Effect of Mfd and Rho on the translocation state of RNAP during RNA release. Transcription complexes were stopped on the JP-CA74-R1 template by EcoRI Gln-111 protein or by nucleotide deprivation at +74 through synthesis with only CTP and ATP. The EcoRI Gln-111 block stops RNAP at +72 in these conditions (50 μM ATP and CTP), although at higher NTP concentrations, the stop is at +74, in agreement with ref. 17. Released and retained RNA were measured after incubation with Mfd or Rho and the indicated NTP. In the last six lanes, 7 μM GTP was included, allowing continued synthesis if the EcoRI Gln-111 block could be removed during the reaction.