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Purification and base composition analysis of phage lambda early promoters.

J Y Le Talaer, P Jeanteur

    Proceedings of the National Academy of Sciences of the United States of America
    |December 1, 1971
    PubMed
    Summary
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    Researchers identified specific DNA fragments protected by Escherichia coli RNA polymerase during phage lambda DNA binding. These fragments, particularly those dependent on the sigma factor, are enriched in A-T sequences and likely involved in promoter recognition.

    Area of Science:

    • Molecular Biology
    • Genetics
    • Biochemistry

    Background:

    • Escherichia coli RNA polymerase (RNAP) is crucial for gene transcription.
    • Understanding RNAP-DNA interactions is key to deciphering gene regulation.
    • Phage lambda DNA serves as a model system for studying these interactions.

    Purpose of the Study:

    • To identify DNA sequences protected by E. coli RNAP during binding to phage lambda DNA.
    • To investigate the role of the sigma factor in this protection mechanism.
    • To characterize the sequence and properties of protected DNA fragments.

    Main Methods:

    • RNAP-DNA complex formation in the absence of precursors.
    • Nuclease digestion to excise the protected complex.
    • Phenol-extraction and ethanol precipitation for DNA recovery.

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  • Acrylamide gel electrophoresis for fragment analysis.

    • Main Results:

    • Two distinct oligonucleotide peaks (45-52 and 7-10 nucleotides) were observed.
    • Peak I fragments (45-52 nt) require the sigma factor for their existence.
    • Peak I fragments are significantly enriched in A-T base pairs (up to 67%).
    • Peak II fragments (7-10 nt) exhibit base composition similar to phage lambda DNA.

    Conclusions:

    • Peak I DNA fragments are likely involved in promoter recognition by E. coli RNAP.
    • The sigma factor plays a critical role in selecting these promoter-proximal DNA sequences.
    • These findings contribute to understanding the initial steps of transcription initiation.