NUSA CONTACTS NASCENT RNA IN ESCHERICHIA-COLI TRANSCRIPTION COMPLEXES

We have examined the interaction between NusA and the nascent RNA in E scherichia coli transcription complexes on four different templates. P hotocrosslinking CTP and UTP analogs were incorporated internally and at the 3' end of the RNA. Identical templates with and without boxA se quences were compared. We found that NusA did not contact the ten nucl eotides nearest to the 3' end of the RNA in complexes containing RNA u p to 20 nucleotides long. Longer RNA did crosslink to NusA with all fo ur templates examined, however. We reported that RNA 80 nucleotides lo ng from the bacteriophage T7 Al promoter substituted in two RNA stem-l oops with photocrosslinking UMP analogs did not crosslink to NusA, eve n though interaction between NusA and the transcription complex were d emonstrated. Here, we report that when this same RNA is substituted at CMP residues, it does crosslink to NusA. Templates containing the E. coli ribosomal RNA promoter rrnG P2, with and without a boxA sequence downstream, were compared. Long RNAs from both crosslinked to NusA, an d thus boxA RNA sequences are not required for interaction with NusA. NusA did not interact with the free RNA containing boxA once released from the transcription complex, nor did it interact with RNA in a bina ry complex containing only RNA polymerase and RNA, without the DNA tem plate.