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In vivo mapping of the human adenine nucleotide translocator-2 (ANT2) promoter provides support for regulation by a pair of proximal Sp1-activating sites and an upstream silencer element.

ABSTRACT: Regulatory factors bound to the human adenine nucleotide translocator-2 (ANT2) promoter have been mapped in HeLa cells by in vivo DNase I protection and ligation-mediated PCR amplification. Protein binding was detected at only three sites within the extended promoter region (to nt -703). One, starting at nt -61 and covering the TATA box and transcription start, most probably represents occupation by the transcription-initiation machinery. A repeated Sp1 element determined by in vitro studies to be the major activation element for the promoter was also protected in vivo on nucleotides responsible for strong binding to the zinc fingers. Occupation of two additional upstream Sp1 elements was not observed. The third site occupied in vivo was identified previously by in vitro studies as a unique silencer element. Treatment of cells with trichostatin A to induce hyperacetylation released the silencer-binding protein after 1 h, but had no effect on the Sp1-activating elements. Prolonged treatment (24 h) displaced Sp1 from the activating elements. These findings confirm and extend in vitro studies indicating that regulation of the ANT2 promoter is most probably exerted through a single pair of proximal Sp1-activating elements and an upstream silencer, and that chromatin organization plays a role in the interaction between the two.

SUBMITTER: Luciakova K

PROVIDER: S-EPMC1221484 | biostudies-other | 2000 Dec

REPOSITORIES: biostudies-other

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Project description:Conformational changes are important in RNA for binding and catalysis and understanding these changes is important for understanding how RNA functions. Computational techniques using all-atom molecular models can be used to characterize conformational changes in RNA. These techniques are applied to an RNA conformational change involving a single base pair within a nine base pair RNA duplex. The Adenine-Adenine (AA) non-canonical pair in the sequence 5'GGUGAAGGCU3' paired with 3'PCCGAAGCCG5', where P is Purine, undergoes conformational exchange between two conformations on the timescale of tens of microseconds, as demonstrated in a previous NMR solution structure [Chen, G., et al., Biochemistry, 2006. 45: 6889-903]. The more populated, major, conformation was estimated to be 0.5 to 1.3 kcal/mol more stable at 30 °C than the less populated, minor, conformation. Both conformations are trans-Hoogsteen/sugar edge pairs, where the interacting edges on the adenines change with the conformational change. Targeted Molecular Dynamics (TMD) and Nudged Elastic Band (NEB) were used to model the pathway between the major and minor conformations using the AMBER software package. The adenines were predicted to change conformation via intermediates in which they are stacked as opposed to hydrogen-bonded. The predicted pathways can be described by an improper dihedral angle reaction coordinate. Umbrella sampling along the reaction coordinate was performed to model the free energy profile for the conformational change using a total of 1800 ns of sampling. Although the barrier height between the major and minor conformations was reasonable, the free energy difference between the major and minor conformations was the opposite of that expected based on the NMR experiments. Variations in the force field applied did not improve the misrepresentation of the free energies of the major and minor conformations. As an alternative, the Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) approximation was applied to predict free energy differences between the two conformations using a total of 800 ns of sampling. MM-PBSA also incorrectly predicted the major conformation to be higher in free energy than the minor conformation.

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In vivo mapping of the human adenine nucleotide translocator-2 (ANT2) promoter provides support for regulation by a pair of proximal Sp1-activating sites and an upstream silencer element.

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