The carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) consists of 26 and 52 heptad-repeats in yeast and mammals, respectively. Studies in yeast showed that the strong periodicity of the YSPTSPS heptads is dispensable for cell growth and that di-heptads interspersed by spacers can act as minimal functional units (MFUs) to fulfil all essential CTD functions. Here, we show that the MFU of mammalian cells is significantly larger than in yeast and consists of penta-heptads. We further show that the distance between two MFUs is critical for the functions of mammalian CTD. Our study suggests that the general structure of the CTD remained largely unchanged in yeast and mammals; however, besides the number of heptad-repeats, also the length of the MFU significantly increased in mammals.