酵母基因上游区与内含子可能的短程和长程转录协同增效作用

根据实验观察到的DNA成环和弯折机制,以140bp为分界点,探讨高频转录基因上游区与内含子之间可能存在的短程和长程转录协同增效作用（synergy）。用与随机序列做对比的方法,抽提出最近距离在140bp以下的寡核苷酸对,以及最近距离在140bp以上的寡核苷酸对。仔细分析两种距离下的可能的协同寡核苷酸对的位置特征和碱基组分,发现短程协同作用的寡核苷酸对的平均最近距离都在110bp以下,位于上游区的CCAA是一个很明显的特征;而长程协同作用的寡核苷酸对的平均最近距离集中在250-400bp,并且在多数寡核苷酸对中,位于上游区的寡核苷酸是GC丰富的正调控元件。     

The transmission of stability or instability from site specific protein-DNA complexes.

Theoretical calculations were made to determine the influence of side specific 'melting' and 'stabilizing' proteins on the thermal stability of nearby base pairs (bp). A DNA sequence 999bp. long containing the 123 bp. lactose operon control region in the center was examined. Melting curves of base pairs near the binding sites of the catabolite activator protein, CAP, the lactose repressor, and RNA polymerase were calculated in the absence and presence of each protein. The empirical loop entropy model of the helix-coil transition of DNA was employed. Calculations show that melting and stabilizing proteins alter the tm of base pairs 20 to 100 bp-away. The magnitude and range of the effect is strongly influenced by the base pair composition and sequence of the protein site and the immediately adjacent DNA regions.     

Influence of DNA aptamer structure on the specificity of binding to Taq DNA polymerase

The secondary structure of DNA aptamer to Taq DNA polymerase was established as a hairpin. Both stem and loop structures of DNA ligand were shown to be involved in the interaction with Taq DNA polymerase. Moreover, the structure and sequence of DNA aptamer that was the most effective inhibitor of DNA polymerase activity were established. This crucial structure was evaluated as a GC-rich stem longer than 17 bp, and a loop consisting of 12 bases with strictly determined nucleotide sequence. It was demonstrated that nucleotide in position 23 counting from the 5"-end of DNA ligand was involved in direct contact with Taq DNA polymerase. The ability of optimized DNA aptamer TQ21-11 to form a complex with the enzyme was increased 5-fold in comparison to the initial aptamer.     

Binding leverage as a molecular basis for allosteric regulation

Allosteric regulation involves conformational transitions or fluctuations between a few closely related states, caused by the binding of effector molecules. We introduce a quantity called binding leverage that measures the ability of a binding site to couple to the intrinsic motions of a protein. We use Monte Carlo simulations to generate potential binding sites and either normal modes or pairs of crystal structures to describe relevant motions. We analyze single catalytic domains and multimeric allosteric enzymes with complex regulation. For the majority of the analyzed proteins, we find that both catalytic and allosteric sites have high binding leverage. Furthermore, our analysis of the catabolite activator protein, which is allosteric without conformational change, shows that its regulation involves other types of motion than those modulated at sites with high binding leverage. Our results point to the importance of incorporating dynamic information when predicting functional sites. Because it is possible to calculate binding leverage from a single crystal structure it can be used for characterizing proteins of unknown function and predicting latent allosteric sites in any protein, with implications for drug design.     

Molecular Characterization of Equine APRIL and its Expression Analysis During the Adipogenic Differentiation of Equine Adipose-Derived Stem Cell In Vitro

A proliferation inducing ligand (APRIL) is a member of the TNF superfamily. It shares two receptors with B-cell activating factor (BAFF), B-cell maturation antigen (BCMA), and transmembrane activator and CAML interactor (TACI). Herein, the equine APRIL was identified from equine adipose-derived stem cell (ASC), and the protein expression of APRIL and its related molecules were detected during the adipogenic differentiation of equine ASC in vitro. The equine APRIL gene was located on chromosome 11, spans 1852 base pairs (bp). Its open reading frame covers 753 bp, encoding a 250-amino acid protein with the typical TNF structure domain. During the two weeks’ adipogenic differentiation of equine ASC, although the protein expression of APRIL and TACI had an insignificant change, that of BCMA increased significantly. Moreover, with the addition of recombinant protein His₆-sAPRIL, a reduced differentiation of equine ASC toward adipocyte was detected. These results may provide the basis for investigating the role of APRIL in ASC adipogenic differentiation.     

Modeling H3 histone N-terminal tail and linker DNA interactions

Molecular dynamics computer simulations were performed for the 25-residue N-terminal tail of the H3 histone protein in the proximity of a DNA segment of 10 base pairs (bp), representing a model for the linker DNA in chromatin. Several least biased configurations were used as initial configurations. The secondary structure content of the protein was increased by the presence of DNA close to it, but the locations of the secondary motifs were different for different initial orientations of the DNA grooves with respect to the protein. As a common feature to all simulations, the electrostatic attraction between negatively charged DNA and positively charged protein was screened by the water solvent and counterbalanced by the intrinsic compaction of the protein due to hydrophobic effects. The protein secondary structure limited the covering of DNA by the protein to 4-5 bp. The degree of compaction and charge density of the bound protein suggests a possible role of H3 tail in a nonspecific bending and plasticity of the linker DNA when the protein is located in the crowded dense chromatin.     

DNA structure requirements for the Escherichia coli gamma complex clamp loader and DNA polymerase III holoenzyme

The Escherichia coli chromosomal replicase, DNA polymerase III holoenzyme, is highly processive during DNA synthesis. Underlying high processivity is a ring-shaped protein, the beta clamp, that encircles DNA and slides along it, thereby tethering the enzyme to the template. The beta clamp is assembled onto DNA by the multiprotein gamma complex clamp loader that opens and closes the beta ring around DNA in an ATP-dependent manner. This study examines the DNA structure required for clamp loading action. We found that the gamma complex assembles beta onto supercoiled DNA (replicative form I), but only at very low ionic strength, where regions of unwound DNA may exist in the duplex. Consistent with this, the gamma complex does not assemble beta onto relaxed closed circular DNA even at low ionic strength. Hence, a 3'-end is not required for clamp loading, but a single-stranded DNA (ssDNA)/double-stranded DNA (dsDNA) junction can be utilized as a substrate, a result confirmed using synthetic oligonucleotides that form forked ssDNA/dsDNA junctions on M13 ssDNA. On a flush primed template, the gamma complex exhibits polarity; it acts specifically at the 3'-ssDNA/dsDNA junction to assemble beta onto the DNA. The gamma complex can assemble beta onto a primed site as short as 10 nucleotides, corresponding to the width of the beta ring. However, a protein block placed closer than 14 base pairs (bp) upstream from the primer 3' terminus prevents the clamp loading reaction, indicating that the gamma complex and its associated beta clamp interact with approximately 14-16 bp at a ssDNA/dsDNA junction during the clamp loading operation. A protein block positioned closer than 20-22 bp from the 3' terminus prevents use of the clamp by the polymerase in chain elongation, indicating that the polymerase has an even greater spatial requirement than the gamma complex on the duplex portion of the primed site for function with beta. Interestingly, DNA secondary structure elements placed near the 3' terminus impose similar steric limits on the gamma complex and polymerase action with beta. The possible biological significance of these structural constraints is discussed.