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Structural basis for substrate selection by t7 RNA polymerase

Dmitry Temiakov1, Vsevolod Patlan, Michael Anikin

  • 1Morse Institute for Molecular Genetics, Department of Microbiology, SUNY Health Science Center, 450 Clarkson Avenue, Brooklyn, New York 11203, USA.

Cell
|March 16, 2004
PubMed

Insights

RNA polymerases (RNAPs) select substrates in an open conformation, unlike DNA polymerases. This discovery reveals a novel mechanism for substrate selection before catalytic activation, potentially universal across RNAPs.

Area of Science:

  • Molecular Biology
  • Structural Biology
  • Biochemistry

Background:

  • Nucleotide polymerases are crucial for DNA replication and transcription fidelity.
  • Substrate selection in pol I DNA polymerases is linked to the transition from an open to a closed conformation.
  • The determinants of substrate selection in DNA polymerases are associated with the closed state.

Purpose of the Study:

  • To investigate if substrate selection mechanisms are conserved in single-subunit RNA polymerases (RNAPs).
  • To determine the structure of the T7 RNAP elongation complex with an incoming substrate analog.

Main Methods:

  • X-ray crystallography to determine the structure of the T7 RNAP elongation complex.
  • Structural analysis of substrate binding in the open conformation.
  • Modeling of multisubunit RNAPs.

Main Results:

  • T7 RNAP binds substrate in an open conformation, unlike DNA polymerases.
  • Substrate binding involves base pairing with the template base in the open state.
  • Tyrosine 639 (Tyr639) distinguishes between ribose and deoxyribose substrates.

Conclusions:

  • Substrate selection in RNAPs occurs prior to isomerization to the catalytically active conformation.
  • This mechanism represents a novel pathway for substrate selection in nucleotide polymerases.
  • The findings suggest this mechanism may be conserved across all RNAPs, including multisubunit forms.

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