Pausing as a mechanism of nucleosome recovery

Background

Nucleosome survival during transcription is important for the maintenance of chromatin integrity, gene regulation, cell survival, and aging. In according to the studies in vitro and in vivo, Pol II pauses at positions inside of a nucleosome and nucleosome survives after Pol II transcription through chromatin [1]. A key intermediate, Ø-loop (EC+49), formed at +45 region inside of nucleosome and Pol II pausing at +45 nucleosomal region positively correlated to nucleosome survival [2, 3]. A putative electrostatic interaction between Pol II and histone octamer has also been predicted to stabilize the Ø-loop intermediate by structural modeling[3]. In this study, we will provide more evidences to link those characteristics together.

Materials and methods

Protein purification [3][4]

Computational structure modeling [3]

Transcription assay, Nucleosome fate and DNase I footprinting [3, 5]

Results

In structural modeling, three negatively charged surfaces (regions 1, 2, 3) were identified on Rpb1, a large subunit of yeast Pol II, and plausibly interacted to histone octamer, especially H2B N-tail region, by the electrostatic force. Results of transcriptions by Thermus thermophilus (T.th.) and Thermus aquaticus (T.aq.) RNAPs which contained less net negative charges at all three regions showed lower +45 pausing, no Ø-loop formation and nucleosome displacement. Finally, lower nucleosomal barrier was also shown during the transcription by mutated yeast Pol II which contains less negatively charged at region 2 of Rpb1.

Conclusions

Collectively, we demonstrated the positive correlations of higher negative net charge of the interacting region of RNAP, the stronger +45 barrier, more efficient nucleosome survival and more efficient Ø-loop intermediate formation during transcription.