Mammalian mitochondrial tRNASer(UCN) (mt-tRNASer) and pyrrolysine tRNA (tRNAPyl) fold to near-canonical three-dimensional structures despite having noncanonical secondary structures with shortened interhelical loops that disrupt the conserved tRNA tertiary interaction network. How these noncanonical tRNAs compensate for their loss of tertiary interactions remains unclear. Furthermore, in human mt-tRNASer, lengthening the variable loop by the 7472insC mutation reduces mt-tRNASer concentration in vivo through poorly understood mechanisms and is strongly associated with diseases such as deafness and epilepsy. Using simulations of the TOPRNA coarse-grained model, we show that increased topological constraints encoded by the unique secondary structure of wild-type mt-tRNASer decrease the entropic cost of folding by ∼2.5 kcal/mol compared to canonical tRNA, offsetting its loss of tertiary interactions. Further simulations show that the pathogenic 7472insC mutation disrupts topological constraints and hence destabilizes the mutant mt-tRNASer by ∼0.6 kcal/mol relative to wild-type. UV melting experiments confirm that insertion mutations lower mt-tRNASer melting temperature by 6–9 °C and increase the folding free energy by 0.8–1.7 kcal/mol in a largely sequence- and salt-independent manner, in quantitative agreement with our simulation predictions. Our results show that topological constraints provide a quantitative framework for describing key aspects of RNA folding behavior and also provide the first evidence of a pathogenic mutation that is due to disruption of topological constraints.