Interaction of ribonucleoside triphosphates with the gene 4 primase of bacteriophage T7.
The primase fragment of bacteriophage T7 gene 4 protein catalyzes the synthesis of oligoribonucleotides in the presence of ATP, CTP, Mg(2+) (or Mn(2+)), and DNA containing a primase recognition site. During chain initiation, ATP binds with a K(m) of 0.32 mM, and CTP binds with a K(m) of 0.85 mM. Synthesis of the dinucleotides proceeds at a rate of 3.8/s. The dinucleotide either dissociates or is extended to a tetranucleotide. The primase preferentially inserts ribonucleotides forming Watson-Crick base pairs with the DNA template >200-fold more rapidly than other ribo- or deoxynucleotides. 3'-dCTP binds the primase with a similar affinity as CTP and is incorporated as a chain terminator at a rate (1)/(100) that of CTP. ATP analogues alpha,beta-methylene ATP, beta,gamma-methylene ATP, and beta,gamma-imido ATP are incorporated by the primase fragment at the 5'-ends of the oligoribonucleotides but not at the 3'-ends. A model is presented in which the primase fragment utilizes two nucleotide-binding sites, one for the initiating ATP and one for the nucleoside triphosphate which elongates the primer on the 3'-end. The initiation site binds ATP or oligoribonucleotides, whereas the elongation site binds ATP or CTP as directed by the template.
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Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts 02115, USA.
Kumar
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Richardson
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D000255:Adenosine Triphosphate / Q000378:metabolism; D017123:Bacteriophage T7 / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D003570:Cytidine Triphosphate / Q000378:metabolism; D019915:DNA Primase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D009843:Oligoribonucleotides / Q000378:metabolism; D012265:Ribonucleotides / Q000378:metabolism*; D013379:Substrate Specificity; D013698:Templates, Genetic
D009838:Oligodeoxyribonucleotides; D009843:Oligoribonucleotides; D012265:Ribonucleotides; D015636:Magnesium Chloride; D008345:Manganese; D003570:Cytidine Triphosphate; D000255:Adenosine Triphosphate; D019915:DNA Primase