Upstream interactions of functional mammalian tRNA gene transcription complexes probed using a heterologous DNA-binding protein.

An oligonucleotide containing the recognition site for the Escherichia coli lac repressor was inserted at various positions in the 5' flanking region of a human serine tRNA gene, and the consequences of binding lac repressor on in vitro transcription by RNA polymerase III were investigated. lac repressor prebound to operator sites centered at positions -9, -15, -35, and -37 upstream of the mature tRNA coding region completely inhibited transcription by interfering with the formation or stability of transcription complexes. lac repressor also inhibited transcription of tDNA derivatives containing operator sites at -9 and -15 when added following assembly of transcription complexes or during ongoing synthesis, but had no effects on the other tDNA derivatives if added subsequent to complex assembly. lac repressor prebound at position -43 and -46 partially inhibited transcription and redirected initiation to sites farther downstream. These effects required the continued presence of bound repressor protein. Our findings demonstrate that the human RNA polymerase III transcription complex extends at least 35 nucleotides upstream of the coding region and suggest that the spatial constraints imposed by a protein bound this far upstream can alter start site selection. Moreover, the flanking region encompassing the transcription start site remains accessible to DNA-binding proteins following assembly of the initiation complex and throughout multiple rounds of transcription.