The 5S gene internal control region is composed of three distinct sequence elements, organized as two functional domains with variable spacing.

Systematic oligonucleotide-directed mutagenesis within the internal control region of the Xenopus laevis somatic 5S RNA gene identifies three distinct sequence elements that regulate transcription activity: box A, containing the common, conserved class III promoter domain, and two 5S-gene-specific segments, termed intermediate element and box C. Analysis of the individual steps in the formation of the stable initiation complex reveals that the two 5S-gene-specific elements are the main determinants for the stable binding of TFIIIA. In contrast, TFIIIC binding appears to be dependent on interactions with TFIIIA and on direct DNA interactions in box A as well as probably in box C. Alterations of the spacing between the two major promoter domains of from -3 to +10 nucleotides are tolerated, although they reduce transcription activity and were found to prevent the formation of a stable initiation complex.