Man T K, Pease A J, Winkler M E
Department of Microbiology and Molecular Genetics, University of Texas Houston Medical School, 77030-1501, USA.
J Bacteriol. 1997 Jun;179(11):3458-69. doi: 10.1128/jb.179.11.3458-3469.1997.
The arrangement of the Escherichia coli serC (pdxF) and aroA genes into a cotranscribed multifunctional operon allows coregulation of two enzymes required for the biosynthesis of L-serine, pyridoxal 5'-phosphate, chorismate, and the aromatic amino acids and vitamins. RNase T2 protection assays revealed two major transcripts that were initiated from a promoter upstream from serC (pdxF). Between 80 to 90% of serC (pdxF) transcripts were present in single-gene mRNA molecules that likely arose by Rho-independent termination between serC (pdxF) and aroA. serC (pdxF)-aroA cotranscripts terminated at another Rho-independent terminator near the end of aroA. We studied operon regulation by determining differential rates of beta-galactosidase synthesis in a merodiploid strain carrying a single-copy lambda[phi(serC [pdxF]'-lacZYA)] operon fusion. serC (pdxF) transcription was greatest in bacteria growing in minimal salts-glucose medium (MMGlu) and was reduced in minimal salts-glycerol medium, enriched MMGlu, and LB medium. serC (pdxF) transcription was increased in cya or crp mutants compared to their cya+ crp+ parent in MMGlu or LB medium. In contrast, serC (pdxF) transcription decreased in an lrp mutant compared to its lrp+ parent in MMGlu. Conclusions obtained by using the operon fusion were corroborated by quantitative Western immunoblotting of SerC (PdxF), which was present at around 1,800 dimers per cell in bacteria growing in MMGlu. RNase T2 protection assays of serC (pdxF)-terminated and serC (pdxF)-aroA cotranscript amounts supported the conclusion that the operon was regulated at the transcription level under the conditions tested. Results with a series of deletions upstream of the P(serC (pdxF)) promoter revealed that activation by Lrp was likely direct, whereas repression by the cyclic AMP (cAMP) receptor protein-cAMP complex (CRP-cAMP) was likely indirect, possibly via a repressor whose amount or activity was stimulated by CRP-cAMP.
大肠杆菌的serC（pdxF）基因和aroA基因排列成一个共转录的多功能操纵子，使得参与L-丝氨酸、磷酸吡哆醛、分支酸以及芳香族氨基酸和维生素生物合成所需的两种酶能够协同调节。核糖核酸酶T2保护试验揭示了两个主要转录本，它们起始于serC（pdxF）上游的一个启动子。80%至90%的serC（pdxF）转录本存在于单基因mRNA分子中，这些分子可能是由serC（pdxF）和aroA之间的不依赖Rho的终止产生的。serC（pdxF）-aroA共转录本在aroA末端附近的另一个不依赖Rho的终止子处终止。我们通过测定携带单拷贝λ[phi（serC [pdxF]'-lacZYA）]操纵子融合的部分二倍体菌株中β-半乳糖苷酶合成的差异速率，研究了操纵子调控。serC（pdxF）转录在生长于最低盐-葡萄糖培养基（MMGlu）中的细菌中最大，而在最低盐-甘油培养基、富集MMGlu和LB培养基中降低。与它们在MMGlu或LB培养基中的cya+ crp+亲本相比，cya或crp突变体中的serC（pdxF）转录增加。相反，与MMGlu中其lrp+亲本相比，lrp突变体中的serC（pdxF）转录减少。使用操纵子融合获得的结论通过对SerC（PdxF）的定量Western免疫印迹得到证实，在MMGlu中生长的细菌中，SerC（PdxF）每个细胞约有1800个二聚体。对serC（pdxF）终止的转录本和serC（pdxF）-aroA共转录本数量的核糖核酸酶T2保护试验支持了在测试条件下操纵子在转录水平受到调控的结论。对P（serC（pdxF））启动子上游一系列缺失的研究结果表明，Lrp的激活可能是直接的，而环腺苷酸（cAMP）受体蛋白-cAMP复合物（CRP-cAMP）的抑制可能是间接的，可能是通过一种其数量或活性受CRP-cAMP刺激的阻遏物。
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