Qbeta replicase discriminates between legitimate and illegitimate templates by having different mechanisms of initiation.
Qbeta replicase (RNA-directed RNA polymerase of bacteriophage Qbeta) exponentially amplifies certain RNAs (RQ RNAs) in vitro. Here we characterize template properties of the 5' and 3' fragments obtained by cleaving one of such RNAs at an internal site. We unexpectedly found that, besides the 3' fragment, Qbeta replicase can copy the 5' fragment and a number of its variants, although they lack the initiator region of RQ RNA. This copying can occur as a 3'-terminal elongation or through de novo initiation. In contradistinction to RQ RNA and its 3' fragment, initiation on these templates occurs without regard to the 3'-terminal or internal oligo(C) clusters, is GTP-independent, and does not result in a stable replicative complex capable of elongation in the presence of aurintricarboxylic acid. The results suggest that, although Qbeta replicase can initiate and elongate on a variety of RNAs, only some of them are recognized as legitimate templates. GTP-dependent initiation on a legitimate template drives the enzyme to a "closed" conformation that may be important for keeping the template and the complementary nascent strand unannealed, without which the exponential replication is impossible. Triggering the GTP-dependent conformational transition at the initiation step could serve as a discriminative feature of legitimate templates providing for the high template specificity of Qbeta replicase.
Ugarov
Victor I
VI
Institute of Protein Research of the Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia.
Demidenko
Alexander A
AA
Chetverin
Alexander B
AB
D017908:Allolevivirus / Q000201:enzymology / Q000235:genetics; D001312:Aurintricarboxylic Acid / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D004262:DNA Restriction Enzymes / Q000378:metabolism; D015245:Deoxyribonuclease BamHI / Q000378:metabolism; D015246:Deoxyribonuclease EcoRI / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D006160:Guanosine Triphosphate / Q000494:pharmacology; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D011777:Q beta Replicase / Q000378:metabolism*; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins; D013379:Substrate Specificity; D013698:Templates, Genetic
D011994:Recombinant Proteins; D001312:Aurintricarboxylic Acid; D012313:RNA; D006160:Guanosine Triphosphate; D011777:Q beta Replicase; D004262:DNA Restriction Enzymes; D015245:Deoxyribonuclease BamHI; D015246:Deoxyribonuclease EcoRI