Transcription of eukaryotic tRNA genes in vitro. II. Formation of stable complexes.

Suppression of nonsense codons in Schizosaccharomyces pombe by sup3-e tRNASerUGA or sup3-i tRNASerUAA is reduced or abolished by mutations within the suppressor locus. Twenty-five suppressor-inactive sup3-e genes and thirteen mutant sup3-i genes were isolated from S. pombe genomic clone banks by colony hybridization. Sequence analysis of these revertant alleles corroborates genetic evidence for mutational hotspots within the sup3 tRNA gene. Fifteen types of point mutations or insertions were found. Many of these replace bases which are highly or completely conserved in eucaryotic tRNA genes. Transcription of the altered sup3 genes in a Saccharomyces cerevisiae extract enabled the identification of mutations which affect the rate of 5'-end maturation or splicing of the tRNA precursors or both. A total of seven mutations were found which alter transcriptional efficiencies. Of these, five are located outside the internal transcription control regions.

Section: Methodsmentioning

confidence: 94%

Section: Methodsmentioning

confidence: 99%

Mutations Preventing Expression of sup3 tRNA^Ser Nonsense Suppressors of Schizosaccharomyces pombe

Pearson¹,

Willis²,

Hottinger³

et al. 1985

“…Flanking sequences affecting promoter strength are located within 70 base pairs (bp) 5' of transcriptional start sites, and at least some sequence effects are sensitive to precise spacing. Influence of 3'-flanking sequence on promoter strength has also been documented (9,58,59: see additional references in references 26 and 61). Therefore, it is justifiable to think of such Pol III promoters as extending over their entire transcription units.…”

mentioning

confidence: 90%

Transcription Factor IIIB Generates Extended DNA Interactions in RNA Polymerase III Transcription Complexes on tRNA Genes

Kassavetis

Riggs

Negri

et al. 1989

Transcription complexes that assemble on tRNA genes in a crude Saccharomyces cerevisiae cell extract extend over the entire transcription unit and approximately 40 base pairs of contiguous 5'-flanking DNA. We show here that the interaction with 5'-flanking DNA is due to a protein that copurifies with transcription factor TFIIIB through several steps of purification and shares characteristic properties that are normally ascribed to TFIIIB: dependence on prior binding of TFIIIC and great stability once the TFIIIC-TFIIIB-DNA complex is formed. SUP4 gene (tRNATyr) DNA that was cut within the 5'-flanking sequence (either 31 or 28 base pairs upstream of the transcriptional start site) was no longer able to stably incorporate TFIIIB into a transcription complex. The TFIIIB-dependent 5'-flanking DNA protein interaction was predominantly not sequence specific. The extension of the transcription complex into this DNA segment does suggest two possible explanations for highly diverse effects of flanking-sequence substitutions on tRNA gene transcription: either (i) proteins that are capable of binding to these upstream DNA segments are also potentially capable of stimulating or interfering with the incorporation of TFIIIB into transcription complexes or (ii) 5'-flanking sequence influences the rate of assembly of TFIIIB into stable transcription complexes.

“…A motivation for this analysis was the possibility that stable contacts with transcription factors are made only through the A and B boxes and that sequences outside these elements play a kinetic role, perhaps by increasing the likelihood of the initial interaction between template molecules and transcription factors. Such a role has been suggested for sequences upstream of a Drosophila tRNAArg gene (33). An alternative possibility is that sequences throughout the entire promoter make direct and stable contact with transcription factors.…”

Section: Discussionmentioning

confidence: 99%

Sequences Far Downstream from the Classical tRNA Promoter Elements Bind RNA Polymerase III Transcription Factors

Young

Rivier

Sprague

1991

We have examined the interaction of transcription factors TFIIIC and TFIIID with a silkworm alanine tRNA gene. Previous functional analysis showed that the promoter for this gene is unusually large compared with the classical tRNA promoter elements (the A and B boxes) and includes sequences downstream from the transcription termination site. The goal of the experiments reported here was to determine which sequences within the full promoter make stable contacts with transcription factors. We show that when TFIIIC and TFIIID are combined, a complex is formed with the tRNA(Ala)C gene. Neither factor alone can form this complex. DNase I digestion of gene-factor complexes reveals that most of the tRNA(Ala)C promoter is in contact with factors. The protected region extends from -1 to at least +136 and includes both the A and B boxes and the previously identified downstream promoter sequences. Analysis of mutant promoters shows that sequence-specific contacts throughout the protected region are required for binding. The role of 3'-flanking sequences in transcription factor binding explains the contribution of these sequences to the tRNA(Ala)C promoter. We discuss the possibility that such sequences affect promoter strength in other tRNA genes.