Mammalian cells must coordinate their growth with nutrient availability. Energy, amino acids, and growth factors signals are integrated by the mechanistic Target of Rapamycin Complex 1(mTORC1). mTORC1 is tethered to the lysosome by the Rag GTPase heterodimer when amino acids are present, allowing it to be activated. Defining how amino acid signals are transmitted through the Rag GTPases is critical for understanding how cells regulate growth. In this thesis, I utilized a single-molecule Förster Resonance Energy Transfer (smFRET) platform to monitor the conformational states of the Rag GTPases. While each subunit undergoes local changes upon nucleotide binding, their function as obligate heterodimers requires coordinated global rearrangements that govern interactions with regulators and mTORC1. Using smFRET, I determined how nucleotide binding shapes Rag GTPase conformations, examined how mutations linked to dysfunctional mTORC1 activation change their conformation, and investigated the mechanism of mTORC1 recruitment. These studies show that Rag GTPase conformation is a key determinant of mTORC1 signaling. Analysis of follicular lymphoma–associated mutations uncovered how the conformations and functions of the Rag GTPases can be altered in cancer. A structure of the upstream regulator, GATOR1, showed noncanonical interactions with the RagC subunit of the Rag GTPases. Using biochemical and cell signaling techniques, I showed that this interaction is essential for amino acid signals to be properly transmitted to mTORC1. Together, this work defines how Rag GTPase conformations integrate nucleotide and regulatory inputs to control mTORC1 activation, advancing our understanding of how cells couple nutrient sensing to growth control.