TFIIIB placement on a yeast U6 RNA gene in vivo is directed primarily by TFIIIC rather than by sequence-specific DNA contacts

Gerlach, Valerie L.; Whitehall, Simon K.; Geiduschek, E. Peter; Brow, David A.

doi:10.1128/mcb.15.3.1455

Cited by 68 publications

(89 citation statements)

References 61 publications

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“…Together, these data show that transcription start sites do not occur efficiently on SUP4 templates at positions upstream of Ϫ1 or downstream of ϩ3. The position of this 4-bp initiation window is likely determined by the precise placement of initiation factor TFIIIB on the upstream DNA by the A-block binding component of TFIIIC (3,12,20,28,37).…”

Section: Discussionmentioning

confidence: 99%

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: Discussionmentioning

confidence: 99%

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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“…If the start site is defined by its distance from factors bound upstream or by a polymerase scanning from the TATA box, we should detect primarily upstream starts. In contrast, if the start site is measured from factors bound to intragenic promoter elements, we should detect primarily downstream starts (36).…”

Section: Conserved Sequence Elementsmentioning

confidence: 99%

Quantitative Analysis of in Vivo Initiator Selection by Yeast RNA Polymerase II Supports a Scanning Model

Kuehner

Brow

2006

Journal of Biological Chemistry

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Initiation of transcription by RNA polymerase II (RNAP II) onSaccharomyces cerevisiae messenger RNA (mRNA) genes typically occurs at multiple sites 40 -120 bp downstream of the TATA box. The mechanism that accommodates this extended and variable promoter architecture is unknown, but one model suggests that RNAP II forms an open promoter complex near the TATA box and then scans the template DNA strand for start sites. Unlike most proteincoding genes, small nuclear RNA gene transcription starts predominantly at a single position. We identify a highly efficient initiator element as the primary start site determinant for the yeast U4 small nuclear RNA gene, SNR14. Consistent with the scanning model, transcription of an SNR14 allele with tandemly duplicated start sites initiates primarily from the upstream site, yet the downstream site is recognized with equivalent efficiency by the diminished population of RNAP II molecules that encounter it. A quantitative in vivo assay revealed that SNR14 initiator efficiency is nearly perfect (ϳ90%), which explains the precision of U4 RNA 5 end formation. Initiator efficiency was reduced by cis-acting mutations at ؊8, ؊7, ؊1, and ؉1 and trans-acting substitutions in the TFIIB B-finger. These results expand our understanding of RNAP II initiation preferences and provide new support for the scanning model. Eukaryotes rely on RNA polymerase II (RNAP II)2 to synthesize all messenger RNAs (mRNAs) and most of the small nuclear RNAs (snRNAs) and small nucleolar RNAs (snoRNAs) encoded within their nuclear genomes. Efficient and accurate transcription initiation is vital to ensure the proper expression and function of these RNAs. The recruitment of RNAP II to gene promoters is mediated through the assembly of a pre-initiation complex (PIC). RNAP II accessory proteins provide promoter specificity and the structural core for assembly of the PIC. These accessory proteins include the general transcription factors TFIID, TFIIB, TFIIF, TFIIE, and TFIIH (1-3). Some transcription factors engage in sequence-specific contacts with core promoter elements (4); one of the most fundamental interactions for PIC assembly is between the TATA-binding protein subunit of TFIID and the TATA box (5-6). In a stepwise model for PIC assembly, TATA-binding protein binding is followed by the addition of TFIIB, RNAP II-TFIIF, TFIIE, and TFIIH (7).In metazoans, the assembly of a PIC at the TATA box results in start site selection 25-30 bp downstream (4). The architecture of the PIC is such that the transcription start site is placed precisely within the active center of RNAP II (8 -9). In the yeast Saccharomyces cerevisiae, RNAP II initiation typically occurs at multiple sites at variable distances from the TATA box, with most start sites ranging from 40 to 120 bp downstream of the TATA box (10). The initiation mechanism that accommodates this extended and variable promoter architecture is unknown, but it does not appear to be dependent on assembling the yeast PIC in a manner different from that of metazoans. Yeast p...

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“…A synergistic action of box A and upstream sequence elements in transcription initiation has previously been observed. For example, it has been shown that SNR6 transcription initiation in vivo is insensitive to mutation of the upstream TATA box, unless the box A is simultaneously mutated (59). It is also known that TSS selection on tRNA genes is co-directed by a TFIIIB component, TBP, and by the most upstream, A box-interacting portion of TFIIIC (60).…”

Section: Discussionmentioning

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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TFIIIB placement on a yeast U6 RNA gene in vivo is directed primarily by TFIIIC rather than by sequence-specific DNA contacts

Cited by 68 publications

References 61 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

Quantitative Analysis of in Vivo Initiator Selection by Yeast RNA Polymerase II Supports a Scanning Model

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

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