Biochemical and physical studies of the role of intrinsic metal ions in RNA polymerase from Escherichia Coli

Abstract A variety of nucleotidyl transferases including DNA and RNA polymerases from both prokaryotic and eukaryotic sources are Zn-metalloenzymes. E. coli RNA polymerase (RPase) contains 2 mol of Zn/mol of holoenzyme having a subunit composition of α2ββ′σ. We have identified that one Zn is located in the β subunit which contains the substrate nucleotide binding site and the other in the β′ subunit which possesses the template DNA binding site. While the presence of Zn as an integral part of RPase has been well established, the precise function of the intrinsic metal remains to be elucidated. We have used both in vivo and in vitro metal substitution methods to replace intrinsic Zn with other paramagnetic metals and to probe the functional and structural role of intrinsic metals in RPase. In the in vivo metal substitution studies, the CoCo RPase isolated from E. coli cells grown in Zn-depleted and Co-enriched media was as active as the native ZnZn enzyme. These two RPases are very similar both physically and biochemically except their efficiencies in starting RNA chains at different promoters suggesting that the intrinsic metals may be involved in promoter recognition and RNA chain initiation. Furthermore, the Co enzyme but not the Zn enzyme exhibits a characteristics absorption spectrum in the visible region which can be perturbed by the addition of substrate or template indicating that the intrinsic metals may be involved in substrate or template binding. Recently, we have developed an in vitro metal substitution procedure to selectively replace one of the two Zn ions located in the β subunit with other metals. The method involves a sequential denaturation in urea followed by reconstitution in the presence of 10−5M of other metals such as Co, Mn, Ni or Cu. We have obtained various metal hybrid RPases (CoZn, MnZn, NiZn or CuZn) which retained different enzyme activity. Co and Ni RPases possess absorption spectrum which can be perturbed only by the addition of purine nucleotide (ATP) but not by pyrimidine nucleotide (UTP) in the absence of template and exogenous Mg2+. This is consistent with the contention that the substituted metal on β subunit is located at the initiation site of the enzyme. One question that may be raised is whether these spectral changes are due to a direct interaction of the metal with substrate or an indirect effect such as the conformational change of the enzyme induced by substrate binding at a site distant from the metal. This question has been answered by the distance measurements between Co in CoZn RPase and substrate ATP bound at the initiation site of the enzyme by 1H and 31P NMR spectroscopies. The results indicate that Co is accessible to solvent and there are two fast-exchanging water molecules in the inner coordination sphere of Co ion. ATP or ApA but nto UTP can replace one water molecule. The effects of ATP and ApA do not require the presence of DNA or Mg2+, and their Kd values are estimated to be 0.15 and 0.075 mM, respectively, confirming the earlier conclusion that Co ion is at the initiation site. The base moiety of ATP, sugar (H1′), and three phosphorus α, β, γ, are ca. 4, 6.8, 10, 15, and 14 A, respectively, away from the Co ion. These spatial relationships indicate that the Co ion is directly coordinated to the base moiety of ATP. Thus the intrinsic metal in the β subunit of RPase may play a role in the recognition of the initiating nucleotide and orientation of the nucleotide in a stereospecific position for the catalysis. Similar NMR studies performed in the presence of DNA template and Mg2+ show that the distances from Co to base or sugar protons do not alter significantly from those described above, while the three phosphorus atoms move closer to metal center within 10 A range. Furthermore, substrate ATP binds tighter to the enzyme in the presence of DNA (Kd = 0.09 mM) than in its absence (Kd = 0.15 mM). The proximity relationship between metal binding site and other active sites of CoZn enzyme has also been studied by fluorescence energy transfer technique. When equimolar ratio of CoZn core RPase is added to isolated σ subunit covalently labeled with N-pyrene maleimide (PM-σ), 50 and 39% quenching of the PM-σ fluorescence was observed, respectively, in the absence and presence of DNA, while the corresponding fluorescence quenching caused by ZnZn core RPase was 17 and 14%. The distance between the Co in the β subunit and a specific SH residue in the σ subunit is calculated to be 22 A, which increases to 33 A in the presence of template decreases to 29 A by further addition of substrate indicating a template- and a substrate-induced conformational change of the enzyme.