Magnetic Resonance Studies of the Conformation of Enzyme-Bound Adenylyl(3′ → 5′)uridine and Adenosine 5′-Triphosphate on RNA Polymerase from Escherichia coli

Bruce Bean, Ruth Koren, Albert Mildvan*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

Kinetic studies of Mn(II)-activated RNA polymerase show that ApU stimulates the incorporation of [3H]ATP into polyribonucleotides and that [3H] ApU is itself incorporated into the polyribonucleotide product, indicating that ApU can function as an initiator of RNA synthesis. The paramagnetic effects of RNA polymerase-Mn(II) on the relaxation rates of the protons and phosphorus of ApU bound at the initiation site and of the protons of ATP bound at the elongation site have been investigated. The binary Mn(II)-ApU complex has also been studied as a control. Using correlation times determined from the frequency dependence of the longitudinal relaxation rates of water protons and of ApU protons in the enzyme complexes, distances from the enzyme-bound Mn(II) to the protons and 31P of ApU (9.0–10.5 Å) and to the protons of ATP (4.0–5.7 Å) have been calculated. The smaller distances to ATP than to ApU strongly support the tight binding of the divalent cation activator Mn(II) at the elongation site. The distances from enzymebound Mn(II) to the protons of enzyme-bound ATP are indistinguishable from those in the binary Mn(II)-ATP complex. Upon binding to the enzyme-ApU-Mn(II) complex, ATP decreases the number of fast exchanging water ligands on Mn(II), indicating the displacement or occlusion of water ligands. The seven distances from the enzyme-bound Mn(II) to the protons and phosphorus of enzyme-bound ApU exclude direct coordination of ApU and can best be fit by a nonhelical S1 conformation with glycosidic χ angles of 30° (adenosine) and 60° (uridine). Since an RNA A-type helical conformation for bound ApU is excluded by the distances, the enzyme may play a catalytic role in separating the newly synthesized RNA chain from the enzyme-bound DNA template as the growing RNA chain passes through the initiation site. Unlike the ternary complex, in the weak binary Mn(II)-ApU complex the distances suggest the direct coordination of a phosphoryl oxygen to Mn(II) and a helical P3-type conformation for ApU. Mn(II) and its residual water ligands appear to be intercalated between the parallel planes of the bases which are 7.5 ± 1.0 Å apart.

Original languageEnglish
Pages (from-to)3322-3333
Number of pages12
JournalBiochemistry
Volume16
Issue number15
DOIs
StatePublished - 1 Jul 1977
Externally publishedYes

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