Nucleotide sequence of the Escherichia coli recJ chromosomal region and construction of recJ-overexpression plasmids.
The nucleotide sequence of the recJ gene of Escherichia coli K-12 and two upstream coding regions was determined. Three regions were identified within these two upstream genes that exhibited weak to moderate promoter activity in fusions to the galK gene and are candidates for the recJ promoter. recJ appeared to be poorly translated: the recJ nucleotide sequence revealed a suboptimal initiation codon GUG, no discernible ribosome-binding consensus sequence, and relatively nonbiased synonymous codon usage. Comparison of the sequence of this region of the chromosome with DNA data bases identified the gene immediately downstream of recJ as prfB, which encodes translational release factor 2 and has been mapped near recJ at 62 min. No significant homology between recJ and other previously sequenced regions of DNA was detected. However, protein sequence comparisons with a gene upstream of recJ, denoted xprB, revealed significant homology with several site-specific recombination proteins. Its genetic function is presently unknown. Knowledge of the nucleotide sequence of recJ allowed the construction of a plasmid from which overexpression of RecJ protein could be induced. Supporting the notion that translation of recJ is limiting, a strong T7 bacteriophage promoter upstream of recJ did not, by itself, allow high-level expression of RecJ protein. The addition of a ribosome-binding sequence fused to the initiator GTG of recJ in this construction was necessary to promote expression of high levels of RecJ protein.
Lovett
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Division of Cellular and Molecular Biology, Dana-Farber Cancer Institute, Boston, Massachusetts
Kolodner
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D000595:Amino Acid Sequence; D001483:Base Sequence; D002876:Chromosomes, Bacterial; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D005838:Genotype; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010957:Plasmids; D011401:Promoter Regions, Genetic; D016364:Reading Frames; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
D015345:Oligonucleotide Probes