A split binding site for transcription factor tau on the tRNA3Glu gene.

Camier, Sylvie; Gabrielsen, Odd S.; Baker, Richard E.; Sentenac, André

doi:10.1002/j.1460-2075.1985.tb03655.x

Cited by 112 publications

(78 citation statements)

References 40 publications

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“…The observed sequence context effects on Pol III termination at T 5 might in principle be mediated by TFIIIC, a transcription factor that usually covers a large part of the transcription unit, extending up to the terminator region of tRNA genes (37,38). TFIIICassociated activities have previously been proposed to influence Pol III termination in humans (39), and yeast TFIIIC has recently been shown to facilitate transcription reinitiation by Pol III on long transcription units (13).…”

Section: Distinguishing Between Weak and Strong T 5 Terminator Sequenmentioning

confidence: 99%

Sequence Context Effects on Oligo(dT) Termination Signal Recognition by Saccharomyces cerevisiae RNA Polymerase III

Braglia

Percudani

Dieci

2005

Journal of Biological Chemistry

102

112

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Eukaryotic RNA polymerase (Pol) III terminates transcription at short runs of T residues in the coding DNA strand. By genomic analysis, we found that T 5 and T 4 are the shortest Pol III termination signals in yeasts and mammals, respectively, and that, at variance with yeast, oligo(dT) terminators longer than T 5 are very rare in mammals. In Saccharomyces cerevisiae, the strength of T 5 as a terminator was found to be largely influenced by both the upstream and the downstream sequence context. In particular, the CT sequence, which is naturally present downstream of T 5 in the 3-flank of some tDNAs, was found to act as a terminator-weakening element that facilitates translocation by reducing Pol III pausing at T 5 . In contrast, tDNA transcription termination was highly efficient when T 5 was followed by an A or G residue. Surprisingly, however, when a terminationproficient T 5 signal was taken out from the tDNA context and placed downstream of a fragment of the SCR1 gene, its termination activity was compromised, both in vitro and in vivo. Even the T 6 sequence, acting as a strong terminator in tRNA gene contexts, was unexpectedly weak within the SNR52 transcription unit, where it naturally occurs. The observed sequence context effects reflect intrinsic recognition properties of Pol III, because they were still observed in a simplified in vitro transcription system only consisting of purified RNA polymerase and template DNA. Our findings strengthen the notion that termination signal recognition by Pol III is influenced in a complex way by the region surrounding the T cluster and suggest that read-through transcription beyond T clusters might play a significant role in the biogenesis of class III gene products.Eukaryotic RNA polymerase (Pol) 1 III is unique among DNAdependent RNA polymerases in recognizing a simple run of T residues on the coding strand as a termination signal, in the apparent absence of accessory factors. The minimal signal thought to be sufficient to provoke Pol III termination varies among different eukaryotes. T 4 suffices for termination by Xenopus and human Pol III (1, 2), T 4 /T 5 for Schizosaccharomyces pombe Pol III (3), and T 5 /T 6 for Saccharomyces cerevisiae Pol III (4). Pol III termination within T clusters is generally heterogeneous and progressive (5), and termination efficiency tends to increase with the length of the T run (4). Mechanistically, Pol III termination involves extensive pausing, dictated by the T cluster itself (5, 6). Even during elongation, the addition of three UMP residues in succession is particularly slow (5). Pol III pausing at T clusters sets cycles of hydrolytic RNA chain retraction (7). Both pausing and the associated hydrolytic RNA cleavage are affected by mutations in the C160, C128, and C11 subunits of yeast Pol III (7-9). In the case of C128 and C11, the same mutations have also been shown to affect the termination properties of Pol III, thus suggesting a functional interplay between hydrolytic RNA cleavage and transcription termination (9, 10). The t...

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Section: Distinguishing Between Weak and Strong T 5 Terminator Sequenmentioning

confidence: 99%

Sequence Context Effects on Oligo(dT) Termination Signal Recognition by Saccharomyces cerevisiae RNA Polymerase III

Braglia

Percudani

Dieci

2005

Journal of Biological Chemistry

102

112

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“…Factor r purification and complex formation Factor r was purified according to Camier et al (1985) with the modifications recently described (Gabrielsen et al, 1989), which include a phosphocellulose adsorption step and heparin and DEAE-Sephadex chromatography, followed by affinity chromatography on a tDNA-agarose column. The purified factor preparation contained several polypeptide chains, two of which (145 and 100 kd) were shown to be involved in DNA binding (Gabrielsen et al, 1989).…”

Section: Plasmids and Dna Fragmentsmentioning

confidence: 99%

“…Yeast factor r and its interaction with tDNA have been extensively studied (Klementz et al, 1982;Ruet et al, 1984). Nuclease protection and methylation experiments (Camier et al, 1985;Stillman et al, 1985a), as well as point mutation analyses (Baker and Hall, 1984;Baker et al, 1986), have demonstrated the specific binding of r to the A and B blocks, the involvement of critical bases and the prominent role of the B block sequence in complex formation. Analysis of a series of tRNA3L&u gene recombinants having variable A to B distances have demonstrated the remarkable flexibility of i--DNA interaction (Baker et al, 1987;Fabrizio et al, 1987).…”

Section: Introductionmentioning

confidence: 99%

The two DNA-binding domains of yeast transcription factor tau as observed by scanning transmission electron microscopy.

et al. 1989

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Yeast transcription factor X interacts with the intragenic promoter of tRNA genes, binding to both the A and B block elements. Affinity-purified r factor and r-tDNA complexes were examined by scanning transmission electron microscopy to analyze the structural features of free and DNA bound factor. The free factor appeared as two tightly associated globular domains of roughly similar size (10 nm in diameter) and mass ( -300 kd).A combination of these two domains results in a mass for the factor of 510-670 kd. When r was allowed to interact with recombinant tRNA3Leu genes with variable A block-B block spacing, different structures were observed. With short genes, the two globular domains were not resolved and T appeared as a large particle covering the A and B block region. On the other hand, with genes having a larger A-B distance (53 or 74 bp), mostly dumb-bell-shaped complexes were formed with individualized factor domains bound separately to the A and B blocks. A smaller proportion of the complexes appeared to consist of a large particle bound at only one site, essentially on the B block. Mapping of the binding domains in the DNA showed a good correlation with the respective positions of the A and B promoter elements. Factor binding did not induce a noticeable DNA bending, although with extended genes apparent DNA shortening and cases of DNA looping were observed. Upon cleavage of the tRNA3 uu gene between the A and B blocks after or prior to complex formation, the two factor domains remained attached to the same DNA fragment (mostly the B-DNA fragment). In addition, images of proteinlinked, reconstituted full-length genes were also observed. These different conformational states of the r-tDNA complexes probably reflect the dynamic aspect of the interaction of the factor with its DNA target.

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“…TFIIIC contains an activity that binds to both the A and B regions in vitro (7,8,44) and remains bound through multiple rounds of gene transcription (3,17,34). Experiments with altered tRNA gene templates have shown that binding is primarily directed by the B block, although A block mutations lower the overall binding efficiency and drastically reduce promoter strength (2,7,31,44). The function of TFIIIB is unclear, although it has been shown to be necessary for the formation of stable preinitiation complexes in some cases (3).…”

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confidence: 99%

Comparison of tRNA gene transcription complexes formed in vitro and in nuclei.

1987

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The nucleoprotein structure of single-copy tRNA genes in yeast nuclei was examined by DNase I footprinting and compared with that of complexes formed in vitro between the same genes and transcription factor C.Transcription factor C bound to both the 5' and 3' intragenic promoters of the tRNASUP53 gene in vitro, protecting approximately 30 base pairs at the 3' promoter (B block) and 40 base pairs at the 5' promoter (A block) and causing enhanced DNase I cleavages between the protected regions. Binding to the two sites was independent of the relative orientation of the two sites on the helix and was eliminated by a single point mutation in the 3' promoter. The chromosomal tRNAsU'P53 and tRNAUCG genes showed a pattern of protection and enhanced cleavages similar to that observed in vitro, indicating that the stable complexes formed in vitro accurately reflect at least some aspects of the nucleoprotein structure of the genes in chromatin.The transcription of eucaryotic tRNA genes is controlled by two highly conserved intragenic sequence elements located near the 5' and 3' ends of the genes, termed the A and B blocks, and by more variable sequences near the site of transcription initiation (9,16,18,38,40; reviewed in reference 39). The internal elements are also essential to tRNA processing and function, even in procaryotes, and may therefore have evolved into ubiquitous eucaryotic promoters as tRNA genes became independent transcription units. The upstream sequences have been shown to influence transcription in a species-specific manner (10, 12, 40) and have been postulated to impose differential regulation on classes of tRNA genes within an organism (14,23,29,47). The precise mechanism by which these promoter elements direct RNA polymerase III initiation is not clear, but an early step in gene activation appears to be the association of one or more transcription factors with the A and B blocks.Fractionation of cellular extracts has identified at least two components, transcription factor B (TFIIIB) and TFIIIC, that are required in addition to RNA polymerase III for tRNA transcription in vitro (17,28,37). TFIIIC contains an activity that binds to both the A and B regions in vitro (7,8,44) and remains bound through multiple rounds of gene transcription (3,17,34). Experiments with altered tRNA gene templates have shown that binding is primarily directed by the B block, although A block mutations lower the overall binding efficiency and drastically reduce promoter strength (2,7,31,44). The function of TFIIIB is unclear, although it has been shown to be necessary for the formation of stable preinitiation complexes in some cases (3).The spacing between the A and B block promoter regions varies from 30 to 80 base pairs in naturally occurring tRNA genes (9, 39, 41). The binding of a single TFIIIC complex to two sites separated by such a variable length of DNA would require considerable flexibility in the structure of the factor. It has been proposed instead that the DNA is contorted to allow the promoter elements to attain...

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A split binding site for transcription factor tau on the tRNA3Glu gene.

Cited by 112 publications

References 40 publications

Sequence Context Effects on Oligo(dT) Termination Signal Recognition by Saccharomyces cerevisiae RNA Polymerase III

Sequence Context Effects on Oligo(dT) Termination Signal Recognition by Saccharomyces cerevisiae RNA Polymerase III

The two DNA-binding domains of yeast transcription factor tau as observed by scanning transmission electron microscopy.

Comparison of tRNA gene transcription complexes formed in vitro and in nuclei.

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