Structural Basis for Substrate Selection by T7 RNA Polymerase

The mechanism by which nucleotide polymerases select the correct substrate is of fundamental importance to the fidelity of DNA replication and transcription. During the nucleotide addition cycle, pol I DNA polymerases undergo the transition from a catalytically inactive “open” to an active “closed”...

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Bibliographic Details
Published in:Cell 2004-02, Vol.116 (3), p.381-391
Main Authors: Temiakov, Dmitry, Patlan, Vsevolod, Anikin, Michael, McAllister, William T, Yokoyama, Shigeyuki, Vassylyev, Dmitry G
Format: Article
Language:English
Subjects:
Binding Sites - genetics
Catalytic Domain - genetics
Deoxyribose - metabolism
DNA Polymerase I - genetics
DNA Polymerase I - metabolism
DNA Replication - genetics
DNA-Directed RNA Polymerases - genetics
DNA-Directed RNA Polymerases - metabolism
Evolution, Molecular
Isomerism
Models, Molecular
Nucleotides - metabolism
Phage T7
Protein Conformation
Protein Subunits - genetics
Protein Subunits - metabolism
Ribose - metabolism
RNA, Messenger - biosynthesis
RNA, Messenger - genetics
Structure-Activity Relationship
Substrate Specificity - physiology
Transcription, Genetic - genetics
Tyrosine - metabolism
Viral Proteins
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Summary:The mechanism by which nucleotide polymerases select the correct substrate is of fundamental importance to the fidelity of DNA replication and transcription. During the nucleotide addition cycle, pol I DNA polymerases undergo the transition from a catalytically inactive “open” to an active “closed” conformation. All known determinants of substrate selection are associated with the “closed” state. To elucidate if this mechanism is conserved in homologous single subunit RNA polymerases (RNAPs), we have determined the structure of T7 RNAP elongation complex with the incoming substrate analog. Surprisingly, the substrate specifically binds to RNAP in the “open” conformation, where it is base paired with the acceptor template base, while Tyr639 provides discrimination of ribose versus deoxyribose substrates. The structure therefore suggests a novel mechanism, in which the substrate selection occurs prior to the isomerization to the catalytically active conformation. Modeling of multisubunit RNAPs suggests that this mechanism might be universal for all RNAPs .
ISSN:0092-8674
1097-4172
DOI:10.1016/S0092-8674(04)00059-5