Abstract
The nucleotide sequence of the mtlA gene, which codes for the mannitol-specific Enzyme II of the Escherichia coli phosphotransferase system, is presented. From the gene sequence, the primary translation product is predicted to consist of 637 amino acids (Mr = 67,893). This result is compared to the amino acid composition and molecular weight of the purified mannitol Enzyme II protein. The hydrophobic and hydrophilic properties of the enzyme were evaluated along its amino acid sequence using a computer program (Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132). The computer analysis predicts that the NH2-terminal half of the enzyme resides within the membrane, whereas the COOH-terminal half of the enzyme has the properties of a soluble protein. The possible functions of such a protein structure are discussed. RNA mapping has identified the promoter and mRNA start point for the mtl operon.
Highlights
Codes for the mannitol-specific Enzyme I1 of the Esch- In addition, mutagenesiosf the structuragl ene for theEnzyme erichia coli phosphotransferase system, is presented
Biol. 157, 105-132).The computer analysis predicts that the NHz-terminal half of the enzyme resides within the membrane, whereasthe COOH-terminal half of the enzyme has the properties of a soluble protein
T, polynucleotide kinase was from P-L Biochemicals identified the mtl operon promoter, the mRNA start site, andandthe Klenow fragment of E. coli DNA polymerase was from two RNA 3'-ends, on less than 5% of mtl transcripts, that map in the intercistronic region between the mtlA and mtlD
Summary
Bacterial Strains and Plasmids-Plasmid DNA from E. coli strain L163sr/pCL2.0 was utilized for the nucleotide sequence determination [11]. Nucleotide Sequence of the mtlAGene-A 2-kilobase pair fragment of DNA has been cloned which contains the mtlA gene [11].Fig. 2 shows a detailedmap of the cloned DNAand indicates the restriction sites utilized for the nucleotide sequencing method of Maxam and Gilbert [14]. DNA fragment were Hue111 restrictionsitesdestroyed by ligation to EcoRI linkersduringconstruction of the clone [11].The arrows above and below the restriction map indicate the beginning, direction, and extenoft the nucleotide sequence determination of the noncoding and the coding' strands of the DNA,respectively (Fig. 2). + subsequent nuclease S1 treatment; lane 7,C-specific Maxam-Gilbert sequencing ladder of the DNA probe; lane 8,. The highly conserved nucleotides in the -10 and -35 gins at position 10 and the mtl mRNA start site (T-A-T) is regions are capitalized.
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