Abstract: The Saccharomyces cerevisiae SNR52 gene is unique among the snoRNA coding genes in being transcribed by RNA polymerase III. The primary transcript of SNR52 is a 250 nucleotide precursor RNA from which a long leader sequence is cleaved to generate the mature snR52 RNA. We found that the box A and box B sequence elements in the leader region are both required for the in vivo accumulation of the snoRNA. As expected the box B, but not the box A, was absolutely required for stable TFIIIC binding in vitro. Surprisingly, however, the box B was found to be largely dispensable for in vitro transcription of SNR52, while the box A-mutated template effectively recruited TFIIIB, but nevertheless it was transcriptionally inactive. Even in the complete absence of box B and both upstream TATA-like and T-rich elements, the box A still directed efficient, TFIIIC-dependent transcription. Box B-independent transcription was also observed for two members of the tRNAAsn(GTT) gene family, but not for two tRNAPro(AGG) gene copies. Fully recombinant TFIIIC supported box B-independent transcription of both SNR52 and tRNAAsn genes, but only in the presence of TFIIIB reconstituted with a crude B" fraction. Non-TFIIIB component(s) in this fraction were also required for transcription of wild type SNR52. Transcription of the box B-less tRNAAsn genes was strongly influenced by their 5'-flanking regions, and it was stimulated by TBP and Brf1 proteins synergistically. The box A can thus be viewed as a core TFIIIC-interacting element that, assisted by upstream TFIIIB-DNA contacts, is sufficient to promote class III gene transcription.