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Activation of a chimeric Rpb5/RpoH subunit using library selection.

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A specific mutation in the chimeric RNA polymerase subunit Rpb5 (Rp5H) enabled it to function in yeast, revealing insights into archaeal and eukaryotic RNA polymerase interactions.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Structural Biology

Background:

  • Rpb5 is a universal eukaryotic RNA polymerase subunit with N-terminal and C-terminal domains.
  • The archaeal homolog, RpoH, possesses only the C-terminal domain.
  • A chimera (Rp5H) of yeast Rpb5 N-terminus and Pyrococcus furiosus RpoH C-terminus failed to complement yeast rpb5 deletion (Δrpb5).

Purpose of the Study:

  • To investigate the functional complementation of yeast RNA polymerase by archaeal subunits.
  • To identify specific mutations enabling heterospecific complementation.
  • To elucidate the structural basis for interactions between RNA polymerase subunits.

Main Methods:

  • Random mutagenesis of the chimeric Rp5H construct.
  • Functional complementation assays in a yeast Δrpb5 strain.
  • In vivo stability and growth assays in Pyrococcus.
  • In vitro transcription assays with purified RNA polymerases.
  • Sequence alignments and analysis of crystal structures.

Main Results:

  • The E197K mutation in the Rp5H C-terminal domain enabled complementation of yeast Δrpb5.
  • The corresponding E62K mutation in archaeal RpoH was stable in vivo and did not impair in vitro transcription activity.
  • Structural analysis suggests the E197K mutation may stabilize interactions with neighboring subunits via salt bridges.

Conclusions:

  • A single amino acid substitution in the C-terminal domain can confer heterospecific function to RNA polymerase subunits.
  • The findings highlight conserved structural and functional principles in archaeal and eukaryotic RNA polymerases.
  • The study provides a structural basis for subunit interactions within the RNA polymerase complex.