Abstract
The internal control elements of Neurospora crassa 5 S genes include an A box and a C box as in Xenopus and Saccharomyces cerevisiae, plus a novel element, the Ribo box at position +18 to +34. The Ribo box is also found in the 40 S rRNA promoter and a ribosomal protein gene but is absent from tRNA genes in N. crassa. The 5 S A box diverges from the tRNA A box consensus at two positions. We tested whether replacement of the 5 S A box with a tRNALeu A box sequence would increase 5 S gene transcription in vitro or would remove the requirement for the Ribo box. The 5 S gene with the tRNALeu A box was transcribed poorly, and the Ribo box and the C box are still required for transcription. We tested the function of the Ribo box and 5 S A box in a tRNA-like transcription unit by constructing hybrids between a 5 S gene and a tRNALeu gene. In the tRNA-like context, the 5 S A box supported a lower level of transcription than the tRNA A box, and the Ribo box was not required at all. Therefore, in N. crassa, all of the 5 S internal control elements are gene-specific. In particular, the 5 S and tRNA A box sequences are not functionally interchangeable and may bind different transcription factors. Transcription of the hybrids was initiated at the 5 S initiation site, suggesting that the mechanism of initiation site selection is the same in the 5 S and tRNA genes. Competition experiments with the tRNA B box suggested that the N. crassa 5 S and tRNA genes require at least one common transcription factor such as TFIIIC.
Highlights
All Internal Promoter Elements oNf eurospora crmsa 5 S rRNA and tRNA Genes, Includingthe A Boxes, Are FunctionallyGene-specific*
Certain A box mutations interfere with found in the40 S rRNA promoter anad ribosomal the bindingof TFIIIC to the TFII5IAS.DNA complex (Pieler proteingenebut is absentfrom tRNA genes in N . et al, 1987), but no binding of TFIIIC to the A box can be crassa
We tested whether replacement of the 5 S A box with a tRNALe“A box sequence would increase5 S gene transcriptionin vitroor would remove the requirement for tRheib0 box
Summary
In these studies were the 5 S gene a52 (Selker et al, 1981) and a cloned tRNALe"gene (Huiet et al, 1984). The tRNA portion of the hybrid extends from the HpaI site a t +49 to a ClaI site 200 bp beyond the 3' end of the gene, and includes the B box and the transcription terminator (Fig. l E , template e ). In the case of competition experiments, the competitor DNA was premixed with all other components including the transcription extract on ice for 5-10 min before addition of template base pair insertion allowed by the consensus (415-13 in Fig. 1A) (the term"altered A box" will beused to refer to the41513 A box sequence). The Rib0 box mutation reduced transcription of the wild type gene 33-fold(Fig. 2, lane d ) and abolished the remaining transcription of the 5 S gene containing the tRNA A box (lane c).
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