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 ∼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∼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 ∼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∼P and FNR, the transcripts generated by promoters 6 and 7 are subject to differential temporal and post-transcriptional regulation.
ApirionD, GegenheimerP.
1984; Molecular biology of RNA processing in prokaryotic cells. In Processing of RNA pp 36–52 Edited by
ApirionD.
Boca Raton, FL: CRC Press;
AristarkhovA, MikulskisA, BelascoJ. G, LinE. C. C.
1996; Translation of the adhE transcript to produce ethanol dehydrogenase requires RNaseIII cleavage in Escherichia coli . J Bacteriol 178:4327–4332
BeggY. A, WhyteJ. N, HaddockB. A.
1977; The identification of mutants of Escherichia coli deficient in formate dehydrogenase and nitrate reductase activities using dye indicator plates. FEMS Microbiol Lett 2:47–50[CrossRef]
CallaghanA. J, MarcaidaM. J, SteadJ. A, McDowallK. J, ScottW. G, LuisiB. F.
2005; Structure of Escherichia coli RNase E catalytic domain and implications for RNA turnover. Nature 437:1187–1191[CrossRef]
CarpousisA. J.
2002; The Escherichia coli RNA degradosome: structure, function and relationship in other ribonucleolytic multienzyme complexes. Biochem Soc Trans 30:150–155
CasadabanM. J, CohenS. N.
1979; Lactose genes fused to exogenous promoters in one step using Mu-lac bacteriophage: in vivo probe for transcriptional control sequences. Proc Natl Acad Sci U S A 76:4530–4533[CrossRef]
ChristiansenL, PedersenS.
1981; Cloning, restriction endonuclease mapping and post-transcriptional regulation of rpsA , the structural gene for ribosomal protein S1. Mol Gen Genet 181:548–551[CrossRef]
DrapalN, SawersG.
1995; Promoter 7 of the Escherichia coli pfl operon is a major determinant in the anaerobic regulation of expression by ArcA. J Bacteriol 177:5338–5341
HigginsC. F, SmithN. H.
1986; Messenger RNA processing, degradation and the control of gene expression. In Regulation of Gene Expression – 25 Years On (Society for General Microbiology Symposium no. 39) pp 179–198 Edited by
BoothI. R., HigginsC. F.
Cambridge: Cambridge University Press;
KnappeJ, SawersG.
1990; A radical route to acetyl-CoA: the anaerobically induced pyruvate formate-lyase system of Escherichia coli . FEMS Microbiol Rev 75:383–398
LiZ, PanditS, DeutscherM. P.
1999; RNase G (CafA protein) and RNase E are both required for the 5′ maturation of 16S ribosomal RNA. EMBO J 18:2878–2885[CrossRef]
MuddE. A, KrischH. M, HigginsC. F.
1990; RNase E, an endoribonuclease, has a general role in the chemical decay of Escherichia coli mRNA: evidence that rne and ams are the same genetic locus. Mol Microbiol 4:2127–2135[CrossRef]
OwM. C, LiuQ, KushnerS. R.
2000; Analysis of mRNA decay and rRNA processing in Escherichia coli in the absence of RNase E-based degradosome assembly. Mol Microbiol 38:854–866[CrossRef]
OwM. C, LiuQ, MohantyB. K, AndrewM. E, MaplesV. F, KushnerS. R.
2002; RNase E levels in Escherichia coli are controlled by a complex regulatory system that involves transcription of the rne gene from three promoters. Mol Microbiol 43:159–171[CrossRef]
Reyes-RamírezF.,
SawersR. G.
2006; Aerobic activation of the anaerobically inducible Escherichia coli focA-pfl operon transcription by FNR. FEMS Microbiol Lett 255:262–267[CrossRef]
SawersG.
1993; Specific transcriptional requirements for positive regulation of the anaerobically inducible pfl operon by ArcA and FNR. Mol Microbiol 10:737–747[CrossRef]
SawersR. G.
2005a; Expression of fnr is constrained by an upstream IS 5 insertion in certain Escherichia coli K-12 strains. J Bacteriol 187:2609–2617[CrossRef]
SawersR. G.
2005b; Evidence for novel processing of the anaerobically inducible dicistronic focA-pfl mRNA transcript in Escherichia coli . Mol Microbiol 58:1441–1453[CrossRef]
SawersG, SuppmannB.
1992; Anaerobic induction of pyruvate formate-lyase gene expression is mediated by the ArcA and FNR proteins. J Bacteriol 174:3474–3478
SawersR. G, ClarkD. P.
2004; Fermentative pyruvate and acetyl CoA metabolism. In EcoSal – Escherichia coli and Salmonella Cellular and Molecular Biology , chapter 3.5.3; (posted July 2004) Editor-in
ChiefR.
Curtiss, III. Washington, DC: American Society for Microbiology; www.ecosal.org
SuppmannB, SawersG.
1994; Isolation and characterisation of hypophosphite-resistant mutants of Escherichia coli : identification of the FocA protein, encoded by the pfl operon, as a putative formate transporter. Mol Microbiol 11:965–982[CrossRef]
TockM. R, WalshA. P, CarrollG, McDowallK. J.
2000; The CafA protein required for the 5′-maturation of 16S rRNA is a 5′-end-dependent ribonuclease that has context-dependent broad sequence specificity. J Biol Chem 275:8726–8732[CrossRef]
TowbinH, StaehelinT, GordonJ.
1979; Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76:4350–4354[CrossRef]
UmitsukiG, WachiM, TakadaA, HikichiT, NagaiK.
2001; Involvement of RNase G in in vivo mRNA metabolism in Escherichia coli . Genes Cells 6:403–410[CrossRef]
WagnerA. F. V, FreyM, NeugebauerF. A, KnappeJ,
SchäferW. 1992; The free-radical in pyruvate formate-lyase is located on glycine-734. Proc Natl Acad Sci U S A 89:996–1000[CrossRef]