Evidence for the Transcript Secondary Structures Predicted to Regulate Transcription Attenuation in the Trp Operon

We have examined the leader transcript of the Serratia marcescens trp operon to determine if the alternate secondary structures postulated to control transcription attenuation actually form in purified trp leader RNA. RNA secondary structures were analyzed by partial digestion of 32P end-labeled leader transcripts with single and double strand-specific nucleases. We found that the 176 nucleotide wild-type transcript formed the predicted hairpin structures designated 1:2 and 3:4; the latter structure is believed to signal transcription termination. We constructed a deletion plasmid, pSm delta 1,4, to determine whether the postulated RNA antiterminator structure 2:3 could also form. This plasmid lacked the DNA regions corresponding to RNA segments 1 and 4. We found that the leader transcript from pSm delta 1,4 formed structure 2:3. This provides the first direct evidence establishing the formation of a predicted antitermination secondary structure.