The mouse thymidylate synthase promoter: essential elements are in close proximity to the transcriptional initiation sites.

Deng, Tiliang; Li, Yue; Jolliff, Keith; Johnson, Lee F.

doi:10.1128/mcb.9.9.4079

Cited by 25 publications

(38 citation statements)

References 27 publications

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“…Table 2 shows that deletions within the region that encompassed the CIII or GC-D box reduced mRNA content 4-to 10-fold, in line with the results obtained with promoters that contained point mutations in this region. In contrast, deletions upstream of -105 (9) or between -72 and -6 had little effect on mRNA content. In most cases, the content of TS mRNA correlated well with TS enzyme level, although some discrepancies were noted.…”

Section: Aagagcgccaggaaggtccimentioning

confidence: 73%

“…TS-deficient (Ts-) V79 Chinese hamster cells (31) were maintained on plastic petri dishes in Dulbecco's modified Eagle's medium (GIBCO) supplemented with 10% NuSerum (Collaborative Research) and 10 p,M thymidine. Transient transfections were performed by the calcium phosphate method (9). Cells were harvested for RNA or enzyme analyses 40 to 60 h after transfection.…”

Section: Methodsmentioning

confidence: 99%

“…Cytoplasmic RNA and poly(A)+ mRNA were isolated by standard procedures (9,22). Almost all analyses were performed with total cytoplasmic RNA.…”

Section: Methodsmentioning

confidence: 99%

“…The other element, GC-D (AGGCGG) at -82, is a weak Spl binding site (17). A second Spl binding site (GC-U) at -132 as well as a USF binding site (38) at -139 can be deleted without significantly affecting promoter activity (9). The goals of the present study were to determine whether there are additional promoter elements downstream of the first transcriptional initiation site and to study the mechanism responsible for the complex pattern of transcriptional initiation sites.…”

mentioning

confidence: 99%

“…lacks a TATA box, and initiates transcription at many sites between -1 and -92 nucleotides relative to the AUG codon. Promoter deletion analyses revealed that sequences that are sufficient for wild-type promoter activity and for generating the normal spectrum of transcriptional start sites are located downstream of -105 (9). Deletion to -85 leads to the loss of expression.…”

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confidence: 99%

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Lack of an initiator element is responsible for multiple transcriptional initiation sites of the TATA-less mouse thymidylate synthase promoter.

Geng¹,

Johnson²

1993

Mol. Cell. Biol.

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The mouse thymidylate synthase promoter lacks a TATA box and initiates transcription at many sites across a 90-nucleotide initiation window. We showed previously that wild-type promoter activity is maintained with a promoter that extends only 13 nucleotides upstream of the first start site. G/A-rich and G/C-rich promoter elements were identified in the vicinity of the first transcriptional start site. The goals of the present study were to determine whether there are additional promoter elements in the initiation window and to determine why transcription initiates across such a broad region. Minigenes containing a variety of substitution, deletion, and insertion mutations in the promoter region were transfected into cultured cells, and the effects on expression and the pattern of start sites were determined. The results indicate that there are no additional promoter elements downstream of the G/C box. The boundaries of the transcription window are established by elements near the 5' end of the window, whereas the pattern of start sites is determined by sequences within the window. The promoter lacks an initiator element. When an initiator element was inserted, transcription initiated predominantly at the position directed by the initiator when it was inserted within the initiation window but not when it was inserted immediately upstream of the window.The promoters of protein-coding genes in higher eukaryotes generally consist of a TATA motif in combination with one or more binding sites for transcriptional regulatory proteins. Transcriptional initiation by RNA polymerase II occurs at a single site 25 to 30 nucleotides downstream of the TATAA sequence. The TATA motif is the binding site for the general transcription factor TFIID (29,34). This factor promotes the assembly of the transcription initiation complex, which includes many additional factors as well as RNA polymerase II (3, 39). The TATA box also plays an important role in the selection of the site of transcriptional initiation, since alterations in the distance between the TATA box and the normal start site can lead to the creation of a new initiation site approximately 30 nucleotides downstream of the TATA box (24).Although most class II promoters contain a TATA box, there are many examples of promoters which initiate transcription at a unique site but which lack an A/T-rich sequence 30 nucleotides upstream of the initiation site (13,16,41). A special DNA sequence at the transcriptional start site, which has been designated the initiator element, is responsible for specifying the transcriptional start site in such promoters (2,4,26,41,47). Initiator elements have also been identified in some TATA-containing promoters (6, 30). Many initiator elements serve as binding sites for trans-acting factors and are able to function autonomously to initiate transcription (4,26,32,33,36,40,42).Finally, there are several examples of TATA-less promoters that have multiple, widely dispersed sites of transcriptional initiation (14,15,27,35). Little is known about the me...

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Section: Aagagcgccaggaaggtccimentioning

confidence: 73%

Section: Methodsmentioning

confidence: 99%

“…Cytoplasmic RNA and poly(A)+ mRNA were isolated by standard procedures (9,22). Almost all analyses were performed with total cytoplasmic RNA.…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Lack of an initiator element is responsible for multiple transcriptional initiation sites of the TATA-less mouse thymidylate synthase promoter.

Geng¹,

Johnson²

1993

Mol. Cell. Biol.

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Inactivation of MED-1 elements in the TATA-less, initiator-less mouse thymidylate synthase promoter has no effect on promoter strength or the complex pattern of transcriptional start sites

Rudge

Johnson

1999

J. Cell. Biochem.

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The mouse thymidylate synthase (TS) promoter is a member of a family of promoters that lack a TATA box as well as an initiator element and that initiate transcription at many sites over a broad initiation window. An element (MED-1) downstream of the initiation window of almost all promoters of this family has been proposed to be important for promoter activity, as well as for multiple start site utilization. Two consensus MED-1 elements are located downstream of the initiation window of the TS promoter. To determine the role of the MED-1 elements in the TS promoter, one or both elements were inactivated by site-directed mutagenesis and the effects on promoter function were determined. We found that inactivation of the MED-1 elements had no measurable effect on promoter strength, the boundaries of the initiation window, or the pattern of transcriptional start sites. Furthermore, inactivation of the elements did not affect the ability of the TS promoter to direct S phase-specific expression of the gene in growth-stimulated cells. We conclude that the MED-1 element does not play a significant role in TS promoter function and therefore is not an essential component of all TATA-less promoters with complex transcriptional initiation patterns.

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Transcriptional control elements and complex initiation pattern of the TATA-less bidirectional human thymidylate synthase promoter

2000

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The nucleotide sequences that are important for transcription of the human thymidylate synthase gene were analyzed by deletion and site-directed mutagenesis of the promoter region. Deletion analyses from the 5' and 3' ends indicated the presence of multiple positive and negative elements. The promoter had approximately the same strength in the normal or inverted orientation. The region between 161 and 141 nt upstream of the translational start codon was found to be both necessary and sufficient for high-level promoter activity in both directions and was designated the essential promoter region. This region, which is highly conserved in human, mouse and rat TS promoters, contains potential binding sites for Ets, Sp1, and LSF transcription factors. Site directed mutagenesis of each of these elements led to large decreases in promoter strength. However, inactivation of potential Sp1 and E2F elements adjacent to the essential promoter region led to increases in promoter strength. The transcriptional start site pattern was analyzed by S1 nuclease protection assays of mRNA isolated from cells transiently transfected with TS minigenes. Multiple start sites were detected, most of which were between 160 and 120 nt upstream of the AUG codon.

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The mouse thymidylate synthase promoter: essential elements are in close proximity to the transcriptional initiation sites.

Cited by 25 publications

References 27 publications

Lack of an initiator element is responsible for multiple transcriptional initiation sites of the TATA-less mouse thymidylate synthase promoter.

Lack of an initiator element is responsible for multiple transcriptional initiation sites of the TATA-less mouse thymidylate synthase promoter.

Inactivation of MED-1 elements in the TATA-less, initiator-less mouse thymidylate synthase promoter has no effect on promoter strength or the complex pattern of transcriptional start sites

Transcriptional control elements and complex initiation pattern of the TATA-less bidirectional human thymidylate synthase promoter

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