Stable transcription complex formation of eukaryotic tRNA genes is dependent on a limited separation of the two intragenic control regions.

Abstract

We have examined the transcriptional role of the DNA region which lies between the two intragenic control sequences (D-control and T-control) of tRNA genes. Deletion templates (3' and 5') of a Drosophila tRNAArg gene, which contain either the D- or T-control region, were joined together through XhoI linkers such that the mutant tRNA genes formed now contain an internal cloning site. DNA fragments of different lengths were inserted into the newly formed cloning site to create a series of mutant tRNAArg genes which have an increased separation between the two intragenic control regions of 12 to 1530 nucleotides. Increased separation of the two intragenic control regions did not alter the transcription initiation or termination sites from those of the wild type tRNAArg gene. Transcription, while most efficient in the wild type gene, is still efficient when the two regions are further separated by a distance of 12-77 nucleotides. However, any further increase in length of the sequence separating the control regions resulted in a decreased transcription efficiency and in a decreased ability to compete in the binding of transcription factors in Drosophila Kc cell extracts. The reduction in transcription efficiency is directly related to the decreased ability of the insertion mutant tDNAs to form stable transcription complexes. The tRNAArg gene forms detectable stable complexes up to a separation of the two intragenic control regions by approximately 200 to 400 base pairs. These results suggest a model for tRNA gene transcription that involves factor recognition of sequences within each control region and that the control regions interact only via these factors.