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Three GPN-loop GTPases, GPN1-GPN3, are central to the maturation and trafficking of eukaryotic RNA polymerase II. This GTPase family is widely represented in archaea but typically occurs as single paralogs. Structural analysis of the GTP- and GDP-bound states of theSulfolobus acidocaldariusGPN enzyme (SaGPN) showed that this central GPN-loop GTPase adopts two distinct quaternary structures. In the GTP-bound form the γ-phosphate induces a tensed dimeric arrangement by interacting with the GPN region that is relaxed upon hydrolysis to GDP. Consequently, a rocking-like motion of the two protomers causes a major allosteric structural change towards the roof-like helices. Using a lock-switch-rock (LSR) mechanism, homo- and heterodimeric GPN-like GTPases are locked in the GTP-bound state and undergo large conformational changes upon GTP hydrolysis. AΔsaGPNstrain ofS. acidocaldariuswas characterized by impaired motility and major changes in the proteome underscoring its functional relevance forS. acidocaldarius in vivo.Significance StatementGPN-loop GTPases have been found to be crucial for eukaryotic RNA polymerase II assembly and nuclear trafficking. Despite their ubiquitous occurrence in eukaryotes and archaea the mechanism by which these self-activating GTPases mediate their function is unknown. Our study on an archaeal representative fromSulfolobus acidocaldariusshowed that these dimeric GTPases undergo large-scale conformational changes upon GTP hydrolysis, which can be summarized as a lock-switch-rock mechanism. The observed requirement ofSaGPN for motility appears to be due to its large footprint on the archaeal proteome.