3P118
マルチカノニカル法による RNA ポリメラーゼ C 末端領域の研究（近大先端技研）○米澤 康滋
Study on the C-terminal Domain of RNA Polymerase II by using
Multicanonical Molecular Dynamics Simuation.
（Kinki.Univ. Inst.Adv.Tech.）○Yasushige Yonezawa
In eukaryotic cells, the C-terminal domain (CTD) of the RNA polymerase II plays animportant roles to regulate the transcription of genes. The CTD consists of 26-52 tandem
heptapeptide repeats with the consensus sequence YSPTSPS. The structure of the CTD in
the RNA polymerase has not been observed by X-ray crystal diffraction experiment,suggesting the CTD has intrinsically undefined structure. The serine residues at secondand fifth position in the CTD consensus are known to be major phosphorylation sites. Thus,
the regulated phosphorylation and de-phosphorylation of the CTD switch theconformation states and carry out a vital role in the recruitment and assembly oftranscription complexes. However, so far, the nature of conformational states of the CTD
is never elucidated in experimentally and theoretically. Knowledge of the conformationstates corresponding to the different phosphorylation must be useful for understanding
the CTD mechanisms.
Here, we have tackled the issue by using an advanced computational methodology.
Recently, molecular simulations are considered to be a useful tool for supporting
experiments even in molecular biology. Indeed, molecular simulations provide atomic
detail of protein and peptide under a variety of physiological conditions. However, proteinsand peptides have the huge number of degree of freedoms. Then, normal standard
molecular simulation hardly searches the conformation space sufficiently. To address thisdifficulty, we employed an extended ensemble method, multi canonical molecular
dynamics simulation (McMD). McMD is an umbrella sampling method using thetemperature space as a reaction coordinate, significantly increasing the conformational
search ability.
To investigate phosphorylation effects on the CTD conformation, we studied foursystems: unit-repeat-heptapeptide (h-peptide) without phosphorylation, h-peptide

phosphorylated on 2Ser, h-peptide phosphorylated on 5Ser, and h-peptide phosphorylated
on simultaneously 2 and 5Ser. The phosphorylated Ser residue which is absent in thestandard force field parameter was created according to the amber general procedure.
We then conducted the McMD simulations for the unphospholylated andphosphorylated h-peptides centered at cubic box in the periodic boundary conditions with
explicit waters and added ions for neutralizing the systems as seen in Fig. 1. All of thesystems were potential energy minimized for reducing hard contacts among atoms andthen constant atom-number, temperature, and volume simulations were conducted until
reaching the equilibrium condition, followed by constant atom-number, temperature,pressure simulations.
After fixed the volumes to their averaged vales, the upper limit temperature of theMcMD simulations was set to 600K which is found to ensure efficient conformation
searches for those systems. McMD simulation needs an accurate density-of-state toperform a random walk in the temperature space. To build the density-of-state accurately,
it is necessary to iteratively carry out preparation molecular dynamics-runs until thedensity-of-state converged. After over 20-times preparation-runs, we obtained theaccurate density-of-state for all systems. Snap-shots of atomic trajectory of the systemwere taken at every 10psec interval for analysis. All of the simulations are performed byusing myPresto program package.
We then analyzed the h-peptide system from the McMD simulations. First of all, theMcMD data was re-weighted to obtain ensemble data at room-temperature. Using the reweighted data, we study the conformation space of the h-peptides. Backbone dihedral
angles, φ and ψ are well suitable to characterize a peptide conformation, so we analyzed
the thermodymanically conformational properties dependence on the phosphorylation
with respect to the dihedral angles space. The details of procedures of the analysis results
would be explained and the biological significance including cis-trans isomerase enzyme
Pin1 would be also discussed.