Mechanism of ribose 2'-group discrimination by an RNA polymerase.
The mechanism by which T7 RNA polymerase (RNAP) discriminates between rNTP and dNTP substrates has been characterized. During transcript elongation T7 RNAP uses rNTPs 70-80-fold more efficiently than dNTPs. Discrimination of the hydrogen-bonding character of the ribose 2'-substituent contributes a largely Km-mediated factor of approximately 20 to this preference for rNTPs. Discrimination of 2'-substituent H-bonding character appears to be made through a hydrogen bond to the hydroxyl group of tyrosine 639. This hydrogen bond makes little net contribution to either rNTP ground or transition state binding energy apparently because it is balanced by the energy of desolvation of the tyrosine hydroxyl. This mechanism may reflect a strategy to facilitate translocation by minimizing contributions from polymerase-NMP moiety interactions to NTP binding energy so as to minimize the affinity of the NTP binding site for the 3'-NMP of the product nucleic acid.
Huang
Y
Y
Department of Biochemistry, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7760, USA.
Eckstein
F
F
Padilla
R
R
Sousa
R
R
D017123:Bacteriophage T7 / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000378:metabolism; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis; D009841:Oligonucleotides / Q000096:biosynthesis; D011401:Promoter Regions, Genetic; D012265:Ribonucleotides / Q000378:metabolism; D012266:Ribose / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014764:Viral Proteins
D004279:DNA, Viral; D003854:Deoxyribonucleotides; D009841:Oligonucleotides; D012265:Ribonucleotides; D014764:Viral Proteins; D012266:Ribose; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases