Catabolite repression of the Bacillus subtilis a-amylase gene (amyE) involves an operator sequence located just downstream of the promoter (amyR), overlapping the transcription start site. Oligonucleotide site-directed mutagenesis of this sequence identified bases required for catabolite repression. Two mutations increased both the 2-fold symmetry of the operator and the repression ratio. Although many mutations reduced the repression ratio 3-to 11-fold, some also caused a 2-fold or greater increase in amylase production. Others caused hyperproduction without affecting catabolite repression. Homologous sequences in other catabolite-repressed B. subtilis promoters suggest a common regulatory site may be involved in catabolite repression.Enzymes involved in the metabolism of complex carbon and energy sources are unnecessary under conditions of abundant, readily metabolized alternatives such as glucose. The repression of these enzymes by glucose has been termed catabolite repression (1). In Escherichia coli, catabolite repression is mediated through the cAMP receptor protein (CRP, also called CAP, catabolite gene activator protein), which in the presence of cAMP binds specific DNA sites near promoters and activates transcription (for reviews, see refs. 2-4). Specific contacts are made between CRP, which functions as a dimer, and the CRP-binding sites on the DNA. The binding sites are roughly homologous and have a partial 2-fold symmetry in their consensus sequence (5-7). Recent evidence suggests that transcriptional activation is mediated by protein-protein interaction between CRP and RNA polymerase (8).Like E. coli, Bacillus subtilis is subject to catabolite repression but by a different mechanism. cAMP cannot normally be found in Bacillus species (9,10), except under conditions of oxygen limitation (11) BRE (trpC2, recE4, amyE). The plasmids used were p5'aGR10 (24), pAMY10 (25), pARED, and pGEMR1F (23). Plasmid pARED is a derivative of pAMY10 from which the EcoRI fragment containing the 5' end ofthe amylase structural gene and the amyR region has been deleted. The E. coli phagemid pGEMR1F (Fig. 1) contains the EcoRI fragment deleted in the generation of pARED. Subcloning the EcoRI fragment containing operator mutations from pGEMR1F back into pARED reconstitutes the amylase gene and creates a plasmid identical to pAMY10 except for the mutations. In a similar way, pAR1GR10 was created by subcloning this EcoRI fragment from p5'aB10, which contains the gralO mutation. The operator mutant plasmids were named pGEMRlMx or pARlMx, where x equals the identification number of the mutant, as noted in Tables 1 and 2. All strains were grown as described (23). Antibiotic concentrations were 10 ,ug/ml for chloramphenicol and 50 ,g/ml for ampicillin.Enzymes, Reagents, and Amylase Assays. All chemicals and reagents were at least reagent grade and were purchased from Sigma. Restriction enzymes, EcoRI and Hpa I, and DNA ligase were purchased from Promega Biotec. Amylase assays were done as reported (24).Plasmid Isolation...

SummaryGalactose transport and metabolism in Escherichia coli involves a multicomponent amphibolic pathway. Galactose transport is accomplished by two different galactose-specific transport systems. At least four of the genes and operons involved in galactose transport and metabolism have promoters containing similar regulatory sequences. These sequences are recognized by at least three regulators, Gal repressor (GaIR), Gal isorepressor (GalS) and cAMP receptor protein (CRP), which modulate transcription from these promoters. The negative regulators, GaIR and GalS, discriminate between utilization of the high-affinity (regulated by GalS) and low-affinity (regulated by GaIR) transport systems, and modulate the expression of genes for galactose metabolism in an overlapping fashion. GalS is itself autogenously regulated and CRP dependent, while the gene for GalR is constitutive. The gal operon encoding the enzymes for galactose metabolism has two promoters regulated by CRP in opposite ways; one [P^) is stimulated and the other [Pî nhibited by CRP. Both promoters are strongly repressed by GaIR but weakly by GalS. All but one of the constituent promoters of the gal regulon have two operators. The gal regulon has the potential to coordinate galactose metabolism and transport in a highly efficient manner, under a wide variety of conditions of galactose availability.

We report here a novel finding that norvaline can be incorporated in place of leucine in recombinant human hemoglobin expressed in Escherichia coli. The presence of the norvaline was confirmed by several analytical methods such as amino acid analysis, peptide mapping, electrospray mass spectrometry, and Edman protein sequencing. It appears that substitution is distributed across both the ␤-and di-␣-globins in purified recombinant hemoglobin. The level of misincorporation correlated with the ratio of the free norvaline/leucine pool available in the cell culture. This suggests that the incorporation of norvaline for leucine occurs through misaminoacylation of tRNA Leu , similar to the misincorporation of norleucine for methionine found in many recombinant proteins expressed in E. coli.

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