DNA bases are numbered from the RNA 3' end in the post-translocated TEC. (A) An overview: β’ LL and the structurally conserved five amino acid loop of NusG NTD form a channel accommodating the exiting upstream DNA. (B) The template DNA nucleotide at position ten can be modeled to pair with the non-template DNA in a partially unstacked conformation (top) or interact with the cleft between the β’ RL and β’ LL (bottom). DNA and RNA bases are shown as spheres, sugar-phosphate backbones as cartoons. The β subunit is omitted for clarity. (C) The superimposition of Aquifex aeolicus NusG NTD (PDB ID 1M1G) and Pyrococcus furiosus Spt5 NTD (PDB ID 3P8B). The cartoon color changes from magenta in well superimposed regions to cyan in poorly superimposed or unaligned regions. The β’ clamp helices (yellow, from PDB ID 3QQC), the upstream DNA (grey, from the model in A) and Spt4 (cyan cartoon, from PDB ID 3P8B) are included to present the superimposition in the context of the TEC. (D) Multiple sequence alignment of the structurally conserved five amino acid loop of NusG family proteins and the flanking secondary structure elements. Species names are abbreviated as follows: Eco, E. coli, Bsu, Bacillus subtilis, Mtu, Mycobacterium tuberculosis, Tma, Thermotoga maritima, Syn, Synechocystis sp. PCC 6803, Tth, T. thermophilus, Aae, Aquifex aeolicus, Mja Methanocaldococcus jannaschii, Pfu, Pyrococcus furiosus, Sce, Saccharomyces cerevisiae, Hsa, Homo sapiens. Amino acid residues are shaded as follows: hydrophobic –green, polar –olive, Pro and Gly –yellow, Asp and Glu –red, Arg, Lys and His –cyan.