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. 2014 Jan 6;42(6):3884–3893. doi: 10.1093/nar/gkt1356

Figure 5.

Figure 5.

The upstream promoter DNA region contours mtRNAP molecule in the pre-IC. (A) DNA cross-linking at −49 base is TFAM-dependent. Pre-initiation complexes (150 nM of 32P-labeled template containing 4-thio UMP at position −49, 150 nM mtRNAP and 0–400 nM TFAM) were UV-irradiated for 15 min. (B) Mutant mtRNAP lacking N-terminal TFAM-binding site does not cross-link to the upstream promoter DNA. The cross-link was performed using WT or Δ150 mtRNAP in the presence (lanes 2,4) or absence (lanes 1,3) of TFAM by UV-irradiation for 15 min. (C) Promoter sequence is required for DNA-TFAM cross-linking. The reaction contained WT mtRNAP, TFAM (where indicated) and templates with (lanes 1,2) or without (lanes 3,4) LSP promoter sequence. (D) The far upstream promoter region (−60 to −40) is important for efficient transcription. Transcription activity was measured using synthetic template having LSP promoter from −40 (‘−40’, lane 1), nonspecific sequence from −40 to −60 (‘−60NS’, lane 2) or LSP promoter from −60 (‘−60’, lane 3).