TFIIH XPB mutants suggest a unified bacterial-like mechanism for promoter opening but not escape

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Abstract

DNA helicases open the duplex during DNA replication, repair and transcription. However, RNA polymerase II is the only member of its family with this requirement; RNA polymerases I and III and bacterial RNA polymerases open DNA without a helicase. In this report, characterization of XPB mutants indicates that its helicase activity is not used for RNA polymerase II promoter opening, which is instead driven by its ATPase activity. The mutants have parallels in σ54 bacterial transcription and this suggests a similar mode of opening DNA for both RNA polymerases, involving ATP-dependent enzyme conformational changes. Promoter escape is defective in these XPB mutants, suggesting that the XPB helicase acts as an ATP-driven motor to reorganize the tightly wrapped multiprotein eukaryotic preinitiation complex during the remodeling that precedes elongation and the coupling to RNA processing events.

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Figure 1: Helicase-defective mutants are severely deficient in transcription.
Figure 2: ATP-independent promoter opening of motif III and motif VI XPB mutants in extracts.
Figure 3: K354R, T478A and Q647A are defective in a 3′ → 5′ helicase activity assay.
Figure 4: T478A and Q647A are defective in promoter escape.
Figure 5: Helicase motif III and VI mutants can open the inv1 promoter in the absence of nucleotide but cannot clear from the promoter and enter elongation.

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Acknowledgements

We thank D. Vargas for technical assistance and M.S. Fenton for discussions. The research was supported by grant GM49048 from the US National Institutes of Health.

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Correspondence to Jay D Gralla.

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