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Related Experiment Videos

Conformational changes in E. coli RNA polymerase during promoter recognition

K L Brodolin1, V M Studitsky, A D Mirzabekov

  • 1W.A. Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow.

Nucleic Acids Research
|December 11, 1993
PubMed
Summary

Researchers studied E. coli RNA polymerase interactions with the lacUV5 promoter using cross-linking. They identified specific DNA-protein contacts and protein-protein interactions in different promoter complexes, revealing how lac repressor affects these interactions.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • Bacterial transcription initiation involves RNA polymerase (RNAP) binding to promoter DNA.
  • Understanding the structural dynamics of RNAP-promoter complex formation is crucial for deciphering gene regulation.
  • The lacUV5 promoter and E. coli RNAP serve as a model system for studying transcription initiation.

Purpose of the Study:

  • To investigate the DNA-protein and protein-protein interactions during the recognition of the lacUV5 promoter by E. coli RNA polymerase.
  • To characterize the structural differences between closed (RPC), intermediate (RPI), and open (RPO) complexes.
  • To determine the effect of lac repressor on these interactions.

Main Methods:

  • Utilized formaldehyde as a cross-linking reagent to stabilize transient DNA-protein and protein-protein interactions.

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  • Analyzed cross-linked complexes using gel electrophoresis and other biochemical techniques.
  • Compared cross-linking patterns across different functional states of the RNAP-promoter complex.

    • Main Results:

    • Identified specific DNA regions (-50 to -49, -5 to -10, +5 to +8, +18 to +21) cross-linked to the RNAP beta' subunit in the open complex (RPO).
    • Observed strong sigma-beta' and beta-beta' protein-protein interactions in solution and RPO, but only beta-beta' in closed (RPC) and intermediate (RPI) complexes.
    • Demonstrated that lac repressor presence, before or after RPO formation, results in cross-linking patterns similar to RPI/RPC complexes.

    Conclusions:

    • The beta' subunit plays a key role in DNA binding within the open complex at the lacUV5 promoter.
    • Distinct protein-protein interaction profiles characterize different stages of RNAP-promoter complex formation.
    • Lac repressor binding alters RNAP-promoter interactions, shifting the complex towards an earlier, less open state.