Figure 7.

Rpb5 incorporated into the archaeal RNAP cross-links at the same position in elongation complexes as H. Archaeal ΔH RNAP complemented in transcription reactions with Rpb5 was cross-linked in ECs stalled at position +20 (lane 7). Reactions contained transcription factors TFB and TBP (TFs), RNAP purified from Pyrococcus cells (lanes 2, 5 and 8), reconstituted ΔH RNAP and subunit Rpb5 as indicated on top of the lanes. The template DNA was derivatized with APB at position +23. Note that the presence of Rpb5 is essentially required for the formation of cross-linkable stalled ECs by the ΔH RNAP (compare lanes 6 and 7). Subunits of reconstituted ΔH RNAP run at higher molecular weight because they are His-tagged.