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A novel method for prokaryotic promoter prediction based on DNA stability.

ABSTRACT: BACKGROUND: In the post-genomic era, correct gene prediction has become one of the biggest challenges in genome annotation. Improved promoter prediction methods can be one step towards developing more reliable ab initio gene prediction methods. This work presents a novel prokaryotic promoter prediction method based on DNA stability. RESULTS: The promoter region is less stable and hence more prone to melting as compared to other genomic regions. Our analysis shows that a method of promoter prediction based on the differences in the stability of DNA sequences in the promoter and non-promoter region works much better compared to existing prokaryotic promoter prediction programs, which are based on sequence motif searches. At present the method works optimally for genomes such as that of Escherichia coli, which have near 50 % G+C composition and also performs satisfactorily in case of other prokaryotic promoters. CONCLUSIONS: Our analysis clearly shows that the change in stability of DNA seems to provide a much better clue than usual sequence motifs, such as Pribnow box and -35 sequence, for differentiating promoter region from non-promoter regions. To a certain extent, it is more general and is likely to be applicable across organisms. Hence incorporation of such features in addition to the signature motifs can greatly improve the presently available promoter prediction programs.

SUBMITTER: Kanhere A

PROVIDER: S-EPMC545949 | biostudies-literature | 2005

REPOSITORIES: biostudies-literature

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A novel method for prokaryotic promoter prediction based on DNA stability.

Kanhere Aditi A Bansal Manju M

BMC bioinformatics 20050105

<h4>Background</h4>In the post-genomic era, correct gene prediction has become one of the biggest challenges in genome annotation. Improved promoter prediction methods can be one step towards developing more reliable ab initio gene prediction methods. This work presents a novel prokaryotic promoter prediction method based on DNA stability.<h4>Results</h4>The promoter region is less stable and hence more prone to melting as compared to other genomic regions. Our analysis shows that a method of pr ...[more]

PMID: 15631638

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Project description:BackgroundTo facilitate the screening of large quantities of new antimicrobial peptides (AMPs), we describe a cost-effective method for high throughput prokaryotic expression of AMPs. EDDIE, an autoproteolytic mutant of the N-terminal autoprotease, Npro, from classical swine fever virus, was selected as a fusion protein partner. The expression system was used for high-level expression of six antimicrobial peptides with different sizes: Bombinin-like peptide 7, Temporin G, hexapeptide, Combi-1, human Histatin 9, and human Histatin 6. These expressed AMPs were purified and evaluated for antimicrobial activity.ResultsTwo or four primers were used to synthesize each AMP gene in a single step PCR. Each synthetic gene was then cloned into the pET30a/His-EDDIE-GFP vector via an in vivo recombination strategy. Each AMP was then expressed as an Npro fusion protein in Escherichia coli. The expressed fusion proteins existed as inclusion bodies in the cytoplasm and the expression levels of the six AMPs reached up to 40% of the total cell protein content. On in vitro refolding, the fusion AMPs was released from the C-terminal end of the autoprotease by self-cleavage, leaving AMPs with an authentic N terminus. The released fusion partner was easily purified by Ni-NTA chromatography. All recombinant AMPs displayed expected antimicrobial activity against E. coli, Micrococcus luteus and S. cerevisia.ConclusionsThe method described in this report allows the fast synthesis of genes that are optimized for over-expression in E. coli and for the production of sufficiently large amounts of peptides for functional and structural characterization. The Npro partner system, without the need for chemical or enzymatic removal of the fusion tag, is a low-cost, efficient way of producing AMPs for characterization. The cloning method, combined with bioinformatic analyses from genome and EST sequence data, will also be useful for screening new AMPs. Plasmid pET30a/His-EDDIE-GFP also provides green/white colony selection for high-throughput recombinant AMP cloning.

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A novel method for prokaryotic promoter prediction based on DNA stability.

Publications

A novel method for prokaryotic promoter prediction based on DNA stability.

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