Abstract
RNA polymerases (RNAPs) synthesize RNA from NTPs, whereas DNA polymerases synthesize DNA from 2′dNTPs. DNA polymerases select against NTPs by using steric gates to exclude the 2′OH, but RNAPs have to employ alternative selection strategies. In single-subunit RNAPs, a conserved Tyr residue discriminates against 2′dNTPs, whereas selectivity mechanisms of multi-subunit RNAPs remain hitherto unknown. Here, we show that a conserved Arg residue uses a two-pronged strategy to select against 2′dNTPs in multi-subunit RNAPs. The conserved Arg interacts with the 2′OH group to promote NTP binding, but selectively inhibits incorporation of 2′dNTPs by interacting with their 3′OH group to favor the catalytically-inert 2′-endo conformation of the deoxyribose moiety. This deformative action is an elegant example of an active selection against a substrate that is a substructure of the correct substrate. Our findings provide important insights into the evolutionary origins of biopolymers and the design of selective inhibitors of viral RNAPs.
Highlights
RNA polymerases (RNAPs) synthesize RNA from NTPs, whereas DNA polymerases synthesize DNA from 2′dNTPs
We systematically investigated the effects of individual substitutions of the active site residues on the discrimination against 2′dNTPs in single nucleotide addition (SNA) assays and during processive transcript elongation by the E. coli RNAP
We performed a systematic analysis of the contributions of the active site residues of the multi-subunit RNAP to selecting NTPs over 2′dNTPs
Summary
WT 2’dGTP β’R425K 2’dGTP at-C pre-G at-G at-C pre-G pre-G at-C pre-G at-G at-C at-C pixel counts. To test if the unavailability of the 3′OH group was responsible for the destabilization of the TL folding, we solved the X-ray crystal structure of the initially transcribing complex of the T. thermophilus RNAP with 3′dCTP at 3.0 Å resolution. The comparative analysis of RNAP structures with CMPCPP, 2′dCTP, and 3′dCTP in combination with in silico docking experiments suggested that β′Arg[425] inhibited the incorporation of 2′dNTPs by interacting with their 3′OH group and favoring the 2′-endo conformation of the deoxyribose moiety. Β′Arg[425] could inhibit the incorporation of the 2′dNMP solely by slowing down the initial steps of the TL folding, by sequestering the 3′OH group and preventing its interaction with the TL β′Gln[929] To test this hypothesis, we determined the incorporation rate of 3′dGMP by the WT RNAP (Supplementary Fig. 2c). The remaining 10-fold inhibition of the overall 50-fold inhibitory effect was contributed by some other features of the 2′-endo binding pose, as discussed below
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