New non-detrimental DNA-binding mutants of the Escherichia coli initiator protein DnaA.
The initiator protein DnaA has several unique DNA-binding features. It binds with high affinity as a monomer to the nonamer DnaA box. In the ATP form, DnaA binds cooperatively to the low-affinity ATP-DnaA boxes, and to single-stranded DNA in the 13mer region of the origin. We have carried out an extensive mutational analysis of the DNA-binding domain of the Escherichia coli DnaA protein using mutagenic PCR. We analyzed mutants exhibiting more or less partial activity by selecting for complementation of a dnaA(Ts) mutant strain at different expression levels of the new mutant proteins. The selection gave rise to 30 single amino acid substitutions and, including double substitutions, more than 100 mutants functional in initiation of chromosome replication were characterized. The analysis indicated that all regions of the DNA-binding domain are involved in DNA binding, but the most important amino acid residues are located between positions 30 and 80 of the 94 residue domain. Residues where substitutions with non-closely related amino acids have very little effect on protein function are located primarily on the periphery of the 3D structure. By comparison of the effect of substitutions on the activity for initiation of replication with the activity for repression of the mioC promoter, we identified residues that might be involved specifically in the cooperative interaction with ATP-DnaA boxes.
Asklund
M
M
Department of Life Sciences and Chemistry, Roskilde University, DK-4000 Roskilde, Denmark.
Atlung
T
T
D000483:Alleles; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D001710:Biotin / Q000378:metabolism; D002876:Chromosomes, Bacterial / Q000235:genetics; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics; D005420:Flavoproteins / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D010641:Phenotype; D011401:Promoter Regions, Genetic / Q000235:genetics; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011995:Recombination, Genetic / Q000235:genetics; D018741:Replication Origin / Q000235:genetics; D016415:Sequence Alignment; D013696:Temperature
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D029968:Escherichia coli Proteins; D005420:Flavoproteins; C109225:MioC protein, E coli; D001710:Biotin; D004247:DNA