title: Differential turnover of the multiple processed transcripts of the Escherichia coli focA-pflB operon.
abstract: Expression of the anaerobically inducible focA-pflB operon of Escherichia coli is subject to complex transcriptional and post-transcriptional control, which generates eight transcripts whose 5' ends span approximately 1.2 kb. All eight transcripts have the same 3' end. The 5' ends of three of the transcripts, termed 6, 6a and 7, are located upstream of the operon. The promoters generating transcripts 6 and 7 are anaerobically regulated by FNR and ArcA approximately P, while promoter 6a is constitutively active. The 5' ends of the other five transcripts are all located within the operon. Most of the 5' ends of these operon-internal transcripts result from RNA polymerase-dependent processing of the three longer primary transcripts, 6, 6a and 7. Here, it is demonstrated that subsequent to, and distinct from, these processing events, post-transcriptional modification of these transcripts also occurs through the action of the endoribonuclease RNase E. Transcripts 6 and 7 exhibit differential stability with half-lives of 1 and 5 min, respectively. Transcript 7, which has the longer half-life, is the longest transcript of the operon and has a approximately 340 base untranslated leader. Two of the operon-internal transcripts, 4 and 5, also have comparatively short half-lives in the wild-type, which are significantly increased in a mutant with impaired RNase E activity. A precursor-product relationship is observed between the longer transcripts 3-7 and transcripts 1 and 2. The 5' ends of transcripts 1 and 2 are closest to the pflB gene and have half-lives of approximately 7-8 min. The consequence of this regulation is an accumulation of full-length pflB transcript and comparably low levels of dicistronic transcript. This ensures different levels of synthesis of the formate transporter FocA and pyruvate formate-lyase during anaerobic growth, while maintaining coordinate regulation. Transcript analysis throughout the growth phase revealed that maximal anaerobic expression of the focA-pflB operon was restricted to exponentially growing cells. Expression of transcript 7 peaked in early to mid-exponential phase, while the levels of transcript 6 steadily accumulated toward the late-exponential phase of growth. Taken together, these findings indicate that although subject to common positive control by ArcA approximately P and FNR, the transcripts generated by promoters 6 and 7 are subject to differential temporal and post-transcriptional regulation.
authors.affiliation: Department of Molecular Microbiology, John Innes Centre, Norwich, UK.
authors.forename: R Gary
authors.initials: RG
authors.lastname: Sawers
mesh_terms: D004722:Endoribonucleases / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D006207:Half-Life; D026901:Membrane Transport Proteins / Q000235:genetics*; D009876:Operon; D012333:RNA, Messenger / Q000032:analysis
chemical_list: D029968:Escherichia coli Proteins; C088103:FocA protein, E coli; D026901:Membrane Transport Proteins; D012333:RNA, Messenger; D004722:Endoribonucleases; C022537:ribonuclease E