Expression of the Bacillus subtilis polC gene in Escherichia coli

Ott, Russell W.; Goodman, Laurie; Ganesan, A. T.

doi:10.1007/bf00331598

Mol Gen Genet

1987

DOI: 10.1007/bf00331598

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Expression of the Bacillus subtilis polC gene in Escherichia coli

Russell W. Ott

Laurie Goodman

A. T. Ganesan

Abstract: We have cloned a 14 kb DNA segment containing the coding sequence (polC) for DNA polymerase III (PolIII) from the Bacillus subtilis chromosome. The plasmid carrying the sequence, pRO10, directs the synthesis of the 160 kDa PolIII protein and three additional polypeptides in Escherichia coli maxicells from strain CSR603. A set of deletion derivatives of pRO10 was constructed in vitro. The location of the PolIII coding sequence and the direction of transcription through the polC gene were determined by analysis … Show more

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Cited by 6 publications

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“…The promoter and encoded proteins have been analyzed. The relation between genetic and restriction maps has been established (7,8 Fig. 1.…”

mentioning

confidence: 99%

“…The promoter and encoded proteins have been analyzed. The relation between genetic and restriction maps has been established (7,8). Recently, a holoenzyme form of pol III, similar to that of E. coli, has been purified from B. subtilis (9).…”

mentioning

confidence: 99%

“…The poIC gene does not hybridize to its cognate gene of E. coli nor can it complement the thermosensitive polC mutant ofE. coli (8). The pol III ofB.…”

mentioning

confidence: 99%

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DNA polymerase III gene of Bacillus subtilis.

Sanjanwala¹,

Ganesan²

1989

Proc. Natl. Acad. Sci. U.S.A.

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The Baillus subtilis dnaF (poiC) 2). In E. coli it is an asymmetric dimer with two active sites and has nine different polypeptides in a 1:1 stoichiometry. The mass ofthis complex is 900 kDa (3). The cofactors contribute to the "fidelity, processivity, and rapidity" of the replicative synthesis. One of the subunits (e) encoded by the dnaQ gene has the proofreading 3' --5' nuclease activity (4).The pol III of B. subtilis has an intrinsic 3' -) 5' exonuclease activity and is larger than E. coli pol III (151 kDa vs. 132 kDa). Mapping of several intragenic mutations (5) and the isolation of the gene were enabled by a mutation (azp-12) that confers resistance to 6-(p-hydroxyphenylazo)uracil (HPUra). The interaction ofthe reduced form ofthe drug with the enzyme is by binding to the active site (6).The polC gene ofB. subtilis has been cloned. The promoter and encoded proteins have been analyzed. The relation between genetic and restriction maps has been established (7,8 Fig. 1. For the fragments larger than 600-700 base pairs (bp), sequencing was done by producing a series of overlapping deletions according to Dale et al. (11). The overlaps were 60-100 nucleotides between deletions. Singlestranded templates were made from an overnight culture of TG1 grown in 12 ml of2x YT broth [containing (wt/vol) Sequencing gels (6% acrylamide/7.7 M urea/90 mM Tris/ 90 mM boric acid/3.3 mM EDTA) or buffer gradient sequencing gels (6% acrylamide/7.7 M urea/10% sucrose/450 mM Trizma base/450 mM boric acid/16.5 mM EDTA/50 ,ug of bromophenol blue dye per ml at the bottom and at the top 6% acrylamide/7.7 M urea/45 mM Tris/45 mM boric acid/1.6 mM EDTA) were run at constant power to maintain surface temperature between 55°C and 65°C.

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“…The promoter and encoded proteins have been analyzed. The relation between genetic and restriction maps has been established (7,8 Fig. 1.…”

mentioning

confidence: 99%

mentioning

confidence: 99%

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DNA polymerase III gene of Bacillus subtilis.

Sanjanwala¹,

Ganesan²

1989

Proc. Natl. Acad. Sci. U.S.A.

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Genetic structure and domains of DNA polymerase III of Bacillus subtilis

Sanjanwala

Ganesan

1991

Molec. Gen. Genet.

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We have determined the nucleotide sequence of the polC gene of Bacillus subtilis which codes for DNA polymerase III. Our recent analysis has revealed that the gene comprises 4311 nucleotides, from the start to the stop codon, 306 nucleotides more than we reported earlier. The plasmid reported by us and by N.C. Brown's laboratory contained a sequence at the end of the gene which is not related to the polC region of B. subtilis. We have isolated the rest of the gene, the sequence of which is presented in this paper. The new stop codon is followed by a hyphenated palindromic sequence of 13 nucleotides. The C-terminus of the coding region contains the novel mutation, dnaF, which results in a defect in the initiation of replication due to a change in the codon TCC to TTC (serine to phenylalanine). The hypermutator mutation mut-1 is due to two point mutations in the 3' to 5' exonuclease domain, the proof reading function. The codon changes are GGA to GAA (glycine to glutamic acid) and AGC to AAC (serine to asparagine). The elongation defective mutation, polC26, affecting the catalytic site that adds nucleotides to the growing chain, is due to a change in the codon GTC to GAC (valine to aspartic acid). It is separated from the mutation reported earlier, azp-12, by 306 nucleotides. Knowing the locations of the mutational sites allowed us to deduce the domains of the gene and the enzyme it encodes, and permitted us to present a precise map of the gene at the molecular level.

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Leader region of the gene encoding DNA polymerase III of Bacillus subtilis

Sanjanwala

Ganesan

1993

Molec. Gen. Genet.

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Previously we cloned and sequenced the polC gene of Bacillus subtilis and identified regions corresponding to various catalytic domains of DNA polymerase III, the enzyme it encodes. In the present study, by using primer extension, we have identified the transcription start site and a 139 nucleotide leader region upstream of the first codon. This region contains a DnaA box in the non-transcribed DNA strand. An RNA transcript of the leader would contain a sequence that could form a 29 bp stem-loop secondary structure followed by a strong terminator sequence, rich in uracil, before the ribosome binding site. Plasmids were constructed containing either the intact leader region or deletion mutations of the leader, fused to the Escherichia coli lacZ gene in an expression vector. Single copies of the fusions were then integrated into the B. subtilis genome by transformation. Studies of the expression of beta-galactosidase by the transformed cells supported the idea that the leader region is important in regulating polC gene expression.

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Expression of the Bacillus subtilis polC gene in Escherichia coli

Cited by 6 publications

References 23 publications

DNA polymerase III gene of Bacillus subtilis.

DNA polymerase III gene of Bacillus subtilis.

Genetic structure and domains of DNA polymerase III of Bacillus subtilis

Leader region of the gene encoding DNA polymerase III of Bacillus subtilis

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