Mechanism of polynucleotide phosphorylase.
The de novo polymerization of RNA initiated by polynucleotide phosphorylase from nucleoside diphosphates was examined. End group analysis performed under conditions designed to specifically end label the polymer revealed no evidence for a 5'-pyrophosphate-terminated polymer. However, we observed preferential incorporation of the ADP alpha S(RP) diastereomer into the 5' end (Marlier & Benkovic, 1982) in chain initiation, suggesting that the enzyme incorporates a nucleoside diphosphate specifically into the 5' end of the product, with subsequent enzymatic removal of the polyphosphate linkage. No evidence could be obtained for a covalent adduct between the enzyme and the 5' end of the polymer chain, despite the high processivity of the polymerization reaction. Gel electrophoretic analysis showed the polymer to be highly disperse, varying from 1 to 30 kb. Scanning transmission electron microscopy supported this product analysis and further suggested that (i) each subunit can produce an RNA polymer and (ii) both 5' and 3' ends of the RNA can be bound simultaneously to the same or differing enzyme molecules.
Sulewski
M
M
Department of Chemistry, Pennsylvania State University, University Park 16802.
Marchese-Ragona
S P
SP
Johnson
K A
KA
Benkovic
S J
SJ
D000227:Adenine Nucleotides / Q000494:pharmacology; D000244:Adenosine Diphosphate; D001665:Binding Sites; D004789:Enzyme Activation / Q000187:drug effects; D008837:Micrococcus / Q000201:enzymology; D008854:Microscopy, Electron; D008970:Molecular Weight; D009843:Oligoribonucleotides / Q000494:pharmacology; D011117:Polyribonucleotide Nucleotidyltransferase / Q000378:metabolism*; D012313:RNA / Q000096:biosynthesis / Q000648:ultrastructure
D000227:Adenine Nucleotides; D009843:Oligoribonucleotides; C061075:adenosine diphosphate adenosine monophosphate; D000244:Adenosine Diphosphate; D012313:RNA; D011117:Polyribonucleotide Nucleotidyltransferase