Identification, isolation, and characterization of the structural gene encoding the delta' subunit of Escherichia coli DNA polymerase III holoenzyme.
The gene encoding the delta' subunit of DNA polymerase III holoenzyme, designated holB, was cloned by a strategy in which peptide sequence was used to derive a DNA hybridization probe. The gene maps to 24.95 centisomes of the chromosome. Sequencing of holB revealed a 1,002-bp open reading frame predicted to produce a 36,936-Da protein. The gene has a ribosome-binding site and promoter that are highly similar to the consensus sequences and is flanked by two potential open reading frames. Protein sequence analysis of delta' revealed a high degree of similarity to the dnaX gene products of Escherichia coli and Bacillus subtilis, including one stretch of 10 identical amino acid residues. A lesser degree of similarity to the gene 44 protein of bacteriophage T4 and the 40-kDa protein of the A1 complex (replication factor C) of HeLa cells was seen. The gene, when placed into a tac promoter-based expression plasmid, directed expression of two proteins of similar size. By immunodetection with anti-holoenzyme immunoglobulin G, both proteins are judged to be products of holB.
University of Colorado Health Sciences Center, Department of Biochemistry, Biophysics and Genetics, Denver, Colorado 80262.
J R
JR
Carter
M A
MA
Franden
R
R
Aebersold
C S
CS
McHenry
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D003062:Codon; D004258:DNA Polymerase III / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping
D001426:Bacterial Proteins; D003062:Codon; D004269:DNA, Bacterial; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D004258:DNA Polymerase III