Mechanism of Polyadenylate-Polyuridylate Synthesis by RNA Polymerase Holoenzyme II of Escherichia coli

Abstract

Poly(A)•poly(U) synthesis in the absence of template DNA is a unique reaction catalyzed by the RNA polymerase [EC 2.7.7.6] holoenzyme II of Escherichia coli. As one approach to investigating the physiological role of the enzyme, the molecular mechanism of poly(A)•poly(U) synthesis was studied.
Streptolydigin, an inhibitor of the elongation of RNA chains, was shown to inhibit poly(A)•poly(U) synthesis, and the inhibition was released by a streptolydigin-resistant mutation on the β subunit. These observations indicate that the active site for the reaction might be located on the β subunit. Another antibiotic, rifampicin, which is known to be a specific inhibitor of the initiation of DNA-dependent RNA synthesis, effectively inhibited both initiation and elongation steps in poly(A)•poly(U) synthesis. This suggests that the enzyme conformation during the chain elongation reaction might be different in this case from that in DNA-dependent RNA synthesis. Analysis of the products formed during the initiation reaction indicated that the rate-determining reaction in poly(A)•poly(U) synthesis was the formation of primers of short chain length, and that holoenzyme I was unable to form the first phosphodiester bond in this reaction. Functional properties of holoenzyme II are discussed in connection with these observations.