Experimental microbial evolution (EME) provides the opportunity to study the mutations that drive adaptation. We recently completed an EME that subjected 115 independent Escherichia coli populations to thermal stress (42.2C) for a year. At the end of the experiment, we identified two major, negatively epistatic adaptive pathways. One pathway had mutations in rpoB, the gene encoding the RNA polymerase beta subunit, and the other that had mutations in rho, which encodes a transcriptional terminator. Here we focused on four putatively beneficial rho mutations by introducing them into the ancestral genotype and by measuring their effects on fitness, gene expression, protein structure and termination efficiency. Results were heterogenous among mutations; two conferred adaptive effects at levels similar to four beneficial rpoB mutations, without any discernible effect on transcriptional termination. The other two enhanced transcriptional read-through and were not adaptive, suggesting they are beneficial only in the context of epistatic interactions. Fitness had no discernible relationship with the predicted effect of mutations on the free energy of protein folding. Overall, the rho mutations had similar effects on gene expression as rpoB mutations. Of 1726 genes that were differentially expressed between rpoB mutations and the EME ancestor, 83% were also affected by rho mutations. Moreover, both rho and rpoB mutations tended to restore gene expression to that of the unstressed (37.7C) ancestor, and the extent of restoration correlated quantitatively with fitness. This rare opportunity to compare beneficial mutations within and across adaptive pathways suggests the pathways traverse similar fitness landscapes.