Beyond kinetic traps in RNA folding.

Large RNAs often have rugged folding energy landscapes that result in severe misfolding and slow folding kinetics. Several interdependent parameters that contribute to misfolding are now well understood and examples of large RNAs and ribonucleoproteins that avoid kinetic traps have been reported. These advances have facilitated the exploration of fundamental RNA folding processes that were previously inaccessible.

Concerted kinetic folding of a multidomain ribozyme with a disrupted loop-receptor interaction.

The free energy landscape for the folding of large, multidomain RNAs is rugged, and kinetically trapped, misfolded intermediates are a hallmark of RNA folding reactions. Here, we examine the role of a native loop-receptor interaction in determining the ruggedness of the energy landscape for folding of the Tetrahymena ribozyme. We demonstrate a progressive smoothing of the energy landscape for ribozyme folding as the strength of the loop-receptor interaction is reduced. Remarkably, with the most severe mutation, global folding is more rapid than for the wild-type ribozyme and proceeds in a concerted fashion without the accumulation of long-lived kinetic intermediates. The results demonstrate that a complex interplay between native tertiary interactions, divalent ion concentration, and non-native secondary structure determines the ruggedness of the energy landscape. Furthermore, the results suggest that kinetic folding transitions involving large regions of highly structured RNAs can proceed in a concerted fashion, in the absence of significant stable, preorganized tertiary structure.

An optimal Mg(2+) concentration for kinetic folding of the tetrahymena ribozyme.

Divalent metal ions, such as Mg(2+), are generally required for tertiary structure formation in RNA. Although the role of Mg(2+) binding in RNA-folding equilibria has been studied extensively, little is known about the role of Mg(2+) in RNA-folding kinetics. In this paper, we explore the effect of Mg(2+) on the rate-limiting step in the kinetic folding pathway of the Tetrahymena ribozyme. Analysis of these data reveals the presence of a Mg(2+)-stabilized kinetic trap that slows folding at higher Mg(2+) concentrations. Thus, the Tetrahymena ribozyme folds with an optimal rate at 2 mM Mg(2+), just above the concentration required for stable structure formation. These results suggest that thermodynamic and kinetic folding of RNA are cooptimized at a Mg(2+) concentration that is sufficient to stabilize the folded form but low enough to avoid kinetic traps and misfolding.

Exposing the kinetic traps in RNA folding.

Large ribozymes fold on a 'glacial' timescale compared to the folding of their protein counterparts. The sluggish folding exhibited by large RNAs results from the formation of kinetically trapped, misfolded intermediates, which are nonessential features of the folding mechanism. Newly developed mutant ribozymes that avoid kinetic traps should facilitate the study of the RNA folding problem.

Fast folding mutants of the Tetrahymena group I ribozyme reveal a rugged folding energy landscape.

A model for the kinetic folding pathway of the Tetrahymena ribozyme has been proposed where the two main structural domains, P4-P6 and P3-P7, form in a hierarchical manner with P4-P6 forming first and P3-P7 folding on the minute timescale. Recent studies in our laboratory identified a set of mutations that accelerate P3-P7 formation, and all of these mutations appear to destabilize a native-like kinetic trap. To better understand the microscopic details of this slow step in the Tetrahymena ribozyme folding pathway, we have used a previously developed kinetic oligonucleotide hybridization assay to characterize the folding of several fast folding mutants. A comparison of the temperature dependence of P3-P7 folding between the mutant and wild-type ribozymes demonstrates that a majority of the mutations act by decreasing the activation enthalpy required to reach the transition state and supports the existence of the native-like kinetic trap. In several mutant ribozymes, P3-P7 folds with biphasic kinetics, indicating that only a subpopulation of molecules can evade the kinetic barrier. The rate of folding of the wild-type increases in the presence of urea, while for the mutants urea merely shifts the distribution between the two folding populations. Small structural changes or changes in solvent can accelerate folding, but these changes lead to complex folding behavior, and do not give rise to rapid two-state folding transitions. These results support the recent view of folding as an ensemble of molecules traversing a rugged energy landscape to reach the lowest energy state.

Kinetic intermediates trapped by native interactions in RNA folding.

In the magnesium ion-dependent folding of the Tetrahymena ribozyme, a kinetic intermediate accumulates in which the P4-P6 domain is formed, but the P3-P7 domain is not. The kinetic barriers to P3-P7 formation were investigated with the use of in vitro selection to identify mutant RNA molecules in which the folding rate of the P3-P7 domain was increased. The critical mutations disrupt native tertiary interactions within the P4-P6 domain and increase the rate of P3-P7 formation by destabilizing a kinetically trapped intermediate. Hence, kinetic traps stabilized by native interactions, and not simply by mispaired nonnative structures, can present a substantial barrier to RNA folding.

A CCHC metal-binding domain in Nanos is essential for translational regulation.

The Drosophila Nanos protein is a localized repressor of hunchback mRNA translation in the early embryo, and is required for the establishment of the anterior-posterior body axis. Analysis of nanos mutants reveals that a small, evolutionarily conserved, C-terminal region is essential for Nanos function in vivo, while no other single portion of the Nanos protein is absolutely required. Within the C-terminal region are two unusual Cys-Cys-His-Cys (CCHC) motifs that are potential zinc-binding sites. Using absorption spectroscopy and NMR we demonstrate that the CCHC motifs each bind one equivalent of zinc with high affinity. nanos mutations disrupting metal binding at either of these two sites in vitro abolish Nanos translational repression activity in vivo. We show that full-length and C-terminal Nanos proteins bind to RNA in vitro with high affinity, but with little sequence specificity. Mutations affecting the hunchback mRNA target sites for Nanos-dependent translational repression were found to disrupt translational repression in vivo, but had little effect on Nanos RNA binding in vitro. Thus, the Nanos zinc domain does not specifically recognize target hunchback RNA sequences, but might interact with RNA in the context of a larger ribonucleoprotein complex.

Cisplatin-DNA adducts are molecular decoys for the ribosomal RNA transcription factor hUBF (human upstream binding factor).

The toxicity of DNA-damaging agents is widely believed to result from the formation of lesions that block polymerases or disrupt the integrity of the genome. A mechanism heretofore not addressed is that DNA damage may titrate essential DNA-binding proteins away from their natural sites of action. This report shows that the ribosomal RNA (rRNA) transcription factor hUBF (human upstream binding factor) binds with striking affinity (Kd(app) approximately 60 pM) to the intrastrand cis-[Pt(NH3)2](2+-d(GpG) crosslink formed by the anticancer drug cis-diamminedichloroplatinum(II) (cisplatin). When protein blots of human cell extracts are probed with cisplatin-modified DNA, 97- and 94-kDa proteins are detected, consistent with the known sites of hUBF species. A similar analysis of blots containing in vitro translated hUBF confirmed that the protein binds cisplatin adducts with high specificity. By contrast, DNA adducts of the clinically ineffective trans isomer of cisplatin, trans-diamminedichloroplatinum(II), are not recognized by hUBF. DNase I inhibition patterns of hUBF bound to a 100-base-pair DNA fragment containing a centrally located cis-[Pt(NH3)2](2+)-d(GpG) crosslink reveal specific protein-DNA interactions in a 14-base-pair region flanking the adduct. The affinity of hUBF for the rRNA promoter is similar (Kd(app) approximately 18 pM) to that measured for the cisplatin adduct. In addition, we observe that the hUBF-promoter interaction is highly sensitive to the antagonistic effects of cisplatin-DNA adducts. These results suggest that a cisplatin-mediated transcription-factor-hijacking mechanisms could disrupt rRNA synthesis, which is stimulated in proliferating cells.

An ultraviolet light-damaged DNA recognition protein absent in xeroderma pigmentosum group E cells binds selectively to pyrimidine (6-4) pyrimidone photoproducts.

The binding specificity was defined of a human ultraviolet light-damaged DNA recognition protein (UV-DRP), the activity of which is absent in some xeroderma pigmentosum complementation group E cells. Our results suggest that cyclobutane pyrimidine dimers (CPDs) are not high affinity UV-DRP binding sites--a finding consistent with other reports on this protein (Hirschfeld et al., (1990) Mol. Cell Biol., 10, 2041-2048). A major role for 6-4 photoproducts in UV-DRP binding was suggested in studies showing that irradiated oligonucleotides containing a T4C UV box sequence, which efficiently forms a TC 6-4 photoproduct, was a superior substrate for the UV-DRP when compared to a similar irradiated oligonucleotide having a T5 sequence. The latter sequence forms CPDs at a much higher frequency than 6-4 photoproducts. In a more direct approach, T4C-containing oligonucleotides complexed with the UV-DRP were separated from the unbound oligonucleotide fraction and the frequencies of 6-4 photoproducts in the two DNA populations were compared. The UV-DRP-bound fraction was highly enriched for the 6-4 lesion over the unbound fraction supporting the conclusion that 6-4 photoproducts are the principal binding cues for the UV-DRP.

Characterization of a DNA damage-recognition protein from mammalian cells that binds specifically to intrastrand d(GpG) and d(ApG) DNA adducts of the anticancer drug cisplatin.

A factor has been identified in extracts from human HeLa and hamster V79 cells that retards the electrophoretic mobility of several DNA restriction fragments modified with the antitumor drug cis-diamminedichloroplatinum(II) (cisplatin). Binding of the factor to cisplatin-modified DNA was sensitive to pretreatment with proteinase K, establishing that the factor is a protein. Gel mobility shifts were observed with probes containing as few as seven Pt atoms per kilobase of duplex DNA. By competition experiments the dissociation constant, Kd, of the protein from cisplatin-modified DNA was estimated to be (1-20) X 10(-10) M. Protein binding is selective for DNA modified with cisplatin, [Pt(en)Cl2] (en, ethylenediamine), and [Pt(dach)Cl2] (dach, 1,2-diaminocyclohexane) but not with chemotherapeutically inactive trans-diamminedichloroplatinum(II) or monofunctionally coordinating [Pt(dien)Cl]Cl (dien, diethylenetriamine) complexes. The protein also does not bind to DNA containing UV-induced photoproducts. The protein binds specifically to 1,2-intrastrand d(GpG) and d(ApG) cross-links formed by cisplatin, as determined by gel mobility shifts with synthetic 110-bp duplex oligonucleotides; these modified oligomers contained five equally spaced adducts of either cis-[Pt(NH3)2d(GpG) or cis-[Pt(NH3)2d(ApG)]. Oligonucleotides containing the specific adducts cis-[Pt(NH3)2d(GpTpG)], trans-[Pt(NH3)2d(GpTpG)], or cis-[Pt(NH3)2(N3-cytosine)d(G)] were not recognized by the protein. The apparent molecular weight of the protein is 91,000, as determined by sucrose gradient centrifugation of a preparation partially purified by ammonium sulfate fractionation. Binding of the protein to platinum-modified DNA does not require cofactors but is sensitive to treatment with 5 mM MnCl2, CdCl2, CoCl2, or ZnCl2 and with 1 mM HgCl2.(ABSTRACT TRUNCATED AT 250 WORDS)