In vivo analyses of upstream promoter sequence elements in the 5 S rRNA gene from saccharomyces cerevisiae 1 1Edited by S.Reed

Lee, Yoon; Wong, William; Guyer, David; Erkine, Alexander M.; Nazar, Ross N.

doi:10.1006/jmbi.1997.1071

Cited by 14 publications

(7 citation statements)

References 42 publications

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“…RNA size markers were generated by T7 RNA polymerase (Amersham Biosciences) in vitro transcription (23) of linearized pBlueScript-KS constructs bearing inserts of different sizes in the SmaI site: the S. cerevisiae I(TAT)LR1 tRNA gene and flanking regions (302 bp (6)) and the sequences coding for yeast ribosomal proteins L13 (600 bp (23)) and S24 (408 bp). 3 DNase I Footprinting and Gel Retardation Assays-For the DNase I footprinting experiment in Fig. 4A, a 992-bp SCR1 fragment, 5Ј-endlabeled on the sense strand, was generated by PCR using 5Ј-labeled SCR1fw and unlabeled SCR1rev as primers and pBlueScript-SCR1 as a template.…”

Section: Methodsmentioning

confidence: 99%

Intragenic Promoter Adaptation and Facilitated RNA Polymerase III Recycling in the Transcription of SCR1, the 7SL RNA Gene ofSaccharomyces cerevisiae

Dieci

Giuliodori

Catellani

et al. 2002

Journal of Biological Chemistry

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The SCR1 gene, coding for the 7SL RNA of the signal recognition particle, is the last known class III gene of Saccharomyces cerevisiae that remains to be characterized with respect to its mode of transcription and promoter organization. We show here that SCR1 represents a unique case of a non-tRNA class III gene in which intragenic promoter elements (the TFIIIC-binding Aand B-blocks), corresponding to the D and T⌿C arms of mature tRNAs, have been adapted to a structurally different small RNA without losing their transcriptional function. In fact, despite the presence of an upstream canonical TATA box, SCR1 transcription strictly depends on the presence of functional, albeit quite unusual, A-and B-blocks and requires all the basal components of the RNA polymerase III transcription apparatus, including TFIIIC. Accordingly, TFIIIC was found to protect from DNase I digestion an 80-bp region comprising the A-and B-blocks. B-block inactivation completely compromised TFIIIC binding and transcription capacity in vitro and in vivo. An inactivating mutation in the A-block selectively affected TFIIIC binding to this promoter element but resulted in much more dramatic impairment of in vivo than in vitro transcription. Transcriptional competition and nucleosome disruption experiments showed that this stronger in vivo defect is due to a reduced ability of A-block-mutated SCR1 to compete with other genes for TFIIIC binding and to counteract the assembly of repressive chromatin structures through TFIIIC recruitment. A kinetic analysis further revealed that facilitated RNA polymerase III recycling, far from being restricted to typical small sized class III templates, also takes place on the 522-bp-long SCR1 gene, the longest known class III transcriptional unit.The most represented RNA polymerase III (Pol III) 1 -transcribed genes, those coding for the tRNAs and the 5 S rRNA, have a highly conserved intragenic promoter comprising the binding sites for the general transcription factor TFIIIC (Aand B-blocks) and for the 5 S-specific factor TFIIIA (C-block). This conservation probably reflects the dual function of the above elements as both nucleation sites for transcription complex assembly and key determinants of tRNA and 5 S rRNA structure. Within the same genes, in fact, an extremely high sequence variability is displayed by the structurally unconstrained, vicinal upstream region. This region provides the binding surface for the initiation factor TFIIIB (1) and can modulate the strength of the intragenic promoter (see Refs. 2-4 and references therein). TFIIIB, which in yeast is minimally composed of the TATA-box-binding protein, the TFIIB-related factor BRF (or TFIIIB70), and the Pol III-specific factor BЉ (or TFIIIB90), is generally assembled on tRNA genes in a TFIIICdependent manner (5). One extreme case of 5Ј-flanking sequence effect, however, has recently been documented for some tRNA genes of Saccharomyces cerevisiae, which, due to the presence of a canonical TATA box in their 5Ј-flanking region, are capable of autonomous ...

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Section: Methodsmentioning

confidence: 99%

Intragenic Promoter Adaptation and Facilitated RNA Polymerase III Recycling in the Transcription of SCR1, the 7SL RNA Gene ofSaccharomyces cerevisiae

Dieci

Giuliodori

Catellani

et al. 2002

Journal of Biological Chemistry

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“…The 5S rRNA genes are transcribed by polymerase III which is strongly inhibited by p53 (12) and depends strongly in eukaryotic cells on the binding of a 40 kDa protein-transcription factor IIIA (TF IIIA)-to the internal control region of 5S rRNA genes (13). There is also evidence for direct interactions of upstream regulatory elements and a new independent upstream promoter element centered about -17 to -20 (14). One of the remarkable features of TF IIIA is that it is capable of specific binding to the 5S rRNA gene and the gene product with high affinity and specificity, although three-dimensional structures of RNA and DNA are clearly different.…”

Section: Introductionmentioning

confidence: 99%

5S Ribosomal RNA data bank

Szymański

Barciszewska²,

Barciszewski³

et al. 1999

Nucleic Acids Research

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This paper presents the updated version of the data base of ribosomal 5S ribonucleic acids (5S rRNA) and their genes (5S rDNA). This edition of the data bank contains 1889 primary structures of 5S rRNA and 5S rDNA. These include 60 archaebacterial, 439 eubacterial, 63 plastid, 9 mitochondrial and 1318 eukaryotic sequences. The nucleotide sequences of 5S rRNAs or 5S rDNAs are divided according to the taxonomic position of organisms. The sequences stored in the database can be viewed and retrieved using the taxonomic browser at the URL: http://rose.man.poznan.pl/5SData/5SRNA.html++ +

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“…In spite of all the available information on the in vitro systems, very little is known about the organization of transcription complexes on 5S genes inside yeast cells. A weak modulation of DNase I cutting has recently been observed in the 5Ј-flanking sequence of 5S genes on episomes in vivo (31,32). Previous attempts to reveal transcription factor-DNA interactions on the 5S chromosomal genes in vivo did not provide conclusive information.…”

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confidence: 98%

RNA Polymerase III Transcription Complexes on Chromosomal 5S rRNA Genes In Vivo: TFIIIB Occupancy and Promoter Opening

Costanzo

Camier

Carlucci

et al. 2001

Molecular and Cellular Biology

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Quantitative analysis of multiple-hit potassium permanganate (KMnO 4 ) footprinting has been carried out in vivo on Saccharomyces cerevisiae 5S rRNA genes. The results fix the number of open complexes at steady state in exponentially growing cells at between 8 and 17% of the 150 to 200 chromosomal copies. UV and dimethyl sulfate footprinting set the transcription factor TFIIIB occupancy at 23 to 47%. The comparison between the two values suggests that RNA polymerase III binding or promoter opening is the rate-limiting step in 5S rRNA transcription in vivo. Inhibition of RNA elongation in vivo by cordycepin confirms this result. An experimental system that is capable of providing information on the mechanistic steps involved in regulatory events in S. cerevisiae cells has been established.Yeast RNA polymerase III transcription machinery components and their interaction with promoter elements are known in detail (for reviews, see references 19, 22, and 45). 5S rRNA transcription requires TFIIIB, which is the initiation factor proper of RNA polymerase III, and two assembly factors, TFIIIA and TFIIIC (25). The in vitro topography of transcription factor complexes is well described thanks to extensive DNA footprinting studies (8,25,26), protein-DNA cross-linking (2, 9, 27), and protein subunit assembly studies (13,29). Analysis of KMnO 4 sensitivity on open complexes and stalled elongation complexes has suggested structural analogies to bacterial polymerase mechanisms (25,28). In spite of all the available information on the in vitro systems, very little is known about the organization of transcription complexes on 5S genes inside yeast cells. A weak modulation of DNase I cutting has recently been observed in the 5Ј-flanking sequence of 5S genes on episomes in vivo (31, 32). Previous attempts to reveal transcription factor-DNA interactions on the 5S chromosomal genes in vivo did not provide conclusive information. These difficulties are primarily due to (i) the low occupancy by transcription complexes of the multiple copies of the genes present in the cell (14), (ii) the lack of studies making use of noninvasive footprinting techniques, and (iii) the lack of proper nontranscribing control conditions allowing clear distinctions to be made between changes due to transcription complexes and those due to chromatin organization. In this study we overcome these problems. UV (3, 5), dimethyl sulfate (DMS) (21), and KMnO 4 (37) footprinting can be performed directly on growing cells. This approach involves short exposure times with minimal impact on the biological structure to be investigated. Histone-DNA interactions have only a small influence on UV irradiation and DMS reactivity patterns, allowing selective visualization of transcription factor footprints (4, 21). KMnO 4 is a highly specific probe for open-complex formation. A system was developed to transcribe 5S rRNA genes in the absence of TFIIIA (12). In this system, yeast cells devoid of TFIIIA, which therefore cannot form transcription complexes on endogenous chromo...

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In vivo analyses of upstream promoter sequence elements in the 5 S rRNA gene from saccharomyces cerevisiae 1 1Edited by S.Reed

Cited by 14 publications

References 42 publications

Intragenic Promoter Adaptation and Facilitated RNA Polymerase III Recycling in the Transcription of SCR1, the 7SL RNA Gene ofSaccharomyces cerevisiae

Intragenic Promoter Adaptation and Facilitated RNA Polymerase III Recycling in the Transcription of SCR1, the 7SL RNA Gene ofSaccharomyces cerevisiae

5S Ribosomal RNA data bank

RNA Polymerase III Transcription Complexes on Chromosomal 5S rRNA Genes In Vivo: TFIIIB Occupancy and Promoter Opening

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