Supplemental Data Mediator-Dependent Recruitment of TFIIH Modules in Preinitiation Complex

Plasmid constructions and cloning All cloning were done using the Gateway Invitrogen cloning method. Wild-type MED11 gene was amplified from YPH499 genomic DNA using oligonucleotides matching the gene sequence initiation codon and following codons for the 5' forward primer and the stop codon and preceding codons for the 3' reverse primer. The oligonucleotides were flanked with attB1 or attB2 sequences, respectively. The amplified sequence was cloned into pDONR201 (Invitrogen) using standard BP reaction. The recombinant plasmid was sequence verified. The cloned sequence was then transferred into pVV208 (CEN URA3 pTetO7) or pVV204 (CEN TRP1 pTetO7) vector (Van Mullem et al., 2003) by the LR reaction. RAD3 was cloned into pVV221 (2µ URA3 pTetO7) and the MED11 alleles into pVV220 (2µ TRP1 pTetO7), pVV212 (derived from pGBT9) and pVV213 (derived from pACTII) using a similar procedure. K7N, V68D and G108S point mutations in Med11 were obtained by PCR overlap extension mutagenesis (Higuchi et al., 1988), cloned using the Gateway standard method and transferred into pVV204.