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Mutations that improve the pRE promoter of coliphage lambda.

M E Mahoney, D L Wulff

    Genetics
    |April 1, 1987
    PubMed
    Summary

    Mutations in the lambda pRE promoter reveal that sequences upstream of the -35 region and near the transcription start site significantly influence promoter activity. Specific thymine bases at positions -42 and -43 are crucial for maximal promoter function.

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    Area of Science:

    • Molecular Biology
    • Genetics
    • Biochemistry

    Background:

    • Promoter activity is regulated by specific DNA sequences.
    • The lambda pRE promoter is a well-studied system for investigating promoter regulation.
    • The roles of sequences outside the canonical promoter elements are not fully understood.

    Purpose of the Study:

    • To investigate the impact of mutations in the lambda pRE promoter on its activity in vivo.
    • To identify specific nucleotide sequences that influence promoter strength.
    • To determine the importance of sequences upstream of the -35 region and near the transcription start site.

    Main Methods:

    • Site-directed mutagenesis was used to create specific mutations in the lambda pRE promoter.
    • Promoter activity was measured in vivo using reporter assays.
    • The dependence of mutant promoters on the cII protein was assessed.

    Main Results:

    • A C-to-T change at position -43 (dya5 mutation) resulted in a twofold increase in pRE activity.
    • Mutations at positions -1 (dya2) and +5 (dya3) caused smaller increases in promoter activity.
    • All mutant promoters remained dependent on the cII protein.
    • Mutations converting G-C pairs to A-T pairs at positions -41, -44, and -45 did not increase activity.
    • A T-to-C change at position -42 decreased promoter activity.

    Conclusions:

    • Nucleotide sequences at least eight nucleotides upstream of the -35 region and near the transcription start site influence pRE-like promoter activity.
    • Thymine bases at positions -42 and -43 are essential for maximal lambda pRE promoter activity.
    • The findings do not support a general hypothesis that A-T richness in the -40 to -45 region correlates with higher promoter activity.

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