difference between helices containing symmetric and asymmetric loops. The measured sequence dependence of internal loop stability is not consistent with approximations used in current algorithms for predicting RNA secondary structure.

Base Sequence↗
PubMed1990

Effects of GA mismatches on the structure and thermodynamics of RNA internal loops.

UV melting, CD and NMR studies indicate rGCGAGCG and rGCAGGCG from unusually stable duplexes of type a and b. The observed delta G degree 37's in 1 M NaCl are -6.7 and -6.3 kcal/mol, respectively. For the related duplex, c, delta G degree 37 is -4.2 kcal/mol. The predicted delta G degree 37 from nearest-neighbor parameters (formula; see text) for all three duplexes is -4.7 kcal/mol (Freier, S.M., Kierzek, R., Jaeger, J.A., Sugimoto, N., Caruthers, M.H., Neilson, T., & Turner, D.H. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 9373-9377). The results suggest a special interaction in the duplexes containing GA mismatches. Presumably, this is hydrogen bonding between G and A. While the thermodynamics for (rGCGAGCG)2 and (rGCAGGCG)2 are similar, CD and the imino region of the proton NMR spectra indicate their structures are different. In particular, (rGCAGGCG)2 exhibits a CD spectrum typical of A-form geometry with a weak negative band at 280 nm. In contrast, the CD spectrum for (rGCGAGCG)2 has an intense positive band at 285 nm. The NMR spectrum of (rGCAGGCG)2 has a resonance corresponding to a hydrogen-bonded GA mismatch, while for (rGCGAGCG)2 no hydrogen-bonded imino proton is observed for the mismatch. The glycosidic torsion angles of the bases in the GA mismatches of (rGCAGGCG)2 and (rCGCAGGCG)2 are anti. Duplexes of type d, where X is A, G, or U, are more stable than e, and the stability differences are similar to those (formula; see text) observed for f versus g. Thus, 3'-dangling ends in this system make contributions to duplex stability that are similar to contributions observed with fully paired duplexes.

Adenosine↗
PubMed1990

Antibodies specific for branched ribonucleic acids.

A chemically synthesized branched tetranucleotide, G3'p5'A [2'p5'G]3'p5'C corresponding to the consensus sequence at the branch point in introns undergoing RNA splicing, was used as a hapten to elicit antibranch antibodies. Binding assays with 32P-labeled hapten and unlabeled structurally related haptens indicated that the antibodies are highly specific for the branch structure and have some specificity for the A2'p5'G sequence at the branch point, but have essentially none for a variety of other 2'p5' or 3'p5' dinucleotides or for the linear trinucleotide G3'p5'A3'p5'C. Purification of these antibodies by binding to A2'p5'G covalently linked to Sepharose followed by covalent attachment of the purified antibodies to protein A-Sepharose has provided an adsorbent that immunospecifically retains branched oligonucleotides as well as branched introns released from RNAs during in vitro splicing.

Animals↗
PubMed1990

Thermodynamic and spectroscopic study of bulge loops in oligoribonucleotides.

Thermodynamic parameters for bulge loops of one to three nucleotides in oligoribonucleotide duplexes have been measured by optical melting. The results indicate bulges Bn of An and Un have similar stabilities in the duplexes, GCGBmGCG + CGCCGC. The stability increment for a bulge depends on more than its adjacent base pairs. For example, the stability increment for a bulge is affected more than 1 kcal/mol by changing two nonadjacent base pairs or by adding terminal unpaired nucleotides (dangling ends) three base pairs away. Thus a nearest-neighbor approximation for helixes with bulges is oversimplified. Many of the non-self-complementary strands used in this study were observed to form homoduplexes. Such duplexes with GA mismatches were particularly stable.

Base Sequence↗
PubMed1989

Laser temperature-jump, spectroscopic, and thermodynamic study of salt effects on duplex formation by dGCATGC.

Salt effects on duplex formation by dGCATGC have been studied with spectroscopic, thermodynamic, and kinetic methods. Circular dichroism spectra indicate different salt conditions have little effect on the structures of the duplex and single strand. NMR chemical shifts indicate the structure of the duplex in 1 M NaCl is similar to that of the B-form determined previously in 0.5 M KCl [Nilges, M., Clore, G. M., Gronenborn, A. M., Brunger, A. T., Karplus, M., & Nilsson, L. (1987) Biochemistry 26, 3718-3733]. Optical melting experiments indicate the effect of Na+ concentration on melting temperature is similar to that expected for a polynucleotide with the same GC content. Laser temperature-jump experiments indicate the effect of Na+ concentration on the rate of duplex formation is much less than is observed for polynucleotides. The observations are consistent with expectations based on a counterion condensation model. This is surprising for a duplex with only 10 phosphates.

Circular Dichroism↗
PubMed1989

Effects of substrate structure on the kinetics of circle opening reactions of the self-splicing intervening sequence from Tetrahymena thermophila: evidence for substrate and Mg2+ binding interactions.

The self-splicing intervening sequence from the precursor rRNA of Tetrahymena thermophila cyclizes to form a covalently closed circle. This circle can be reopened by reaction with oligonucleotides or water. The kinetics of circle opening as a function of substrate and Mg2+ concentrations have been measured for dCrU, rCdU, dCdT, and H2O addition. Comparisons with previous results for rCrU suggest: (1) the 2' OH of the 5' sugar of a dinucleoside phosphate is involved in substrate binding, and (2) the 2' OH of the 3' sugar of a dimer substrate is involved in Mg2+ binding. Evidently, the binding site for a required Mg2+ ion is dependent on both the ribozyme and the dimer substrate. The apparent activation energy and entropy for circle opening by hydrolysis are 31 kcal/mol and 50 eu, respectively. The large, positive activation entropy suggests a partial unfolding of the ribozyme is required for reaction.

Animals↗
PubMed1988

Kinetics for reaction of a circularized intervening sequence with CU, UCU, CUCU, and CUCUCU: mechanistic implications from the dependence on temperature and on oligomer and Mg2+ concentrations.

The self-splicing intervening sequence from the rRNA precursor in Tetrahymena thermophila produces a covalently closed, circularized form (C IVS). Reaction rates for reverse cyclization (linearization) of C IVS by the covalent addition of the oligoncleotides CU, UCU, CUCU, and CUCUCU have been measured. The dependence of the observed rates on oligomer and Mg2+ concentrations indicates the presence of intermediates that are generated by separate binding steps for both oligomer and Mg2+. Linearization of C IVS by OH- hydrolysis is suppressed in the presence of oligomer, suggesting oligomer binds near the active site. The binding constants derived for CU at 30 degrees C in 1 and 10 mM Mg2+ are 5 X 10(3) and 2.5 X 10(4) M-1, respectively. These are roughly 4 orders of magnitude larger than expected for simple Watson-Crick base pairing. The binding constants derived for UCU, CUCU, and CUCUCU at 30 degrees C in 10 mM Mg2+ are 1.2 X 10(5), 4 X 10(5), and greater than 10(7) M-1, respectively. The free energy increments for binding of UCU and CUCU relative to CU are similar to those expected from a nearest-neighbor model for addition of base pairs. This indicates the factors responsible for the unusually strong binding of CU to C IVS are restricted to two nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

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.

Base Composition↗
PubMed1987

Sequence dependence for the energetics of terminal mismatches in ribooligonucleotides.

Stability increments of terminal mismatches on the core helixes AUGCAU and UGCGCA are reported. Enthalpy, entropy, and free energy changes of helix formation were measured spectrophotometrically for 15 oligoribonucleotides containing the core sequences and various mismatches. Free energy increments for mismatches in this series range from -0.5 to -1.1 kcal/mol. These increments for mismatches on AU base pairs are smaller than those measured previously on GC base pairs [Freier, S.M., Kierzek, R., Caruthers, M.H., Neilson, T., & Turner, D.H. (1986) Biochemistry 25, 3209-3213]. The terminal GU mismatches in the sequences GAUGCAUUp and UAUGCAUGp add approximately the same stability increment as the corresponding terminal AU mismatch. The stability increments for pyrimidine-pyrimidine and pyrimidine-purine mismatches can be approximated within 0.3 kcal/mol by adding the stability increments for the corresponding 3' and 5' unpaired nucleotides (dangling ends). Stability increments for purine-purine mismatches are approximated well by the stability increment for the corresponding 3' dangling end made more favorable by 0.2 kcal/mol. These approximations are used to provide a table of stability increments for all 48 possible sequences of mismatches.

Base Sequence↗