Figure 3.

The RtcR CARF domain is repressive, and RtcR is not activated by RtcAB or oligoadenylates in vitro

The transcriptional activity of purified RtcR and its CARF deletion variant was measured in an in vitro transcription assay leading to synthesis of radioactive RNA UpGpGpG following extension of UpG by addition of radioactive GTP from the super-coiled P_rtcBA-nifH hybrid promoter template.

(A–C) The RtcR_ΔCARF variant, in the presence of in trans CARF domain, (B) full length RtcR and cyclic tetra/hexa (4/6) adenylates (cOA), or (C) full length RtcR, RtcA and/or RtcB and cOA. The UpGpGpG was separated from unincorporated GTP by electrophoresis on a 20% (w/v) urea-PAG. In panels (A–C), the %A is percentage of transcription activity versus that seen with the RtcR_ΔCARF variant.