Molecular cloning, sequencing, and overexpression of the structural gene encoding the delta subunit of Escherichia coli DNA polymerase III holoenzyme.
Using an oligonucleotide hybridization probe, we have mapped the structural gene for the delta subunit of Escherichia coli DNA polymerase III holoenzyme to 14.6 centisomes of the chromosome. This gene, designated holA, was cloned and sequenced. The sequence of holA matches precisely four amino acid sequences obtained for the amino terminus of delta and three internal tryptic peptides. A holA-overproducing plasmid that directs the expression of delta up to 4% of the soluble protein was constructed. Sequence analysis of holA revealed a 1,029-bp open reading frame that encodes a protein with a predicted molecular mass of 38,703 Da. holA may reside downstream of rlpB in an operon, perhaps representing yet another link between structural genes for the DNA polymerase III holoenzyme and proteins involved in membrane biogenesis. These and other features are discussed in terms of genetic regulation of delta-subunit synthesis.
Carter
J R
JR
Department of Biochemistry, Biophysics and Genetics, University of Colorado Health Sciences Center, Denver 80262.
Franden
M A
MA
Aebersold
R
R
McHenry
C S
CS
D000595:Amino Acid Sequence; D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D004258:DNA Polymerase III / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D017422:Sequence Analysis, DNA; D013728:Terminator Regions, Genetic / Q000235:genetics
D003062:Codon; D010446:Peptide Fragments; D011994:Recombinant Proteins; D004258:DNA Polymerase III