Discrimination against deoxyribonucleotide substrates by bacterial RNA polymerase

Abstract

Nucleic acid polymerases have evolved elaborate mechanisms that prevent incorporation of the non-cognate substrates, which are distinguished by both the base and the sugar moieties. While the mechanisms of substrate selection have been studied in single-subunit DNA and RNA polymerases (DNAPs and RNAPs, respectively), the determinants of substrate binding in the multisubunit RNAPs are not yet known. Molecular modeling of Thermus thermophilus RNAP-substrate NTP complex identified a conserved beta' subunit Asn(737) residue in the active site that could play an essential role in selection of the substrate ribose. We utilized the Escherichia coli RNAP model system to assess this prediction. Functional in vitro analysis demonstrates that the substitutions of the corresponding beta' Asn(458) residue lead to the loss of discrimination between ribo- and deoxyribonucleotide substrates as well as to defects in RNA chain extension. Thus, in contrast to the mechanism utilized by the single-subunit T7 RNAP where substrate selection commences in the inactive pre-insertion site prior to its delivery to the catalytic center, the bacterial RNAPs likely recognize the sugar moiety in the active (insertion) site.

Fig. 1. A model of the substrate bound to the T. thermophilus RNAP active (insertion) site

The numbering corresponds to the E. coli RNAP. The bridge helix (orange) is shown in the uniform conformation that has been observed in the yeast RNAP (16); in an alternative distorted state the central portion of the helix (dark blue) is flipped out (17). The DNA template strand and the RNA nucleotides in the RNA/DNA hybrid are shown in red and yellow, respectively. Two catalytic Mg²⁺ ions (cMg1 and cMg2, magenta spheres) are coordinated (white dashed lines) by the four catalytic RNAP Asp residues (white) and by the substrate phosphates. β′ Asn⁴⁵⁸ (cyan) located in the β′ active site loop (white ribbon) and β′ Arg⁴²⁵ (cyan) forming internal hydrogen-bonding network in the crystal structure (cyan dashed lines) make putative contacts (cyan dashed lines) with the substrate ribose. A, overall view of the substrate-binding site. B, close-up view of the active site.

Fig. 2. Assay for the incorporation of the r/dNMPs

A, linear pIA349 template used to generate radiolabeled TECs halted at position G37 with the start site indicated by an arrow) (top) and the schematic representation of the assays used to measure the utilization of r/dNTP substrates (bottom). B, representative gel panels illustrating the extension of the nascent RNA in complexes halted at A39 or U38 upon addition of the increasing concentrations of r/dGTP and r/dATP substrates. Selection of rUTP versus dTTP was measured by extension of G37 RNA; selection of r/dCTP was assayed on a similar T7A1 promoter template, pIA171. C, comparison of the discrimination efficiencies between four r/d nucleotide combinations. The assays were repeated two to four times for each enzyme-r/dNTP combination; the discrimination efficiencies varied within 20%; NT, not tested. In E. coli RNAP, all substitutions were in the β′ subunit; the effects of selected substitutions in T7 RNAP (6) are presented for comparison.

Fig. 3. Transcription elongation by the Asn⁴⁵⁸ mutant enzymes

Top, transcript generated from the T7A1 promoter on a linear pIA349 template; transcription start site (+1), transcript end (run-off) and positions of ops (U43) and P_his (U145) pause sites are indicated. Bottom, halted G37 TECs were challenged with heparin at 50 μg/ml and NTPs at low (20 μ_M GTP, 100 μ_M ATP, CTP, and UTP) or high (100 μ_M GTP, 500 μ_M ATP, CTP, and UTP) concentrations. Aliquots were withdrawn at times indicated above each lane, followed by the high NTP chase (1 m_M each NTP; C) and quenched as above. Positions of the DNA size markers are shown on the right (in nt).