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Interaction of Qβ RNA replicase with guanine nucleotides Different modes of inhibition and inactivation

1977; Elsevier BV; Volume: 478; Issue: 2 Linguagem: Inglês

10.1016/0005-2787(77)90183-6

1879-3002

Thomas Blumenthal,

RNA and protein synthesis mechanisms

Qβ replicase, an RNA-dependent RNA polymerase, contains the Escherichia coli protein synthesis elongation factors EF-Tu and EF-Ts. A binding site specific for guanine nucleotides is present on EF-Tu. A chemically modified but enzymatically active form of the enzyme (“cross-linked” replicase) contains a covalently linked EF-Tu · Ts complex in which the guanine nucleotide binding site of the EF-Tu is blocked. Initiation of transcription of poly(C) by both native and “cross-linked” replicase is competitively inhibited by high concentrations of GDP and ppGpp, while elongation of pre-initiated polynucleotide chains is not. When Qβ replicase is denatured with 8 M urea, the rate of its renaturation following dilution in a high-salt buffer is inhibited by relatively low concentrations of GDP, ppGpp and GTP. In this assay the “cross-linked” replicase is not inhibited by guanine nucleotides. Similarly, inactivation of Qβ replicase (but not “cross-linked” replicase) upon incubation at 30°C is accelerated by low concentrations of GDP, ppGpp and GTP. Thus guanine nucleotides inhibit renaturation of denatured replicase and cause inactivation of Qβ replicase by interacting at the binding site on EF-Tu. On the other hand, inhibition of initiation of transcription by guanine nucleotides involves a different site, presumably the site at which GTP binds to initiate transcription.