Figure 1

The RNAP trigger loop makes multiple contributions to substrate selection. The data from Yuzenkova et al. [2] are presented here as a generalized model for nucleotide triphosphate selectivity by multisubunit RNA polymerases, focusing on the trigger loop. A schematic elongation complex is shown with a nascent RNA (red), template DNA (blue), various NTP substrates (orange), catalytic Mg²⁺ (magenta), and the trigger loop (pink). The green star illustrates the relative efficiency of catalysis with the specified substrate. (a) A catalytically favorable alignment or environment of a matched (cognate) NTP (cNTP) substrate relies on numerous trigger loop residues. Biochemical and structural evidence suggests that Met1238 positioning adjacent to the base moiety of the complementary NTP, Arg1239 and His1242 contacts with the triphosphate group, and Gln1235 interactions with the hydroxyls of the ribose moiety are important for rapid catalysis with matched substrates. Structural analyses indicate additional non-trigger loop contacts with triphosphates and ribose hydroxyls (not shown). (b) A 3'-cdNTP may utilize base-pairing with the template for positioning in the active site but is not added efficiently due to loss of particular trigger loop interactions, purportedly Gln1235 and its positioning of Arg1239. 3'-cdNTP substrates do not appear to compete with the trigger loop, consistent with localization of a 3'-cdNTP adjacent (base-paired) to the template in a Pol II crystal structure (K-M Larsson, personal communication). Even in a base-pairing conformation, catalysis is reduced. (c) 2'-cdNTPs may also base-pair with the DNA template (occupancy in the addition site, or 'A site'); however, this occupancy is low at steady state in Pol II crystal structures, probably because of loss of a critical interaction with RNAP and the 2'-hydroxyl. Therefore, the trigger loop is unable to contribute efficiently to catalysis with 2'-cdNTPs. In addition, the 2'-cdNTPs may occupy a conformation at some frequency that is in conflict with a conformation of the intact trigger loop, leading to competition with the 2'-cdNTP and decreasing its affinity for the RNAP active site. This competition is sensitive to substitutions of Met1238, and is interpreted as indicating a requirement for Met1238 for trigger loop folding/movement. (d) Mismatched, non-cognate NTPs (ncNTPs) may not form complementary interactions with the template base, but the precise nature of ncNTP-template interactions will depend on the particular ncNTP-template mismatch. Because of these different orientations, catalysis with ncNTPs also varies by mismatch, but in all cases is reduced greatly by comparison with catalysis with cNTPs. Additionally, as with 2'-cdNTPs, an intact trigger loop may compete with ncNTPs, reducing the affinity of the enzyme for ncNTPs. This competition is also sensitive to substitutions in Met1238, again interpreted as indicating a requirement for Met1238 for trigger loop folding/movement. (e) In the absence of the trigger loop, NTPs are presumed still to base-pair effectively with the template DNA. However, they are not efficiently incorporated because of lack of critical trigger loop contacts. (f) 2'-cdNTPs are not greatly affected by loss of the trigger loop, as the trigger loop does not strongly contribute to their incorporation because of inefficient positioning of the 2'-cdNTP for cooperation with the trigger loop. However, affinity for 2'-cdNTPs increases in the absence of the trigger loop because of lack of competition. (g) Certain ncNTPs may still be misincorporated in the absence of the trigger loop, without major loss of efficiency, because the trigger loop does not contribute greatly to their incorporation. Affinity for such ncNTPs increases, though, because of removal of trigger loop-ncNTP competition. (h) Other ncNTPs are discriminated against efficiently by the RNAP active site, depending on template base, indicating that their incorporation actually requires the trigger loop. In the absence of the trigger loop, they are considered unusable (unNTPs). The trigger loop still contributes to the overall selection for correct cNTPs over ncNTPs, by contributing more to catalysis of cNTPs than of ncNTPs even in the presence of trigger loop-independent discrimination between cNTPs and ncNTPs.