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. 2015 Feb 26;43(5):2625–2637. doi: 10.1093/nar/gkv133

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© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 1. — Rat1/Rai1 terminates RNAPII in vitro in the presence of ATP. (A) In vitro transcription termination assay scheme. The EC was assembled with double-stranded DNA (EC1), 5′ phosphorylated RNA and purified RNAPII and was subsequently coupled to magnetic beads. The 3′-end of RNA was radioactively labeled by RNAPII. Rat1/Rai1 digested RNA from 5′ to 3′ up to the surface of RNAPII. If Rat1/Rai1 failed to terminate RNAPII, the polymerase would elongate further using NTPs. However, if Rat1/Rai1 terminated RNAPII, the remaining RNA, which was protected by RNAPII, would be completely degraded by Rat1/Rai1. The red arrow specifies the position (n) of the first incoming NTP. (B) Purified RNAPII complex and Rat1/Rai1 on gels stained with Coomassie. (C) Representative gel image of the in vitro transcription termination assay with Rat1/Rai1 treatment in the absence of ATP. Rat1/Rai1 did not terminate RNAPII by itself. Black arrows indicate the RNAs predicted in (A). Red arrows show ATP-misincorporated RNAs. (D) digestion by Rat1/Rai1. (E) Extension of RNA after addition of NTPs mixture. Quantification of the remaining RNA compared with the control lacking nuclease (set to 100%) is shown below. (D) Rat1/Rai1 terminates RNAPII efficiently in the presence of 2 mM ATP. (E) Quantification of the remaining RNAs after ATP treatment compared with the no ATP control (set to 100%). (F) Time course showing the degradation of 31-nt single strand RNA substrate by Rat1/Rai1 in the absence or presence of 1 mM ATP. The amount of remaining RNA is presented as a percentage of that from the 0 min reaction after Rat1/Rai1 treatment. Asterisk represents radioactive labeling at 5′end of 31-nt RNA.