The alignment compares full-length NELF-C from Homo sapiens, Mus musculus, Danio rerio, Drosophila melanogaster, Dictyostelium discoideum, Loa loa, Lottia gigantea, and Chlorella variabilis. Residues are colored according to percent conservation with darker shades of blue representing higher conservation. Barrels above the alignment represent α-helices, arrows β-sheets and are colored according to Figure 1B. N- and C-terminal borders of solved crystal structure are indicated. Sequence alignment was done with Mafft (Katoh and Standley, 2013) followed by manual editing and rendered with JALVIEW (Waterhouse et al., 2009). Surprisingly, a part of the NELF-AC complex also exists in the single cell slime mold Dictyostelium discoideum and the green algae Chlorella variabilis. The hypothetical Dictyostelium proteins DDB_G0286295 and DDB_G0268678 share sequence similarity with the crystallized N-terminus of NELF-A (28% identity, 50% similarity) and C-terminal region of NELF-C (33% identity, 53% similarity) (Figure 1B). Similarly, the hypothetical C. variabilis proteins E1ZMT9 and E1Z2I7 also share sequence similarity with the crystallized N-terminus of NELF-A (25% identity, 44% similarity) and C-terminal region of NELF-C (23% identity, 39% similarity). The conservation of many residues in the hydrophobic core and heterodimer interface indicates that the NELF-AC subcomplex exists in this single cell organism. A putative Dictyostelium homolog is also found for a region of human NELF-B comprising residues 1–410 (DDB_G0284195) (Chang et al., 2012). NELF is likely present in both single and multicellular organisms. Plants may also lack NELF, indicating that they may be devoid of promoter proximal pausing or use other mechanisms to regulate gene expression at the level of elongation. Future work would benefit from understanding how NELF evolved and why some organisms either lost or lack the complex.