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. cerevisi… Show more
“…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.…”
Section: Resultsmentioning
confidence: 99%
“…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.…”
Nearly all tRNAHis species have an additional 59 guanine nucleotide (G À1 ). G À1 is encoded opposite C 73 in nearly all prokaryotes and in some archaea, and is added post-transcriptionally by tRNA His guanylyltransferase (Thg1) opposite A 73 in eukaryotes, and opposite C 73 in other archaea. These divergent mechanisms of G À1 conservation suggest that G À1 might have an important cellular role, distinct from its role in tRNA His charging. Thg1 is also highly conserved and is essential in the yeast Saccharomyces cerevisiae. However, the essential roles of Thg1 are unclear since Thg1 also interacts with Orc2 of the origin recognition complex, is implicated in the cell cycle, and catalyzes an unusual template-dependent 39-59 (reverse) polymerization in vitro at the 59 end of activated tRNAs. Here we show that thg1-D strains are viable, but only if histidyl-tRNA synthetase and tRNA His are overproduced, demonstrating that the only essential role of Thg1 is its G À1 addition activity. Since these thg1-D strains have severe growth defects if cytoplasmic tRNA His A 73 is overexpressed, and distinct, but milder growth defects, if tRNA His C 73 is overexpressed, these results show that the tRNA His G À1 residue is important, but not absolutely essential, despite its widespread conservation. We also show that Thg1 catalyzes 39-59 polymerization in vivo on tRNA His C 73 , but not on tRNA His A 73 , demonstrating that the 39-59 polymerase activity is pronounced enough to have a biological role, and suggesting that eukaryotes may have evolved to have cytoplasmic tRNA His with A 73 , rather than C 73 , to prevent the possibility of 39-59 polymerization.
“…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.…”
Section: Resultsmentioning
confidence: 99%
“…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.…”
Nearly all tRNAHis species have an additional 59 guanine nucleotide (G À1 ). G À1 is encoded opposite C 73 in nearly all prokaryotes and in some archaea, and is added post-transcriptionally by tRNA His guanylyltransferase (Thg1) opposite A 73 in eukaryotes, and opposite C 73 in other archaea. These divergent mechanisms of G À1 conservation suggest that G À1 might have an important cellular role, distinct from its role in tRNA His charging. Thg1 is also highly conserved and is essential in the yeast Saccharomyces cerevisiae. However, the essential roles of Thg1 are unclear since Thg1 also interacts with Orc2 of the origin recognition complex, is implicated in the cell cycle, and catalyzes an unusual template-dependent 39-59 (reverse) polymerization in vitro at the 59 end of activated tRNAs. Here we show that thg1-D strains are viable, but only if histidyl-tRNA synthetase and tRNA His are overproduced, demonstrating that the only essential role of Thg1 is its G À1 addition activity. Since these thg1-D strains have severe growth defects if cytoplasmic tRNA His A 73 is overexpressed, and distinct, but milder growth defects, if tRNA His C 73 is overexpressed, these results show that the tRNA His G À1 residue is important, but not absolutely essential, despite its widespread conservation. We also show that Thg1 catalyzes 39-59 polymerization in vivo on tRNA His C 73 , but not on tRNA His A 73 , demonstrating that the 39-59 polymerase activity is pronounced enough to have a biological role, and suggesting that eukaryotes may have evolved to have cytoplasmic tRNA His with A 73 , rather than C 73 , to prevent the possibility of 39-59 polymerization.
“…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.…”
In addition to the well-known internal promoter elements of tRNA genes, 5' flanking sequences can also influence the efficiency of transcription by Saccharomyces cerevilsae extracts in vitro. A consensus sequence of yeast tRNA genes in the vicinity of the transcriptional start site can be derived. To determine whether the activity of this region can be attributed to particular sequence features we studied in vitro mutants of the start site region. We found that the start site can be shifted, but only to a limited extent, by moving the conserved sequence element. We found that both a pyrimidine-purine motif (with transcription initiating at the purine) and a small T:A base pair block upstream are important for efficient transcription in vitro. Thus the sequence surrounding the start site of transcription of the yeast tRNAL1u3 gene does play a role in determining transcription efficiency and fixing the precise site of initiation by RNA polymerase Ill.
“…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).…”
A region within 35 nucleotides upstream of the transcription initiation site of a variety of silkworm Class Ill templates is absolutely required for transcription in vitro. To determine whether the activity of this region can be attributed to a particular sequence element, we systematically replaced 4 -5 bp segments of the region upstream of a silkworm tRNAAla gene. We show that replacement of either of two AT-rich blocks markedly impairs promoter function, whereas replacement of other sequences has little or no effect. Additional mutants were constructed to test whether base composition or sequence is important for function of the AT blocks. We find that some sequences are more effective than others, but that various AT-rich sequences can direct transcription at a high level. Possible mechanisms by which such elements could act are discussed.
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