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Promoter analysis by saturation mutagenesis.

Baliga NS - Biol Proced Online (2001)

Bottom Line: The minimal set of nucleotides that can recruit a protein factor is called a cis-acting element.This article addresses a powerful mutagenesis strategy that can be employed to define cis-acting elements at a molecular level.Technical details including primer design, saturation mutagenesis, construction of promoter libraries, phenotypic analysis, data analysis, and interpretation are discussed.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Systems Biology. 4225 Roosevelt way NE, Suite 200, Seattle, WA 98105. USA. nbaliga@systemsbiology.org

ABSTRACT
Gene expression and regulation are mediated by DNA sequences, in most instances, directly upstream to the coding sequences by recruiting transcription factors, regulators, and a RNA polymerase in a spatially defined fashion. Few nucleotides within a promoter make contact with the bound proteins. The minimal set of nucleotides that can recruit a protein factor is called a cis-acting element. This article addresses a powerful mutagenesis strategy that can be employed to define cis-acting elements at a molecular level. Technical details including primer design, saturation mutagenesis, construction of promoter libraries, phenotypic analysis, data analysis, and interpretation are discussed.

No MeSH data available.


Map of the bop gene cluster, the minimal bop promoter and promoter mutagenesis. (A) The relative sizes and arrangement of the 4 genes, brp, bat, blp, and bop, in a gene cluster on the chromosome is shown in boxes with position and orientation of the promoters (indicated by arrows above). (B) The sequence of nucleotides within the minimal bop promoter and a few surrounding nucleotides with the TATA box, UAS, and RY box, boxed. The start codon is underlined and the transcription start site (indicated by an arrow) is numbered +1. (C) E. coli-Halobacterium shuttle plasmid (pNB series) with the cloned bop gene (black arrow) and promoter (white box). The plasmid contains origins of replication for E. coli (ori ColE1) and Halobacterium (ori pNRC100, repH) in addition to selectable markers (bla for Ampr in E.coli, and mev for Mevr in Halobacterium [shaded arrows]).
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Figure 2: Map of the bop gene cluster, the minimal bop promoter and promoter mutagenesis. (A) The relative sizes and arrangement of the 4 genes, brp, bat, blp, and bop, in a gene cluster on the chromosome is shown in boxes with position and orientation of the promoters (indicated by arrows above). (B) The sequence of nucleotides within the minimal bop promoter and a few surrounding nucleotides with the TATA box, UAS, and RY box, boxed. The start codon is underlined and the transcription start site (indicated by an arrow) is numbered +1. (C) E. coli-Halobacterium shuttle plasmid (pNB series) with the cloned bop gene (black arrow) and promoter (white box). The plasmid contains origins of replication for E. coli (ori ColE1) and Halobacterium (ori pNRC100, repH) in addition to selectable markers (bla for Ampr in E.coli, and mev for Mevr in Halobacterium [shaded arrows]).

Mentions: Simultaneous saturation mutagenesis of multiple nucleotides can yield an astronomical number of mutants making the analysis tedious. Analysis of the mutated-promoter libraries is significantly facilitated if functional sequences yield a scorable phenotype. For example, since the bop gene product provides a colorimetric screen, for mutagenesis of the bop promoter the natural function of the gene was exploited for screening functional sequences (Fig 2).


Promoter analysis by saturation mutagenesis.

Baliga NS - Biol Proced Online (2001)

Map of the bop gene cluster, the minimal bop promoter and promoter mutagenesis. (A) The relative sizes and arrangement of the 4 genes, brp, bat, blp, and bop, in a gene cluster on the chromosome is shown in boxes with position and orientation of the promoters (indicated by arrows above). (B) The sequence of nucleotides within the minimal bop promoter and a few surrounding nucleotides with the TATA box, UAS, and RY box, boxed. The start codon is underlined and the transcription start site (indicated by an arrow) is numbered +1. (C) E. coli-Halobacterium shuttle plasmid (pNB series) with the cloned bop gene (black arrow) and promoter (white box). The plasmid contains origins of replication for E. coli (ori ColE1) and Halobacterium (ori pNRC100, repH) in addition to selectable markers (bla for Ampr in E.coli, and mev for Mevr in Halobacterium [shaded arrows]).
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Related In: Results  -  Collection

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Figure 2: Map of the bop gene cluster, the minimal bop promoter and promoter mutagenesis. (A) The relative sizes and arrangement of the 4 genes, brp, bat, blp, and bop, in a gene cluster on the chromosome is shown in boxes with position and orientation of the promoters (indicated by arrows above). (B) The sequence of nucleotides within the minimal bop promoter and a few surrounding nucleotides with the TATA box, UAS, and RY box, boxed. The start codon is underlined and the transcription start site (indicated by an arrow) is numbered +1. (C) E. coli-Halobacterium shuttle plasmid (pNB series) with the cloned bop gene (black arrow) and promoter (white box). The plasmid contains origins of replication for E. coli (ori ColE1) and Halobacterium (ori pNRC100, repH) in addition to selectable markers (bla for Ampr in E.coli, and mev for Mevr in Halobacterium [shaded arrows]).
Mentions: Simultaneous saturation mutagenesis of multiple nucleotides can yield an astronomical number of mutants making the analysis tedious. Analysis of the mutated-promoter libraries is significantly facilitated if functional sequences yield a scorable phenotype. For example, since the bop gene product provides a colorimetric screen, for mutagenesis of the bop promoter the natural function of the gene was exploited for screening functional sequences (Fig 2).

Bottom Line: The minimal set of nucleotides that can recruit a protein factor is called a cis-acting element.This article addresses a powerful mutagenesis strategy that can be employed to define cis-acting elements at a molecular level.Technical details including primer design, saturation mutagenesis, construction of promoter libraries, phenotypic analysis, data analysis, and interpretation are discussed.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Systems Biology. 4225 Roosevelt way NE, Suite 200, Seattle, WA 98105. USA. nbaliga@systemsbiology.org

ABSTRACT
Gene expression and regulation are mediated by DNA sequences, in most instances, directly upstream to the coding sequences by recruiting transcription factors, regulators, and a RNA polymerase in a spatially defined fashion. Few nucleotides within a promoter make contact with the bound proteins. The minimal set of nucleotides that can recruit a protein factor is called a cis-acting element. This article addresses a powerful mutagenesis strategy that can be employed to define cis-acting elements at a molecular level. Technical details including primer design, saturation mutagenesis, construction of promoter libraries, phenotypic analysis, data analysis, and interpretation are discussed.

No MeSH data available.