Evidence for a pyrimidine‐nucleotide‐specific initiation site (the i site) on Escherichia coli RNA polymerase

Abstract

Escherichia coli RNA polymerase has two sites, the i and i + l , for the binding of the first two substrates. The i site is template-and Mg"-independent and purine-nucleotide-specific, whereas the i + l site is template-and Mg2+-dependent and shows no nucleotide preference. The specificity of the i site for purine nucleotides is well in accord with the fact that most promoters initiate with a purine nucleotide. But there are a few promoters that initiate with a pyrimidine nucleotide. Dinucleotide synthesis at these promoters is completely inhibited by rifampicin. Earlier studies have failed to identify an i site for pyrimidine nucleotides. In this paper, using a fluorescent analog of UTP, namely uridine S-[y-(5-sulfonic acid)naphthylamidate]-triphosphate, abbreviated as UTP[AmNS], we are able to show its binding to RNA polymerase, with a Kd of 0.8 pM, in the absence of Mg2+ and template. This suggests the presence of an i pyrimidine nucleotide site. The fact that UTP-[AmNS] is capable of initiating RNA synthesis from the i site is further evidenced by the abortive transcription analyses at the lac promoter. Fluorescence titration studies performed in the presence and absence of purine initiator molecules indicate that this site is different from the i purine site. Scatchard analysis of the above data indicates the presence of a single binding site for UTP [AmNS] in the absence of Mg". Moreover UTP [AmNS] binds to the core enzyme with a Kd of 3.0 pM implying that, unlike the i purine nucleotide site, the 0 protein confers a tighter binding of UTP-[AmNS] to the low-K, site. Forster's energy transfer measurements using UTP [AmNS] as the donor and rifampicin as the acceptor have been used for estimation of the distance of the i pyrimidine nucleotide site from the rifampicin site. From these measurements, we infer that there is no direct interference of rifampicin with the first phosphodiester bond between two pyrimidine nucleotides.

Template Nucleotide Moieties Required for De Novo Initiation of RNA Synthesis by a Recombinant Viral RNA-Dependent RNA Polymerase

“…Polymerases generally have a purine-specific site for the initiation substrate NTP, the i site, and a second nucleotide binding pocket, the i ϩ 1 site (41). The increased stability of the ternary complex has been demonstrated in the presence of a high initiation substrate (GTP) concentration (15).…”

Selective Stimulation of Hepatitis C Virus and Pestivirus NS5B RNA Polymerase Activity by GTP

“…Perhaps the initiating nucleotide has to be a guanylate as is the case for many RNA polymerases like the one from bacteriophage T7 or the plus strand RNA virus BMV (43,44). For example, the DNA-dependent RNA polymerases of E. coli and T7 possess at least two distinct NTPbinding sites: one that recognizes only the initiating GTP in a Mg 2ϩ -independent manner and the other used for elongation, binding to all four NTPs in a reaction requiring Mg 2ϩ (43). In case of BMV, initiation of RNA synthesis can be stimulated up to ϳ10-fold by GMP or the dinucleotide GpG, corresponding to the penultimate and antepenultimate nucleotide of the viral template (45).…”

Inhibition of bacterial RNA polymerase by the cyanobacterial metabolites 12-epi-hapalindole E isonitrile and calothrixin A

“…coli RNA polymerase has two i sites, and an i+1 site for the binding of the ¢rst two nucleotides [12]. The i sites are template-and Mg 2 -independent and are speci¢c for either a purine or pyrimidine, whereas the i+1 site is template-and Mg 2 -dependent and binds both purine and pyrimidine nucleotides [12]. Rifampicin was shown to have a separate binding site on the enzyme, 3.0 nm from the purine binding site, 2.2 nm from the pyrimidine binding site, and 2.0 nm from the i+1 site.…”