Observing the base-by-base search for native structure along transition paths during the folding of single nucleic acid hairpins

Biomolecular folding involves searching among myriad possibilities for the native conformation, but the elementary steps expected from theory for this search have never been detected directly. We probed the dynamics of folding at high resolution using optical tweezers, measuring individual trajectories as nucleic acid hairpins passed through the high-energy transition states that dominate kinetics and define folding mechanisms. We observed brief but ubiquitous pauses in the transition states, with a dwell time distribution that matched microscopic theories of folding quantitatively. The sequence dependence suggested that pauses were dominated by microbarriers from nonnative conformations during the search by each nucleotide residue for the native base-pairing conformation. Furthermore, the pauses were position dependent, revealing subtle local variations in energy–landscape roughness and allowing the diffusion coefficient describing the microscopic dynamics within the barrier to be found without reconstructing the shape of the energy landscape. These results show how high-resolution measurements can elucidate key microscopic events during folding to test fundamental theories of folding.

Base recognition by l-nucleotides in heterochiral DNA

An l-nucleotide residue in heterochiral oligodeoxynucleotides possesses base pairing selectivity with different effects of the neighboring bases.

Site-Selective Artificial Ribonucleases: Oligonucleotide Conjugates Containing Multiple Imidazole Residues in the Catalytic Domain

Design of site-selective artificial ribonucleases (aRNases) is one of the most challenging tasks in RNA targeting. Here, we designed and studied oligonucleotide-based aRNases containingmultipleimidazole residues in the catalytic part and systematically varied structure of cleaving constructs. We demonstrated that the ribonuclease activity of the conjugates is strongly affected by the number of imidazole residues in the catalytic part, the length of a linker between the catalytic imidazole groups of the construct and the oligonucleotide, and the type of anchor group, connecting linker structure and the oligonucleotide. Molecular modeling of the most active aRNases showed that preferable orientation(s) of cleaving constructs strongly depend on the structure of the anchor group and length of the linker. The inclusion of deoxyribothymidine anchor group significantly reduced the probability of cleaving groups to locate near the cleavage site, presumably due to a stacking interaction with the neighbouring nucleotide residue. Altogether the obtained results show that dynamics factors play an important role in site-specific RNA cleavage. Remarkably high cleavage activity was displayed by the conjugates with the most flexible and extended cleaving construct, which presumably provides a better opportunity for imidazole residues to be correctly positioned in the vicinity of scissile phosphodiester bond.

PcaU, a Transcriptional Activator of Genes for Protocatechuate Utilization in Acinetobacter

ABSTRACT The Acinetobacter pcaIJFBDKCHG operon encodes the six enzymes that convert protocatechuate to citric acid cycle intermediates. Directly downstream from the operon are quiand pob genes encoding sets of enzymes that convert quinate and p-hydroxybenzoate, respectively, to protocatechuate. Prior to this investigation, the only known regulatory gene in thepca-qui-pob cluster was pobR, which encodes a transcriptional activator that responds top-hydroxybenzoate and activates transcription ofpobA. The pca and qui genes were known to be expressed in response to protocatechuate, but a protein that mediated this induction had not been identified. This study was initiated by characterization of a spontaneous mutation that mapped upstream from pcaI and prevented expression of thepca genes. Sequencing of wild-type DNA extending from the translational start of pcaI through and beyond the location of the mutation revealed a 282-bp intergenic region and a divergently transcribed open reading frame, designated pcaU. Downstream from pcaU are two open reading frames encoding proteins similar in amino acid sequence to those associated with the oxidation of acyl thioesters. Inactivation of pcaU reduced the induced expression of pca structural genes by about 90% and impeded but did not completely prevent growth of the mutant cells with protocatechuate. PcaU was expressed in Escherichia coli and shown to bind to a portion of the pcaI-pcaUintergenic region containing a sequence identical in 16 of 19 nucleotide residues to a segment of the pob operator. Further similarity of the two regulatory systems is indicated by 54% amino acid sequence identity in the aligned primary structures of PobR and PcaU. The pob and pca systems were shown to differ, however, in the relative orientations of transcriptional starts with respect to the site where the activator binds to DNA, the size of the intergenic region, and the tightness of transcriptional control. The spontaneous mutation blocking pca gene expression was located in the promoter for the pca operon. The 19-nucleotide residue operator sequences were shown to be parts of a consensus associated with transcriptional activation of genes associated with protocatechuate catabolism. Two different binding sites for Pseudomonas putida PcaR differ from the consensus in only a single nucleotide residue, and DNA directly downstream fromAcinetobacter pcaU contains a 19-bp segment differing from the consensus in only two residues. PcaU was shown to bind to DNA containing this segment as well as to the DNA in thepcaU-pcaI intergenic region.