Transcriptional activation of a moderately expressed tRNA gene by a positioned nucleosome

Abstract

All of the members of a tRNA1Gly multigene family from the mulberry silkworm, Bombyx mori, have identical coding regions and consequently identical internal promoter elements, but are transcribed at different levels. A moderately expressed copy, tRNA1Gly-4 from within this multigene family, which was transcribed to 30–50% of the highly transcribed gene copies harboured two typical TATAA box sequences in the 5′ upstream region at positions −27 nt and −154 nt with respect to the +1 nt of mature tRNA. Deletion of the distal TATAA sequence at −154 nt brought down the transcription more than 70%, whereas mutation of the proximal element did not affect transcription. tRNA1Gly-4 could be readily assembled into chromatin, with a positioned nucleosome in the upstream region, and the assembled nucleosome formed stable complexes with the transcription factors TFIIIC and TFIIIB. Organization of the gene into nucleosomes also enhanced transcription significantly above that of the naked DNA, reaching transcription levels comparable with those of the highly transcribed copies. This nucleosome-mediated enhancement in transcription was absent when the distal TATAA sequences were deleted, whereas mutation of the proximal TATAA element showed no effect. In the absence of the distal TATAA sequences, assembly into the nucleosome inhibited transcription of tRNA1Gly-4. TFIIIB bound directly through the distal TATAA sequence at −154 nt and the positioned nucleosome facilitated its interaction with TFIIIC. The direct binding of TFIIIB to the DNA provided anchoring of the factor to the template DNA which conferred a higher stability on the TFIIIB–TFIIIC–DNA complex. We have proposed a novel mechanism for the nucleosome-mediated stimulation of pol III (RNA polymerase III) transcription of tRNA genes, a model not presented previously.