Asp537 and Asp812 in Bacteriophage T7 RNA Polymerase as Metal Ion-Binding Sites Studied by EPR, Flow-Dialysis, and Transcription

Abstract

Asp537 and Asp812 are essential in the catalytic mechanism of T7 RNA polymerase. The mutants D537N and D812N have no detectable activity whereas the mutants D537E and D812E have significantly reduced activity relative to the wild-type. The hypothesis that these two amino acids act as metal-binding ligands has been tested using EPR with Mn2+ as the metal ion. Mn2+ is able to substitute for Mg2+ in transcription by T7 RNAP on templates containing the T7 promoter. Mg2+ and Mn2+ compete for binding sites, with the former having lower affinity. Mn2+ binding to the wild-type enzyme and the mutants D537N, D812N, D537E, D812E, and Y649F was measured over the concentration range of 25 microM to 1.5 mM. The data were analyzed by nonlinear least-squares fits to the binding isotherms, and the analysis gave approximately two Mn(2+)-binding sites in all cases and a Kd for the wild-type of approximately 340 microM. The Kd value for the mutant Y639F, in which Asp537 and Asp 812 are not mutated, is comparable to the value for the wild-type. Mn2+ binding to the double mutants, D537N/D812N and D537E/D812E, appears to be nonspecific. The Kd values of the Asp-->Asn mutants are only 2-5 times larger than the value for the wild-type, in contrast to the drastic diminution of enzymatic activity in the mutants. The geometry of metal binding to these Asp residues may be crucial in determining the catalytic competence. Mn2+ binding to the wild-type enzyme in the presence of nucleotides, measured by flow dialysis, is characterized by two Mn(2+)-binding sites with a Kd value of ca. 150 microM. The similarity in values of Kd with and without nucleotide suggests that nucleotides do not have a drastic effect on Mn2+ binding. Our results indicate that monodentate carboxylate oxygens of both conserved Asp residues bridge the two metal ions.

N4 RNA Polymerase II, a Heterodimeric RNA Polymerase with Homology to the Single-Subunit Family of RNA Polymerases

“…These motifs occupy similar positions in the various structures: motifs A and C, which contain invariant aspartates, lie in the palm. These aspartates coordinate catalytically essential Mg 2ϩ ions (43,44,59,66). Motif B, which contains an invariant lysine, lies in the fingers subdomain and makes up part of the enzyme's nucleotide binding pocket (27).…”

Template-dependent incorporation of 8-N3AMP into RNA with bacteriophage T7 RNA polymerase

“…It has already been reported that the use of Mn 2+ in place of Mg 2+ appears to result in polymerase activity compatible for a wider range of substrates for both RNA (Conrad et al 1995;Basu et al 1998) and DNA (Tabor and Richardson 1989) polymerases. Other studies suggest that Mn 2+ binds to polymerases with higher affinity, possibly involving two aspartic acid residues (Woody et al 1996;Martin et al 1999). Similar increases in binding affinity for the NTP substrates can reduce the apparent K m for the NTP substrates, which could result in tighter binding for an otherwise unfavorable NTP substrate.…”

Genomic Characterization of the Intron-Containing T7-Like Phage phiL7 ofXanthomonas campestris

“…All important amino acid residues in the large C terminus of T7 RNAP were well conserved in phiL7 RNAP (see Fig. S4 in the supplemental material): Asp507 and Asp741 in phiL7 RNAP, corresponding to Asp537 and Asp812, respectively, in the active site of T7 RNAP; Lys577 and Tyr585 in phiL7 RNAP, corresponding to the catalytic residues Lys631 and Tyr811, respectively, in proximity to the active site of T7 RNAP; and His714 in phiL7 RNAP, corresponding to His784 of T7 RNAP, for holding short transcripts (34,53). However, the amino acid residues at the corresponding sites of major loops varied greatly in sequence and length (see Fig.…”