The genes for mitoribosomal protein S12 (Mrps12) and mitochondrial seryl-tRNA ligase (Sarsm and Sars2) are oppositely transcribed from a conserved promoter region of <200 bp in both human and mouse. Using a dual reporter vector we identified an array of 4 CCAAT box elements required for efficient transcription of the two genes in cultured mouse 3T3 cells, and for enforcing directionality in favour of Mrps12. Electrophoretic mobility shift assay (EMSA) and in vivo footprinting confirmed the importance of these promoter elements as sites of protein-binding, and EMSA supershift and chromatin immunoprecipitation (ChIP) assays identified NF-Y as the key transcription factor involved, revealing a common pattern of protein-DNA interactions in all tissues tested (liver, brain, heart, kidney and 3T3 cells). The inherently bidirectional activity of NF-Y makes it an especially suitable factor to govern promoters of this class, whose expression is linked to cell proliferation.