In an effort to establish a suitable alternative to the widely used 18S rRNA system for molecular systematics of fungi, we examined the nuclear gene RPB2, encoding the second largest subunit of RNA polymerase II. Because RPB2 is a single-copy gene of large size with a modest rate of evolutionary change, it provides good phylogenetic resolution of Ascomycota. While the RPB2 and 18S rDNA phylogenies were highly congruent, the RPB2 phylogeny did result in much higher bootstrap support for all the deeper branches within the orders and for several branches between orders of the Ascomycota. There are several strongly supported phylogenetic conclusions. The Ascomycota is composed of three major lineages: Archiascomycetes, Saccharomycetales, and Euascomycetes. Within the Euascomycetes, plectomycetes, and pyrenomycetes are monophyletic groups, and the Pleosporales and Dothideales are distinct sister groups within the Loculoascomycetes. We confirm the placement of Neolecta within the Archiascomycetes, suggesting that fruiting body formation and forcible discharge of ascospores were characters gained early in the evolution of the Ascomycota. These findings show that a slowly evolving protein-coding gene such as RPB2 is useful for diagnosing phylogenetic relationships among fungi.

We have made specific alterations in the CAACAA element at the transcription start site of a Saccharomyces cerevisiae suppressor tRNA gene. The mutant genes were tested for their ability to suppress the ochre nonsense alleles ade2-1, lys4-1, and met4-1. Many of the mutants showed either no phenotypic change or a weak loss of suppression relative to that of SUP4-o. A 2-bp change, CTCCAA, which alters bases encoding the ؉1 and ؉2 nucleotides of pre-tRNA Tyr , had a strong deleterious effect in vivo, as did the more extensive change CTCCTC. In contrast, mutant genes bearing each of the possible single changes at nucleotide ؉1 retained normal suppression levels. The transcription start point could be shifted in a limited fashion in response to the specific sequences encountered by RNA polymerase III at the start site. ATP was preferentially utilized as the 5 nucleotide in the growing RNA chain, while with start site sequences that precluded utilization of a purine, CTP was greatly preferred to UTP as the ؉1 nucleotide. Short oligopyrimidine RNAs formed on the CTCCTC allele could be repositioned in the active center of the newly formed ternary complex. Early postinitiation complexes containing short nascent RNAs formed on the CTCCTC mutant were more sensitive to the effects of heparin and produced more abortive transcripts than similar complexes formed on SUP4-o. Our results suggest that the purine-rich sequences at the 5 ends of the nascent transcripts of many genes act to stabilize the early ternary complex.The faithful and efficient transcription of DNA into RNA is a primary regulatory point in gene expression. The relative rates at which different genes are transcribed by the same RNA polymerase are strongly influenced by sequences surrounding the transcription start site. Previous studies of these contextual effects have emphasized sequence differences in the formation of the initiation complex and in the selection of the initiation site. RNA polymerases, both bacterial and eukaryotic, preferentially initiate transcription with a purine residue (4,5,8,14,18,29,30). In contrast, pyrimidine-specific transcription start sites are far less frequent as judged by 5Ј-end determination of RNAs synthesized both in vivo and in vitro (5, 18); only 7% of 88 Escherichia coli promoters surveyed by Hawley and McClure (14) directed transcription start sites with either UTP or CTP, with pyrimidine-specific initiation occurring only in the absence of purines within a defined window (36). The specific binding of purines at the i site of E. coli RNA polymerase, both in the presence and in the absence of template DNA, suggests that the interaction of purine ribonucleotides with RNA polymerase is important for productive transcription initiation (1, 47-49). However, the precise role in this process of nucleic acid sequences at and immediately following position ϩ1 remains unclear.As a system for studying small or large quantitative effects upon the level of transcription of a gene, RNA polymerase III transcription of the SUP4 gene ...

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Sequence data are presented for the Saccharomyces cerevisiae TAP1 gene and for a mutant allele, tap1-1, that activates transcription of the promoter-defective yeast SUP4 tRNA(Tyr) allele SUP4A53T61. The degree of in vivo activation of this allele by tap1-1 is strongly affected by the nature of the flanking DNA sequences at 5'-flanking DNA sequences as far away as 413 bp from the tRNA gene and by 3'-flanking sequences as well. We considered the possibility that this dependency is related to the nature of the chromatin assembled on these different flanking sequences. TAP1 encodes a protein 1,006 amino acids long. The tap1-1 mutation consists of a thymine-to-cytosine DNA change that changes amino acid 683 from tyrosine to histidine. Recently, Amberg et al. reported the cloning and sequencing of RAT1, a yeast gene identical to TAP1, by complementation of a mutant defect in poly(A) RNA export from the nucleus to the cytoplasm (D. C. Amberg, A. L. Goldstein, and C. N. Cole, Genes Dev. 6:1173-1189, 1992). The RAT1/TAP1 gene product has extensive sequence similarity to a yeast DNA strand transfer protein that is also a riboexonuclease (variously known as KEM1, XRN1, SEP1, DST2, or RAR5; reviewed by Kearsey and Kipling [Trends Cell Biol. 1:110-112, 1991]). The tap1-1 amino acid substitution affects a region of the protein in which KEM1 and TAP1 are highly similar in sequence.

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