Formation of an active transcription complex in the Drosophila melanogaster 5S RNA gene is dependent on an upstream region.

García, Alonzo D.; O'Connell, Anita; Sharp, Stephen J.

doi:10.1128/mcb.7.6.2046

Cited by 37 publications

(23 citation statements)

References 24 publications

(21 reference statements)

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“…The disruption of base pairing in the 5' stem-loop caused by substitutions in this region [e.g., clones 87U6(1-10 sub) and 87U6 (11)(12)(13)(14)(15)(16)(17)(18)(19)(20) This result is consistent with the hypothesis that the normal A block binding transcription factor TFIIIC can bind to the 87U6 A block region in the context of the 5S gene. However, this conclusion will require further experimental verification.…”

Section: Discussionsupporting

confidence: 80%

“…The level of this transcription is approximately the same as that of the consensus U6 clone that possesses all known 5' promoter elements (pGEM/U6). However, many studies have shown an effect of flanking sequences on the transcription of genes with internal promoters (1,12,26,31 [for review, see reference 13]), and we decided to systematically investigate whether or not this is the case with 87U6. Figure 1 provides a summary of these results.…”

Section: Resultsmentioning

confidence: 99%

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Transcription of a variant human U6 small nuclear RNA gene is controlled by a novel, internal RNA polymerase III promoter.

Tichelaar¹,

Knerer²,

Vrabel³

et al. 1994

Mol. Cell. Biol.

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Promoter elements in the 5' flanking regions of vertebrate U6 RNA genes have been shown to be both necessary and sufficient for transcription by RNA polymerase III. We have recently isolated and characterized a variant human U6 gene (87U6) that can be transcribed by RNA polymerase III in vitro in the absence of any natural 5' or 3' flanking sequences. This gene contains 10 nucleotide differences from the previously characterized human U6 gene (wtU6) within the coding region but has no homology to wtU6 in the upstream promoter region. By constructing chimeras between these two genes, we have shown that mutation of as few as two nucleotides in the coding region of the human U6 RNA gene is sufficient to create an internal promoter that is functional in vitro. A T-to-C transition at position 57 and a single T deletion at position 52 produce an internal U6 promoter that is nearly as active in vitro as the external U6 polymerase III promoter utilized by wtU6. Neither of these residues is absolutely conserved during evolution, and both of these nucleotide changes occur within the previously noted A box homology. Deletion and linker scanning mutations within the coding region of this variant U6 gene suggest that, in addition to the central region including bp 52 and 57, sequences at the extreme 5' end of the gene are critical for efficient transcription. In contrast, flanking sequences have a minor effect on transcriptional efficiency. This arrangement is unique among internal RNA polymerase III promoters and may indicate unique regulation of this gene.The transcription of the small nuclear RNA U6 has been well characterized in both vertebrate (2,8,17,21,22,28) and yeast (6, 7, 9) species. In the three vertebrate species in which a U6 gene has been cloned (humans, mice, and Xenopus tropicalis), transcription has been shown to be entirely dependent on 5' flanking sequences, including a classical TATA box and the U small nuclear RNA specific proximal sequence element (for review, see references 19 and 38). Both of these elements are normally found in RNA polymerase II promoters but have been shown to function in the RNA polymerase III promoter of U6. Indeed, the TATA box has been shown to be the element responsible for conferring RNA polymerase III specificity to the U6 promoter (23,24). In yeast species, the promoter structure is more complex, consisting of both external and internal elements. The U6 gene in Saccharomyces cerevisiae requires a 5' flanking TATA box, but transcription in vivo requires the presence of an internal A block and a B block located 120 nucleotides downstream (4, 6, 7, 9). The fission yeast Schizosaccharomyces pombe also contains a potentially functional B block homology within the intron of its U6 gene (11,32). However, in all cases studied thus far, the transcription of U6 relies wholly or in part on sequences upstream of the coding region.The human genome contains approximately 200 copies of U6-related sequences (14), only five of which have been sequenced and only three of which have been stud...

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Section: Discussionsupporting

confidence: 80%

Section: Resultsmentioning

confidence: 99%

Transcription of a variant human U6 small nuclear RNA gene is controlled by a novel, internal RNA polymerase III promoter.

Tichelaar¹,

Knerer²,

Vrabel³

et al. 1994

Mol. Cell. Biol.

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“…Kassavetis et al {1989} andBraun et al {1989} have shown that the analogous region of tRNA and 5S rRNA genes is bound by TFIIIB in a TFIIIC-dependent fashion. Although many tRNA and 5S rRNA genes do not exhibit a strong upstream sequence dependence, suggesting that TFIIIB can bind in a sequence-independent manner, TATA-like sequences are often present -3 0 bp upstream of tRNA and 5S rRNA genes, and, in some cases, are absolutely required for promoter function (Larson et al 1983;Selker et al 1986;Garcia et al 1987}. Furthermore, movement of the TATA-like sequence with respect to the coding region of a Neurospora 5S rRNA gene can result in a corresponding shift in the transcriptional start site {Tyler…”

Section: Implications For U6 Genes In Other Organismsmentioning

confidence: 99%

Transcription of a yeast U6 snRNA gene requires a polymerase III promoter element in a novel position.

Brow¹,

Guthrie²

1990

Genes Dev.

149

162

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Vertebrate genes coding for U6 small nuclear RNA are transcribed by RNA polymerase III (pol III), using only upstream promoter elements rather than the A and B block internal control regions typical of most pol III transcription units. We show that expression of the U6 gene from the yeast Saccharomyces cerevisiae has two unexpected features: it requires a B block promoter element, and this element is located in a novel position, 120 bp downstream of the coding region. In tRNA genes, the B block is the primary binding site for transcription factor (TF) IIIC, whose function is to promote the subsequent binding of TFIIIB. Both factors are thus implicated in yeast U6 gene transcription. We present a model of the U6 transcription complex based on the structure of yeast and vertebrate U6 promoters.

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“…The 5'-and 3'-deletion mutations were used to construct linker substitution (LS) mutations as described by Garcia et al (19). 5'-and 3'-deletion fragments were ligated via the EcoRI site originally located in the polylinker of M13mp9.…”

Section: Methodsmentioning

confidence: 99%

A discrete region centered 22 base pairs upstream of the initiation site modulates transcription of Drosophila tRNAAsn genes.

1988

Self Cite

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We have studied the mechanism by which 5'-flanking sequences modulate the in vitro transcription of eucaryotic tRNA genes. Using deletion and linker substitution mutagenesis, we have found that the 5'-flanking sequences responsible for the different in vitro transcription levels of three Drosophila tRNA5.' genes are contained within a discrete region centered 22 nucleotides upstream from the transcription initiation site. In conjunction with the A-box intragenic control region, this upstream transcription-modulatory region functions in the selection mechanism for the site of transcription initiation. Since the transcription-modulatory region directs the position of the start site and the actual sequence of the transcription-modulatory region determines the level of tRNAAsn gene transcription, the possibility is raised that the transcription-modulatory region directs a transcription initiation event similar to open complex formation at procaryotic promoters.In procaryotic organisms, initiation of RNA synthesis is controlled by small, conserved DNA sequences located 10 and 35 nucleotides upstream of the transcription initiation site. RNA polymerase holoenzyme recognizes and binds these regions in this first step of the pathway that leads to open-complex formation and transcriptional activation (5, 42). The three nuclear RNA polymerases of eucaryotes do not recognize promoters efficiently and, as a result, are unable to independently initiate gene-specific transcription (for a review, see reference 38). Instead, eucaryotic transcription is dependent upon promoter-specific transcription factors that recognize and bind discrete sequence elements. Current models suggest that the binding of these protein factors activates transcription by allowing each gene to be recognized by the appropriate polymerase. For RNA polymerases I and II, the known sites of transcription factor interaction are 5' of the site of transcription initiation (9,15,45). For RNA polymerase III-directed transcription of eucaryotic 5S RNA, tRNA and adenovirus VA RNA genes, the sites of transcription factor interaction are 3' of the transcription initiation site, within the sequences encoding the RNA (6,16,17,48,50). RNA polymerase III-directed transcription of 7SL RNA, 7SK RNA, and U6 RNA genes requires enhancerlike sequence elements for transcription factor interaction 5' of the transcription initiation site, similar to RNA polymerase II promoters (3,7,30,47).The intragenic control regions (ICRs) in tRNA genes, the A-and B-box ICRs, contain the DNA sequence determinants that direct transcription factor activities (reviewed in reference 40). For tRNA genes, the transcription factors bind to the gene to form a complex that is stable throughout multiple rounds of transcription initiation (4,23,36). This stable complex, comprised of tRNA gene and transcription factors, is considered to form the recognition substrate for RNA polymerase III.Transcriptional activity of tRNA genes is particularly sensitive to mutations in the A-and B-box ICRs. All of the po...

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Formation of an active transcription complex in the Drosophila melanogaster 5S RNA gene is dependent on an upstream region.

Cited by 37 publications

References 24 publications

Transcription of a variant human U6 small nuclear RNA gene is controlled by a novel, internal RNA polymerase III promoter.

Transcription of a variant human U6 small nuclear RNA gene is controlled by a novel, internal RNA polymerase III promoter.

Transcription of a yeast U6 snRNA gene requires a polymerase III promoter element in a novel position.

A discrete region centered 22 base pairs upstream of the initiation site modulates transcription of Drosophila tRNAAsn genes.

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