The priB and priC replication proteins of Escherichia coli. Genes, DNA sequence, overexpression, and purification

The Escherichia coli DNA replication proteins n and n" function in vitro in the assembly of the primosome, a mobile multiprotein replication priming complex thought to operate on the lagging-strand template at the E. coli DNA replication fork. Both proteins have been purified from E. coli HMS83...

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Bibliographic Details
Published in:The Journal of biological chemistry 1991-07, Vol.266 (21), p.13988-13995
Main Authors: ZAVTIZ, K. H, DIGATE, R. J, MARIANS, K. J
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Bacterial Proteins - genetics
Bacterial Proteins - isolation & purification
Base Sequence
Biological and medical sciences
Cloning, Molecular
Codon
DNA
DNA Replication
DNA, Bacterial - genetics
Escherichia coli
Escherichia coli - genetics
Escherichia coli Proteins
Fundamental and applied biological sciences. Psychology
Gene Expression
genes
Genes, Bacterial
Molecular and cellular biology
Molecular genetics
Molecular Sequence Data
n protein
nucleotide sequence
predictions
priB gene
priC gene
Recombinant Proteins - isolation & purification
Replication
role
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Summary:The Escherichia coli DNA replication proteins n and n" function in vitro in the assembly of the primosome, a mobile multiprotein replication priming complex thought to operate on the lagging-strand template at the E. coli DNA replication fork. Both proteins have been purified from E. coli HMS83 cells based on their requirement for the reconstitution of bacteriophage phi X174 complementary strand DNA synthesis in vitro with purified proteins. As a step toward understanding the role of these proteins in vivo, the genes for primosomal proteins n and n", designated priB and priC, respectively, have been cloned molecularly. priB encodes a 104-amino acid 11.4-kDa polypeptide and corresponds to an previously identified open reading frame between rpsF and rps R within a ribosomal protein operon at 95.5 min on the E. coli chromosome. priC encodes a 175-amino acid 20.3-kDa polypeptide. These two gene products were overexpressed at least 1000-fold in E. coli using a bacteriophage T7 transient expression system. Both proteins have been purified to apparent homogeneity from extracts prepared from these overproducing strains.
ISSN:0021-9258
1083-351X
DOI:10.1016/s0021-9258(18)92800-0