Mutational analysis of the transcription start site of the yeast tRNA<sup>Leu</sup><sub>3</sub>gene

Fruscoloni, Paolo; Zamboni, Michela; Panetta, Gianna; Paolis, Augusto De; Tocchini-Valentinl, Glauco P.

doi:10.1093/nar/23.15.2914

Cited by 32 publications

(27 citation statements)

References 31 publications

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“…Our results confirm and extend the findings of Fruscoloni et al (11), who investi- gated in vitro transcription using start site variants of the yeast tRNA 3 Leu gene. The ability of RNA polymerase III to scan the SUP4 initiation region for a preferred start site residue is limited.…”

Section: Discussionsupporting

confidence: 92%

“…These studies suffer from the general uncertainty arising from the creation of novel DNA junction sequences, and the traditional assay for lowered transcription efficiency provides no clue as to the functional lesions produced by these mutations. Recently, specific DNA sequence changes produced within the conserved sequence element at the transcription start site of the yeast LEU3 gene have confirmed the importance of this element for optimal in vitro transcription (11).…”

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confidence: 89%

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Purines Are Required at the 5′ Ends of Newly Initiated RNAs for Optimal RNA Polymerase III Gene Expression

Zecherle

Whelen

Hall

1996

Molecular and Cellular Biology

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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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Section: Discussionsupporting

confidence: 92%

mentioning

confidence: 89%

Purines Are Required at the 5′ Ends of Newly Initiated RNAs for Optimal RNA Polymerase III Gene Expression

Zecherle

Whelen

Hall

1996

Molecular and Cellular Biology

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“…We suggest that the apparent inconsistency between equally effective formation of TFIIIB complexes and inefficient initiation of transcription is mostly attributable to the lack of an efficient start site promoter element around bp -11 (cf. Fruscoloni et al 1995).…”

Section: A 6-bp Tata Box Can Codirect Tfiiib Binding With Tfiiicmentioning

confidence: 99%

“…[Transcription of the yeast U6 gene also initiates 30 bp downstream of the 5' end of the TATA box (Gerlach et al 1995)]. Pol III has a preference for certain DNA sequences around transcription start sites (Fruscoloni et al 1995 and references therein) and can efficiently hunt at least 1 bp upstream and downstream for the best initiating nucleotide, but when forced to initiate 4 bp downstream of the normal site of the tRNA Leu3 gene, it does so very inefficiently, or not at all (Fruscoloni et al 1995). In the context of the wild-type SUP4 gene, we suggest that start sites at bp + 1 and +4 are not attributable to Pol II!…”

Section: Multiple Initiation Sites In Eukaryotic Transcriptionmentioning

confidence: 99%

Alternative outcomes in assembly of promoter complexes: the roles of TBP and a flexible linker in placing TFIIIB on tRNA genes.

Joazeiro¹,

Kassavetis²,

Geiduschek³

1996

Genes Dev.

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Saccharomyces cerevisiae transcription factor (TF) IIIB, a TATA-binding protein (TBP)-containing multisubunit factor, recruits RNA polymerase (Pol) III for multiple rounds of transcription. TFIIIC is an assembly factor for TFIIIB on TATA-less tRNA gene promoters. To investigate the role of TBP-DNA interactions in tRNA gene transcription, we generated sequence substitutions in the SUP4 tRNA TM gene TFIIIB binding site. Purified transcription proteins were used to analyze the selection of transcription initiation sites and the physical structures of the protein complexes formed on these mutant genes. We show that the association of TFIIIB with tRNA genes proceeds through an initial step of binding-site selection that is codirected by its TBP subunit and by TFIIIC. TFIIIB is assembled in a predominantly metric manner with regard to box A, the start site-proximal binding site of TFIIIC, but TFIIIC opens a window within which wild-type TBP can select the TFIIIB-binding site. Despite its clear preference for AT-rich sequences, TBP can mediate TFIIIB assembly at diverse DNA sequences, including stretches containing only G and C. However, a mutant TBP, m3, which recognizes TATAAA and TGTAAA and is active for Pol III transcription, utilizes other sequences only poorly. We also show that alternative alignments between DNA-bound TFIIIB and TFIIIC are possible, implying a remarkably flexible linkage, and suggest that Tfc4, the TFIIIB-assembling subunit of TFIIIC, could be responsible for such elasticity. The relevance of these findings to alternative initiation of Pol II-and other Pol III-transcribed genes is discussed.

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“…1D leaves some ambiguity between the T and the A at the center of a CTAG stretch. Because the general rule for Pol III is that it initiates transcription at a purine preceded by a pyrimidine residue (44,45), we assume that the A within the CTAG stretch is the actual TSS for SNR52. Fig.…”

Section: Resultsmentioning

confidence: 99%

A Minimal Promoter for TFIIIC-dependent in Vitro Transcription of snoRNA and tRNA Genes by RNA Polymerase III

Guffanti

Ferrari

Preti

et al. 2006

Journal of Biological Chemistry

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The Saccharomyces cerevisiae SNR52 gene is unique among the snoRNA coding genes in being transcribed by RNA polymerase III. The primary transcript of SNR52 is a 250-nucleotide precursor RNA from which a long leader sequence is cleaved to generate the mature snR52 RNA. We found that the box A and box B sequence elements in the leader region are both required for the in vivo accumulation of the snoRNA. As expected box B, but not box A, was absolutely required for stable TFIIIC, yet in vitro. Surprisingly, however, the box B was found to be largely dispensable for in vitro transcription of SNR52, whereas the box A-mutated template effectively recruited TFIIIB; yet it was transcriptionally inactive. Even in the complete absence of box B and both upstream TATA-like and T-rich elements, the box A still directed efficient, TFIIIC-dependent transcription. Box B-independent transcription was also observed for two members of the tRNA Asn (GTT) gene family, but not for two tRNA Pro (AGG) gene copies. Fully recombinant TFIIIC supported box B-independent transcription of both SNR52 and tRNA Asn genes, but only in the presence of TFIIIB reconstituted with a crude B؆ fraction. Non-TFIIIB component(s) in this fraction were also required for transcription of wild-type SNR52. Transcription of the box B-less tRNA Asn genes was strongly influenced by their 5-flanking regions, and it was stimulated by TBP and Brf1 proteins synergistically. The box A can thus be viewed as a core TFIIICinteracting element that, assisted by upstream TFIIIB-DNA contacts, is sufficient to promote class III gene transcription.RNA polymerase (Pol) 3 III synthesizes tRNA, 5 S rRNA, and a variety of other types of small nuclear and cytoplasmic RNAs. In general, the transcription of class III genes is under the control of internal control regions (ICR) characterized by discontinuous structures, with essential boxes separated by non-essential nucleotides (1). In the case of tRNA and 5 S rRNA genes, the ICRs are highly conserved and comprise the binding sites for the general transcription factor TFIIIC (box A and box B) and for the 5 S-specific factor TFIIIA (box C). Once assembled, TFIIIC recruits TFIIIB upstream of the transcription start site (TSS); TFIIIB in turn recruits Pol III for transcription initiation. The strong conservation of the ICRs likely reflects their dual function as both nucleation sites for transcription complex assembly and key determinants of tRNA and 5 S rRNA structure. An indication of the constraints imposed on ICR by their overlapping structural and functional roles comes from the variability of promoter organizations displayed by the minority of class III genes not coding for tRNA and 5 S rRNA. In some of these genes, the TFIIIC-interacting control regions (box A and box B) have been maintained within the transcribed region, and adapted to the structure of the small RNA without losing the transcriptional function. For example, in the Saccharomyces cerevisiae SCR1 gene, coding for the 7SL RNA, box A and box B are both intragenic an...

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Mutational analysis of the transcription start site of the yeast tRNA^Leu₃gene

Cited by 32 publications

References 31 publications

Purines Are Required at the 5′ Ends of Newly Initiated RNAs for Optimal RNA Polymerase III Gene Expression

Purines Are Required at the 5′ Ends of Newly Initiated RNAs for Optimal RNA Polymerase III Gene Expression

Alternative outcomes in assembly of promoter complexes: the roles of TBP and a flexible linker in placing TFIIIB on tRNA genes.

A Minimal Promoter for TFIIIC-dependent in Vitro Transcription of snoRNA and tRNA Genes by RNA Polymerase III

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Mutational analysis of the transcription start site of the yeast tRNALeu3gene

Cited by 32 publications

References 31 publications

Purines Are Required at the 5′ Ends of Newly Initiated RNAs for Optimal RNA Polymerase III Gene Expression

Purines Are Required at the 5′ Ends of Newly Initiated RNAs for Optimal RNA Polymerase III Gene Expression

Alternative outcomes in assembly of promoter complexes: the roles of TBP and a flexible linker in placing TFIIIB on tRNA genes.

A Minimal Promoter for TFIIIC-dependent in Vitro Transcription of snoRNA and tRNA Genes by RNA Polymerase III

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Mutational analysis of the transcription start site of the yeast tRNA^Leu₃gene