In holoenzyme and preinitiation complex (PIC) models based on refined EM and X-ray structures, both the middle and head modules make multiple contacts with diverse parts of Pol II and GTFs, explaining the molecular mechanism of Mediator-stimulated CTD phosphorylation by TFIIH and resultant enhancement of basal transcription [15, 16, 19].
For this, we purified recombinant Med6p having ts-2 mutation or internal deletion mutations and examined their capabilities in restoring the transcriptional defects of Pol II holoenzyme caused by med6 ts-2 mutation .
In our previous reports, the Pol II holoenzyme prepared from med6-ts mutants is deficient in the med6-ts protein but the free form of med6-ts protein was not detected during column purifications, possibly due to its rapid degradation [12, 20].
This activator-bypass activation was observed in an artificial recruitment experiment using a holoenzyme devoid of the tail module, which is defective in the Mediator-recruitment step .
Kornberg, "Conserved structures of mediator and RNA polymerase II holoenzyme," Science, vol.
Gopalan et al., "Mediator structure and rearrangements required for holoenzyme formation," Nature, vol.
Young, "Interplay of positive and negative regulators in transcription initiation by RNA polymerase II holoenzyme," Molecular and Cellular Biology, vol.
Our data support a model whereby [T.sup.287] autophosphorylation regulates substrate gating, an intrinsic property of the catalytic domain, which is amplified within the multivalent architecture of the CaMKII holoenzyme.
Conceptually, one might expect that a similar number of activated [T.sup.287] autophosphorylated subunits ([T.sup.286] in alpha isoform) would produce similar levels of substrate phosphorylation within the PSD regardless of whether the catalytic subunits are within a holoenzyme or monomeric.
Postsynaptic densities were phosphorylated (plus [[gamma]-[sup.32]P-] ATP) with [T.sup.287] autophosphorylated holoenzyme ([CaMKII.sub.holo.sup.+P]) or monomer ([CaMKII.sub.m.sup.+P]) in the presence of [Ca.sup.2+]/CaM.
While the catalytic activity of the monomeric kinase was greater than the holoenzyme at the initiation of the PSD phosphorylation reaction, the level of activity at the end of the PSD phosphorylation was similar between monomeric and multimeric CaMKII (Figure 2(e)).
Thus, we tested whether regulating the extent of activation within the CaMKII holoenzyme regulates substrate selectivity.
We expressed maximal catalytic activity as a function of the number of subunits in the holoenzyme (the human CaMKII[delta] holoenzyme is tetradecameric ).