Transcription factor binding is limited by the 5'-flanking regions of a Drosophila tRNAHis gene and a tRNAHis pseudogene.

Abstract

We determined the sequence of a Drosophila tRNA gene cluster containing a tRNAHis gene and a tRNAHis pseudogene in close proximity on the same DNA strand. The pseudogene contains eight consecutive base pairs different from the region of the bona fide gene which codes for the 3' portion of the anticodon stem of tRNAHis. The tRNAHis gene is transcribed efficiently in Drosophila Kc cell extract, whereas the pseudogene is not. The pseudogene is also a much poorer competitor than the real gene in a stable transcription complex formation assay, even though the sequence alteration in the pseudogene does not affect the sequence or spacing of the putative internal transcription control regions. Recombinant clones were constructed in which the 5'-flanking regions are exchanged. The transcription efficiencies and competitive abilities of the recombinant clones resemble those of the genes from which the 5' flank was derived; for example, the tRNAHis pseudogene with the 5'-flanking sequence of the tRNAHis gene is now efficiently transcribed. Deletion analysis of the pseudogene 5' flank failed to uncover an inhibitory element. Deletion analysis of the real gene showed very high dependence on the presence of the wild-type 5'-flanking sequence for factor binding to the internal control regions and stable complex formation. The 5'-flanking sequence of a Drosophila tRNAArg gene active in the Drosophila Kc cell extract does not restore transcriptional activity or stable complex formation. The tRNAHis gene and pseudogene behave atypically in HeLa cell extract. Both genes compete for HeLa transcription factors, but neither of them is efficiently transcribed. Removal of the 5'-flanking sequences of each gene and replacement with various sequences, including the tRNAArg gene 5' flank, does not allow increased transcription in HeLa cell extract.

Modulation of transcriptional activity and stable complex formation by 5'-flanking regions of mouse tRNAHis genes.

“…Another important position may be at -6, which is always a pyrimidine in transcribed clones and, with one exception, a purine in poorly transcribed clones. The importance of these two consensus positions may also explain why a Drosophila tRNAHiS gene is transcribed with much lower efficiency in a HeLa cell extract than in a Drosophila cell extract (3,8). Although the Drosophila gene contains a proximal 5'-flanking region which is similar in some respects to that of the mouse tRNAHis genes, it lacks a G at position -4 and a pyrimidine at -6.…”

“…Several invertebrate tRNA genes contain 5'-flanking sequences that stimulate transcription in homologous extracts (8,11,22,23,38,44). However, in some cases, the 5'-flanking dependence of transcription appears to be reduced in mammalian cell extracts (3,11,32,38,43).…”

“…These "internal" control regions appear to be sites of interaction of specific transcription factors with the gene (12,13,24,43,47). In addition, sequences in the 5'-flanking regions of certain Xenopius (18), Drosophila (8,10,11,38), Bombyx mori (23), and yeast (22,44) tRNA genes can modulate transcription either positively or negatively. Thus, major questions in regard to any specific tRNA gene family are whether modulatory sequences are present in the flanking regions and the mechanisms by which these sequences affect gene activity.…”

Transcription of aDrosophilatRNAArggene in yeast extract: 5′-flanking sequence dependence for transcription in a heterologous system

“…The transcripts were visualized by autoradiography overnight, the bands excised, and Cerenkov radiation determined to quantitate transcription. HeLa cell extract was prepared by the method of Dingermann et al (23), and transcription reactions were performed as previously described (24).…”

Sequence analysis of three tRNAPhe nuclear genes and a mutated gene, and one gene for tRNAAla from Arabidopsis thaliana

“…These approximately correspond to the D loop and the T $ C loop of the tRNA molecule [6]. It was shown also that certain sequences in the 5' flanking region of tRNA genes can modulate the efficiency of transcription in yeast [15], Drosophila [4], Xenopus [9], mouse [19] and rice [22] either positively or negatively. Thus, the study of the molecular structure of the flanking and coding regions of tRNA genes is important for obtaining a full understanding of the regulatory mechanism of transcription of tRNA genes.…”