Either wild-type or med6 ts mutant Pol II holoenzymes were preincubated for 10min at 25[degrees]C or 37[degrees]C with other supplements containing two DNA templates (pGAL4:G- and pGCN4:G-), GTFs, 0.5 mM ATP, and Gal4-VP16, with or without recombinant Med6p derivatives.
Previous biochemical analyses and artificial recruitment experiments using mutant Pol II holoenzymes suggested that facilitated recruitment of a holoenzyme to a promoter is necessary but not sufficient for transcriptional activation and that an unknown biochemical activity is required for transcriptional activation (postrecruitment step) .
Young, "General requirement for RNA polymerase II holoenzymes in vivo," Proceedings of the National Academy of Sciences of the United States of America, vol.
The tight association of Mediator with the CTD of Pol II forms Pol II holoenzyme, which is generally required for the transcription of most class II genes in vivo .
Interestingly, the phosphorylation profile of the non-[T.sup.287] autophosphorylated [CaMKII.sub.m] (Figure 5(b)) was highly similar to the minimally activated [CaMKII.sub.holo] (one subunit/holoenzyme condition in Figure 3(b), suggesting that inactivation (via [T.sup.306] capping) can allow holoenzymes with limited activatable subunits to behave as non-[T.sup.287]-autophosphorylated monomers.
Third, creating mixed holoenzymes of dead or [T.sup.287] A mutants in our insect expression system has an obvious limitation in that quantitatively controlling the mutant to wild-type subunit ratios within individual holoenzymes will be difficult.
Figure 2: Maximally active CaMKII holoenzymes enhance the phosphorylation of PSD proteins over monomeric CaMKII.
The CaMKII holoenzyme is comprised of 12-14 subunits [11-13] that permits coincident CaM binding to support an intraholoenzymeintersubunit autophosphorylation [14, 15] ([T.sup.286] a isoform and [T.sup.287] [beta], [gamma], and [delta] isoforms) reaction (for convenience, we use [T.sup.287] throughout the paper).
Koleske have evidence that, in yeast, several SRB proteins chair a committee - called a holoenzyme - that includes RNA polymerase and several other substances important for reading the genetic code.
But only after the cell convenes this holoenzyme does RNA polymerase proceed to the specific transcription site, Young and Koleske report in the March 31 NATURE.
"The discovery of a holoenzyme clearly has major implications in understanding the mechanism of transcription," says Michael H.
Kornberg and his colleagues at Stanford University School of Medicine suggests that other proteins link with RNA polymerase and form a different sort of holoenzyme that then starts transcribing DNA.