Supershift EMSA with PICs formed in presence or absence of TFEα/β (0.1 µM) being exposed to antisera directed against TFEα, TFEβ and TBP.
In agreement with previous findings (Kolitz et al., 2009; Dong et al., 2014; Martin-Marcos et al., 2014), TC dissociates very little from WT PICs formed with mRNA AUG) over the time course of the experiment, yielding a rate constant of only 0.05 hr−1.
The kon values measured for WT and E144R 40subunitsts were essentially identical, indicating that PICs formed with the mutant ribosomes assemble the POUT complex and rearrange to PIN at the same rates achieved with WT ribosomes, and that rps- E144R primarily reduces the stability of the PIN state.
Although it was well known that perturbation of nucleosome positioning can result in isolated cryptic/unscheduled transcription events [14,15], it is now apparent that even in the unperturbed chromatin context Pol II engages with PICs formed on DNA wherever they find access (Fig. 1A) [1A].
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The results of the expression analysis and biochemical experiments implied that the PIC formed on the promoters of active genes in hESCs would have an atypical TAF composition.
PICs that form with TFIIB R66E are rendered defective prior to pol II CTD serine 5 phosphorylation.
During this time reverse transcription is completed and PICs are formed [34].
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Thus, TFIIB S65A and R66E can form PICs that contain pol II, TFIIF, TFIIE, and TFIIH but fail to form a productive transcription complex.
