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T7 RNA polymerase mutants with altered promoter specificities

C A Raskin1, G A Diaz, W T McAllister

  • 1Morse Institute of Molecular Biology and Genetics, Department of Microbiology and Immunology, State University of New York Health Science Center, Brooklyn 11203-2098.

Proceedings of the National Academy of Sciences of the United States of America
|April 15, 1993
PubMed
Summary

Researchers modified T7 RNA polymerase (RNAP) at position 748, creating new RNAP mutants. Many mutants remained active and showed altered promoter specificities, expanding tools for transcription systems.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • T7 RNA polymerase (RNAP) plays a crucial role in promoter recognition and transcription initiation.
  • Amino acid residues within RNAP are critical for specific DNA sequence recognition.
  • Understanding these interactions is key to engineering RNAPs for biotechnological applications.

Purpose of the Study:

  • To investigate the role of amino acid residue 748 in T7 RNAP's promoter discrimination.
  • To generate and characterize a comprehensive set of T7 RNAP mutants at position 748.
  • To explore the impact of these mutations on RNAP activity and promoter specificity.

Main Methods:

  • Site-directed mutagenesis was employed to introduce all possible amino acid substitutions at position 748 of T7 RNAP.

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  • In vitro assays were performed to assess the transcriptional activity of the resulting mutant RNAPs.
  • Promoter specificity of active mutants was evaluated to identify alterations in DNA recognition.

    • Main Results:

    • A majority of the tested amino acid substitutions (13 out of 19) at position 748 resulted in active T7 RNAP variants.
    • Many of these active mutants displayed altered promoter specificities compared to the wild-type enzyme.
    • This indicates a significant plasticity in the promoter recognition mechanism of T7 RNAP.

    Conclusions:

    • The amino acid at position 748 is not solely responsible for precise promoter discrimination but contributes to specificity.
    • The creation of T7 RNAP mutants with altered specificities broadens the toolkit for synthetic biology and phage RNAP-based transcription systems.
    • These findings underscore the intricate nature of sequence-specific DNA recognition by RNA polymerases.