Top: framework for estimation of residual fidelity phenotype (ϕ). Shown are model logistic curves for incorporation of a triplet, for example GAU (at a fixed concentration, opposite 3’-AUC-5’), versus increasing concentrations of a mismatching triplet, for example GGU. A ribozyme ‘+’ with a fidelity function (e.g. t5a, blue curve) will incorporate equal amounts of the matched and mismatched triplets at a different concentration of the mismatched triplet compared to a ribozyme ‘−’ without a fidelity function (e.g. αβγδ, red curve). x+ and x− represent the lns of these concentrations for + and −; their separation (x+ - x−) is a proxy for the strength of the ‘+’ fidelity phenotype. We measured the relative incorporation (W) of triplet pairs by the two triplet polymerases at test concentrations (t) of mismatched triplet, chosen to maximise the difference in the ribozymes’ resulting fractional incorporations (marked by a blue dot vs. a red dot on respective curves). If a triplet modification (*, right) interferes with mismatch discrimination by ‘+’, it would shift the ‘+’ relative incorporation curve (blue) towards that of ‘−’ (red), reducing the difference in fractional incorporation. Assuming curve steepness (k) remains constant, the residual phenotype (ϕ) for that modification is described by the new separation (x+* - x−*) as a proportion of the original separation (x+ - x−); numerical values and calculations from the measurements described below are supplied in Figure 8—source data 2. Middle: measurements of ratios of incorporation vs. misincorporation (W) for unmodified and modified triplet pairs (at the indicated test concentrations) by the t5a +1 triplet polymerase compared to the ε fidelity domain-truncated αβγδ+1 (at 0.5 μM each). The expected triplet additions are in grey along the left of each gel; the average W from n independent experiments, calculated via densitometry of products containing slower-migrating G misincorporations, is shown below each lane along with the average ϕ. Primers (P), templates (T) and additional triplets were at 0.5/0.5/5 μM, incubated for three days in −7˚C ice. Left: presence of a 2-thiouracil (2SU) at the third position of the triplet abolishes the fidelity domain’s ability to discriminate against a second position wobble pair (P: A10, T: CCCMisGAU, +5 μM pppCCC). Centre-left: the same third position modification (2SU) also abolished the fidelity domain’s preference for G misincorporation at the first position (P: Fγ7, T: TγAGU, +5 μM HOCUG). Centre-right: in contrast, a 2SU at the first position of the triplet exerted no clear influence upon third position wobble discrimination (P: A10, T: CCCMisUGA, +5 μM pppCCC). Right: replacement of a 2’ hydroxyl group with a 2’ fluoro (2’F) at the first triplet position likewise had no effect upon third position mismatch discrimination (P: Fγ7, T: TγAGU, +5 μM HOCUG). Below: Measurement of fidelity phenotype in the presence or absence of a downstream triplet. Residual phenotypes indicate attenuation or abolishment of third position (left panel, P Fγ7, T TγAGU), second position (middle panel, P Fγ7, T TγGAU), and third position (right panel, P Fγ7, T TγAGU) fidelity effects from downstream triplet absence. No effects are seen (upon third position discrimination) from the presence or absence of a 5’ triphosphate on the downstream triplet (ϕppp). In the absence of a triplet bound downstream on the template, the fidelity phenotype is severely compromised, suggesting this adjacent triplet:template duplex plays a critical role in positioning the fidelity domain relative to the incoming triplet.