In vivo modulation of yeast tRNA gene expression by 5′-flanking sequences.

Abstract

A pentadecanucleotide sequence, TTTCAACAAATAAGT, contiguous with the 5′‐end of Saccharomyces cerevisiae tRNA‐Leu3 coding sequence acts as a positive modulator of transcription in a homologous in vitro system. To determine whether modulation also takes place in vivo, the amber suppressor forms of tRNA‐Leu3 genes with different 5′‐flanking sequences were generated by site‐specific mutagenesis and cloned into YCp19, a yeast vector maintained at 1‐2 copies per cell. These plasmids were transformed into S. cerevisiae strains marked with amber mutations lys2‐801, met8‐1, and tyr7‐1. The ability of the tRNA‐Leu3 amber suppressor genes (tDNA‐Leu3A) to suppress functionally lys2‐801 and tyr7‐1 mutations in the yeast host strain correlated well with template activities measured in vitro. We conclude that the plasmid‐borne tRNA gene acts as an effective suppressor from the plasmid and the conserved pentadecanucleotide sequence modulated the expression of yeast tRNA‐Leu3 in vivo as well as in vitro. This regulatory sequence if found associated with genes coding for a number of tRNAs which are abundant in yeast. We postulate that this sequence represents a mechanism by which production of specific tRNAs can be enhanced to match demand created by codon use preferences.

The requirement for the highly conserved G−1 residue of Saccharomyces cerevisiae tRNAHis can be circumvented by overexpression of tRNAHis and its synthetase

“…2B). Since the lys2-801am allele is efficiently suppressed by insertion of serine (Brandriss et al 1976), leucine (Raymond et al 1985), or tryptophan (Kim and Johnson 1988), the lack of suppression by tRNA His am suppressor variants indicates lack of charging of these tRNAs by other amino acids. Furthermore, the dramatically increased lys2-801am suppression upon overexpression of HTS1 indicates that histidine is inserted under these conditions.…”

“…Moreover, suppression almost certainly occurs by insertion of histidine, since the Kex2 serine protease requires histidine at the catalytic H213 position, and since suppression requires overexpression of HTS1. Furthermore, it is unlikely that either tRNA His am C 73 or tRNA His am A 73 is appreciably mischarged, since suppression of a lys2-801am mutation is almost undetectable, although this mutation is known to be suppressed by insertion of any of several different amino acids (Brandriss et al 1976;Raymond et al 1985;Kim and Johnson 1988), and since almost all the observed suppression requires overexpression of HTS1. Second, we showed that thg1-D cells are viable in spite of the lack of detectable G À1 in their tRNA His , but only if both tRNA His and HTS1 are overproduced.…”

Mutational analysis of the transcription start site of the yeast tRNALeu3gene

“…TFIIIB appears not to recognize specific sequences, but to be directed to its 40 bp long DNA binding site, upstream of the transcriptional start site, by intragenically bound TFIIIC (11 We analyzed a large collection of S.cerevisiae tRNA genes to derive a conserved sequence element (CSE) in the neighborhood of the transcriptional start site (Fig. IA), as others have done previously (16)(17). The sequence at the start site ofthe tRNALeu3 gene conforms well with the CSE; the matching block TTTCAAC is located at positions -15 and -9 upstream of the mature coding sequence and includes the start site (the purine A at position -11) indicated by the arrow in Figure lB.…”

Transcription of a silkworm tRNAcAlagene is directed by two AT-rich upstream sequence elements

“…However, some 5' flanking substitutions do result in decreased promoter efficiency (19,20). Moreover, genes coding for a number of abundant yeast tRNAs (tRNALeu, tRNAArg, tRNALys, tRNAser) contain a conserved motif YYCAACAAATAAGT in their 5' flanks (21). Deletion of a DNA segment containing this element from tRNALeU and tRNATYr genes results in decreased transcription (22,5 (9).…”