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1
Means, A. L., and P. J. Farnham. "Transcription initiation from the dihydrofolate reductase promoter is positioned by HIP1 binding at the initiation site." Molecular and Cellular Biology 10, no. 2 (February 1990): 653–61. http://dx.doi.org/10.1128/mcb.10.2.653.
Full textAbstract:
We have identified a sequence element that specifies the position of transcription initiation for the dihydrofolate reductase gene. Unlike the functionally analogous TATA box that directs RNA polymerase II to initiate transcription 30 nucleotides downstream, the positioning element of the dihydrofolate reductase promoter is located directly at the site of transcription initiation. By using DNase I footprint analysis, we have shown that a protein binds to this initiator element. Transcription initiated at the dihydrofolate reductase initiator element when 28 nucleotides were inserted between it and all other upstream sequences, or when it was placed on either side of the DNA helix, suggesting that there is no strict spatial requirement between the initiator and an upstream element. Although neither a single Sp1-binding site nor a single initiator element was sufficient for transcriptional activity, the combination of one Sp1-binding site and the dihydrofolate reductase initiator element cloned into a plasmid vector resulted in transcription starting at the initiator element. We have also shown that the simian virus 40 late major initiation site has striking sequence homology to the dihydrofolate reductase initiation site and that the same, or a similar, protein binds to both sites. Examination of the sequences at other RNA polymerase II initiation sites suggests that we have identified an element that is important in the transcription of other housekeeping genes. We have thus named the protein that binds to the initiator element HIP1 (Housekeeping Initiator Protein 1).
2
Means, A. L., and P. J. Farnham. "Transcription initiation from the dihydrofolate reductase promoter is positioned by HIP1 binding at the initiation site." Molecular and Cellular Biology 10, no. 2 (February 1990): 653–61. http://dx.doi.org/10.1128/mcb.10.2.653-661.1990.
Full textAbstract:
We have identified a sequence element that specifies the position of transcription initiation for the dihydrofolate reductase gene. Unlike the functionally analogous TATA box that directs RNA polymerase II to initiate transcription 30 nucleotides downstream, the positioning element of the dihydrofolate reductase promoter is located directly at the site of transcription initiation. By using DNase I footprint analysis, we have shown that a protein binds to this initiator element. Transcription initiated at the dihydrofolate reductase initiator element when 28 nucleotides were inserted between it and all other upstream sequences, or when it was placed on either side of the DNA helix, suggesting that there is no strict spatial requirement between the initiator and an upstream element. Although neither a single Sp1-binding site nor a single initiator element was sufficient for transcriptional activity, the combination of one Sp1-binding site and the dihydrofolate reductase initiator element cloned into a plasmid vector resulted in transcription starting at the initiator element. We have also shown that the simian virus 40 late major initiation site has striking sequence homology to the dihydrofolate reductase initiation site and that the same, or a similar, protein binds to both sites. Examination of the sequences at other RNA polymerase II initiation sites suggests that we have identified an element that is important in the transcription of other housekeeping genes. We have thus named the protein that binds to the initiator element HIP1 (Housekeeping Initiator Protein 1).
3
Blake, M. C., R. C. Jambou, A. G. Swick, J. W. Kahn, and J. C. Azizkhan. "Transcriptional initiation is controlled by upstream GC-box interactions in a TATAA-less promoter." Molecular and Cellular Biology 10, no. 12 (December 1990): 6632–41. http://dx.doi.org/10.1128/mcb.10.12.6632.
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Numerous genes contain TATAA-less promoters, and the control of transcriptional initiation in this important promoter class is not understood. We have determined that protein-DNA interactions at three of the four proximal GC box sequence elements in one such promoter, that of the hamster dihydrofolate reductase gene, control initiation and relative use of the major and minor start sites. Our results indicate that although the GC boxes are apparently equivalent with respect to factor binding, they are not equivalent with respect to function. At least two properly positioned GC boxes were required for initiation of transcription. Abolishment of DNA-protein interaction by site-specific mutation of the most proximal GC box (box I) resulted in a fivefold decrease in transcription from the major initiation site and a threefold increase in heterogeneous transcripts initiating from the vicinity of the minor start site in vitro and in vivo. Mutations that separately abolished interactions at GC boxes II and III while leaving GC box I intact affected the relative utilization of both the major and minor initiation sites as well as transcriptional efficiency of the promoter template in in vitro transcription and transient expression assays. Interaction at GC box IV when the three proximal boxes were in a wild-type configuration had no effect on transcription of the dihydrofolate reductase gene promoter. Thus, GC box interactions not only are required for efficient transcription but also regulate start site utilization in this TATAA-less promoter.
4
Blake, M. C., R. C. Jambou, A. G. Swick, J. W. Kahn, and J. C. Azizkhan. "Transcriptional initiation is controlled by upstream GC-box interactions in a TATAA-less promoter." Molecular and Cellular Biology 10, no. 12 (December 1990): 6632–41. http://dx.doi.org/10.1128/mcb.10.12.6632-6641.1990.
Full textAbstract:
Numerous genes contain TATAA-less promoters, and the control of transcriptional initiation in this important promoter class is not understood. We have determined that protein-DNA interactions at three of the four proximal GC box sequence elements in one such promoter, that of the hamster dihydrofolate reductase gene, control initiation and relative use of the major and minor start sites. Our results indicate that although the GC boxes are apparently equivalent with respect to factor binding, they are not equivalent with respect to function. At least two properly positioned GC boxes were required for initiation of transcription. Abolishment of DNA-protein interaction by site-specific mutation of the most proximal GC box (box I) resulted in a fivefold decrease in transcription from the major initiation site and a threefold increase in heterogeneous transcripts initiating from the vicinity of the minor start site in vitro and in vivo. Mutations that separately abolished interactions at GC boxes II and III while leaving GC box I intact affected the relative utilization of both the major and minor initiation sites as well as transcriptional efficiency of the promoter template in in vitro transcription and transient expression assays. Interaction at GC box IV when the three proximal boxes were in a wild-type configuration had no effect on transcription of the dihydrofolate reductase gene promoter. Thus, GC box interactions not only are required for efficient transcription but also regulate start site utilization in this TATAA-less promoter.
5
Uprety, Bhawana, Amala Kaja, Jannatul Ferdoush, Rwik Sen, and Sukesh R. Bhaumik. "Regulation of Antisense Transcription by NuA4 Histone Acetyltransferase and Other Chromatin Regulatory Factors." Molecular and Cellular Biology 36, no. 6 (January 11, 2016): 992–1006. http://dx.doi.org/10.1128/mcb.00808-15.
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NuA4 histone lysine (K) acetyltransferase (KAT) promotes transcriptional initiation of TATA-binding protein (TBP)-associated factor (TAF)-dependent ribosomal protein genes. TAFs have also been recently found to enhance antisense transcription from the 3′ end of theGAL10coding sequence. However, it remains unknown whether, like sense transcription of the ribosomal protein genes, TAF-dependent antisense transcription ofGAL10also requires NuA4 KAT. Here, we show that NuA4 KAT associates with theGAL10antisense transcription initiation site at the 3′ end of the coding sequence. Such association of NuA4 KAT depends on the Reb1p-binding site that recruits Reb1p activator to theGAL10antisense transcription initiation site. Targeted recruitment of NuA4 KAT to theGAL10antisense transcription initiation site promotesGAL10antisense transcription. Like NuA4 KAT, histone H3 K4/36 methyltransferases and histone H2B ubiquitin conjugase facilitateGAL10antisense transcription, while the Swi/Snf and SAGA chromatin remodeling/modification factors are dispensable for antisense, but not sense, transcription ofGAL10. Taken together, our results demonstrate for the first time the roles of NuA4 KAT and other chromatin regulatory factors in controlling antisense transcription, thus illuminating chromatin regulation of antisense transcription.
6
Yu, K., and R. T. Elder. "A region internal to the coding sequences is essential for transcription of the yeast Ty-D15 element." Molecular and Cellular Biology 9, no. 9 (September 1989): 3667–78. http://dx.doi.org/10.1128/mcb.9.9.3667.
Full textAbstract:
The major transcript of the yeast transposable element Ty1 has its 5' end in one delta and the 3' end in the opposite delta, the direct repeats of about 335 base pairs (bp) at each end of the element. The transcriptional initiation signals of the Ty-D15 element that give rise to this transcript were found to have a number of unusual characteristics. The 5' delta by itself, which contained the initiation site for Ty transcription, gave no detectable transcription. A region internal to the transcript in a translated part of the element and about 140 bp downstream of the 5' delta was essential for initiation of the major Ty transcript. This internal activating region (IAR) had several interesting properties. When the portion of the delta upstream of the initiation site was replaced with DNA fragments that did not by themselves act as promoters, initiation directed by the IAR still occurred at about the same position, 200 to 400 bp upstream of the IAR. If fragments containing the IAR were inverted, transcription could still occur. When 468 or 636 bp was inserted between the delta and the IAR, initiations occurred near the normal delta initiation site and in the inserted DNA. Therefore, the location and properties of transcription signals for Ty-D15 differ considerably from those expected for a yeast gene transcribed by RNA polymerase II.
7
Yu, K., and R. T. Elder. "A region internal to the coding sequences is essential for transcription of the yeast Ty-D15 element." Molecular and Cellular Biology 9, no. 9 (September 1989): 3667–78. http://dx.doi.org/10.1128/mcb.9.9.3667-3678.1989.
Full textAbstract:
The major transcript of the yeast transposable element Ty1 has its 5' end in one delta and the 3' end in the opposite delta, the direct repeats of about 335 base pairs (bp) at each end of the element. The transcriptional initiation signals of the Ty-D15 element that give rise to this transcript were found to have a number of unusual characteristics. The 5' delta by itself, which contained the initiation site for Ty transcription, gave no detectable transcription. A region internal to the transcript in a translated part of the element and about 140 bp downstream of the 5' delta was essential for initiation of the major Ty transcript. This internal activating region (IAR) had several interesting properties. When the portion of the delta upstream of the initiation site was replaced with DNA fragments that did not by themselves act as promoters, initiation directed by the IAR still occurred at about the same position, 200 to 400 bp upstream of the IAR. If fragments containing the IAR were inverted, transcription could still occur. When 468 or 636 bp was inserted between the delta and the IAR, initiations occurred near the normal delta initiation site and in the inserted DNA. Therefore, the location and properties of transcription signals for Ty-D15 differ considerably from those expected for a yeast gene transcribed by RNA polymerase II.
8
Cano, A. "Characterization of the rat NHE3 promoter." American Journal of Physiology-Renal Physiology 271, no. 3 (September 1, 1996): F629—F636. http://dx.doi.org/10.1152/ajprenal.1996.271.3.f629.
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NHE3, a transmembrane protein involved in transcellular ion transport, is expressed in the apical membrane of renal and gastrointestinal epithelia. Chronic regulation by multiple stimuli, including glucocorticoid-induced transcriptional regulation, has been demonstrated. To study the tissue-specific expression and transcriptional regulation of NHE3, the 5' flanking region of the rat NHE3 gene was cloned. Two genomic libraries were screened with the 5' end of the NHE3 cDNA. The 5' flanking region and first exon were isolated. Primer extension mapped a single transcription start site in stomach, colon and kidney. The NHE3 promoter near the transcription initiation site is characterized by the absence of TATA and CAAT sequences. Two Sp1 sites, one Egr-1 site, and an initiator with the sequence GGGATTAAA mark the area of transcription initiation. Upstream sequences include multiple DNA sequence elements recognized by the glucocorticoid and thyroid receptors, Sp1, atriopeptin-2, and several other transcription factors. Transcriptional regulation by glucocorticoids and chronic acidosis was demonstrated. Promoter activity was present in OKP cells, a renal proximal tubule cell line, but not in fibroblasts. This suggests that the NHE3 promoter contains elements conferring epithelial cell-specific expression.
9
Pal, S. K., S. S. Zinkel, A. A. Kiessling, and G. M. Cooper. "c-mos expression in mouse oocytes is controlled by initiator-related sequences immediately downstream of the transcription initiation site." Molecular and Cellular Biology 11, no. 10 (October 1991): 5190–96. http://dx.doi.org/10.1128/mcb.11.10.5190.
Full textAbstract:
We have employed transient expression assays to analyze the sequences that direct c-mos transcription in mouse oocytes. Plasmids containing the chloramphenicol acetyltransferase (CAT) gene fused to either a 2.4-kb or a 731-bp fragment from the 5'-flanking region of c-mos produced similar levels of CAT activity when injected into nuclei of growing oocytes. BAL 31 deletions revealed that sequences up to 20 bp upstream of the major transcription start site could be removed without any significant loss of CAT activity. Promoter activity only decreased when these deletions closely approached the transcription start site, which was mapped at 53 nucleotides upstream of the first ATG in the c-mos open reading frame. On the other hand, deletion of sequences within 20 nucleotides downstream of the transcription initiation site resulted in a 10-fold reduction in CAT expression. A similar decrease in promoter activity was observed as a result of point mutations in these 5' untranslated sequences. Thus, sequences immediately downstream of the transcription start site, including a consensus sequence (PyPyCAPyPyPyPyPy) present in the initiator elements of several genes, appear to regulate c-mos expression in mouse oocytes. Reverse transcription-polymerase chain reaction analysis of RNA from injected oocytes showed that this regulation is manifest at the transcriptional level. Expression of c-mos in mouse oocytes thus appears to be directed by a simple promoter consisting only of sequences immediately surrounding the transcription start site, including an initiator element in the untranslated leader.
10
Pal, S. K., S. S. Zinkel, A. A. Kiessling, and G. M. Cooper. "c-mos expression in mouse oocytes is controlled by initiator-related sequences immediately downstream of the transcription initiation site." Molecular and Cellular Biology 11, no. 10 (October 1991): 5190–96. http://dx.doi.org/10.1128/mcb.11.10.5190-5196.1991.
Full textAbstract:
We have employed transient expression assays to analyze the sequences that direct c-mos transcription in mouse oocytes. Plasmids containing the chloramphenicol acetyltransferase (CAT) gene fused to either a 2.4-kb or a 731-bp fragment from the 5'-flanking region of c-mos produced similar levels of CAT activity when injected into nuclei of growing oocytes. BAL 31 deletions revealed that sequences up to 20 bp upstream of the major transcription start site could be removed without any significant loss of CAT activity. Promoter activity only decreased when these deletions closely approached the transcription start site, which was mapped at 53 nucleotides upstream of the first ATG in the c-mos open reading frame. On the other hand, deletion of sequences within 20 nucleotides downstream of the transcription initiation site resulted in a 10-fold reduction in CAT expression. A similar decrease in promoter activity was observed as a result of point mutations in these 5' untranslated sequences. Thus, sequences immediately downstream of the transcription start site, including a consensus sequence (PyPyCAPyPyPyPyPy) present in the initiator elements of several genes, appear to regulate c-mos expression in mouse oocytes. Reverse transcription-polymerase chain reaction analysis of RNA from injected oocytes showed that this regulation is manifest at the transcriptional level. Expression of c-mos in mouse oocytes thus appears to be directed by a simple promoter consisting only of sequences immediately surrounding the transcription start site, including an initiator element in the untranslated leader.
11
Loewy, Zvi G., Susan L. Leary, and Howard J. Baum. "Site-directed transcription initiation with a mobile promoter." Gene 83, no. 2 (November 1989): 367–70. http://dx.doi.org/10.1016/0378-1119(89)90123-6.
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12
Geng, Y., and L. F. Johnson. "Lack of an initiator element is responsible for multiple transcriptional initiation sites of the TATA-less mouse thymidylate synthase promoter." Molecular and Cellular Biology 13, no. 8 (August 1993): 4894–903. http://dx.doi.org/10.1128/mcb.13.8.4894.
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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.
13
Geng, Y., and L. F. Johnson. "Lack of an initiator element is responsible for multiple transcriptional initiation sites of the TATA-less mouse thymidylate synthase promoter." Molecular and Cellular Biology 13, no. 8 (August 1993): 4894–903. http://dx.doi.org/10.1128/mcb.13.8.4894-4903.1993.
Full textAbstract:
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.
14
Chang, D. D., and D. A. Clayton. "Precise assignment of the light-strand promoter of mouse mitochondrial DNA: a functional promoter consists of multiple upstream domains." Molecular and Cellular Biology 6, no. 9 (September 1986): 3253–61. http://dx.doi.org/10.1128/mcb.6.9.3253.
Full textAbstract:
Using deletion mutagenesis we localized the promoter for the light strand of mouse mitochondrial DNA to a 97-base-pair region, from -88 to +9 nucleotides of the transcriptional initiation site. Within this region the light-strand promoter could be dissected into at least three different functional domains. The specificity region, a maximum of 19 base pairs between -10 and +9 of the transcriptional initiation site, was essential and sufficient for accurate transcriptional initiation. A second region, extending to -29 nucleotides from the initiation site, facilitated the formation of a preinitiation complex between the template DNA and factor(s) present in the mitochondrial RNA polymerase fraction and was required for efficient transcription. A third, ill-defined upstream region, which extended up to -88 nucleotides from the initiation site, appeared to influence template transcriptional efficiencies in competition assays. Without the specificity domain, the upstream regions were incapable of supporting any transcription. The presence of multiple upstream domains was confirmed by disrupting nucleotide sequences in the upstream region by using linker insertion and linker replacement techniques.
15
Chang, D. D., and D. A. Clayton. "Precise assignment of the light-strand promoter of mouse mitochondrial DNA: a functional promoter consists of multiple upstream domains." Molecular and Cellular Biology 6, no. 9 (September 1986): 3253–61. http://dx.doi.org/10.1128/mcb.6.9.3253-3261.1986.
Full textAbstract:
Using deletion mutagenesis we localized the promoter for the light strand of mouse mitochondrial DNA to a 97-base-pair region, from -88 to +9 nucleotides of the transcriptional initiation site. Within this region the light-strand promoter could be dissected into at least three different functional domains. The specificity region, a maximum of 19 base pairs between -10 and +9 of the transcriptional initiation site, was essential and sufficient for accurate transcriptional initiation. A second region, extending to -29 nucleotides from the initiation site, facilitated the formation of a preinitiation complex between the template DNA and factor(s) present in the mitochondrial RNA polymerase fraction and was required for efficient transcription. A third, ill-defined upstream region, which extended up to -88 nucleotides from the initiation site, appeared to influence template transcriptional efficiencies in competition assays. Without the specificity domain, the upstream regions were incapable of supporting any transcription. The presence of multiple upstream domains was confirmed by disrupting nucleotide sequences in the upstream region by using linker insertion and linker replacement techniques.
16
Chang, D. D., J. E. Hixson, and D. A. Clayton. "Minor transcription initiation events indicate that both human mitochondrial promoters function bidirectionally." Molecular and Cellular Biology 6, no. 1 (January 1986): 294–301. http://dx.doi.org/10.1128/mcb.6.1.294.
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Human mitochondrial DNA is transcribed from two distinct, strand-specific promoters located in the displacement loop region of the genome. The transcriptional control sequences identified by deletion mapping and site-directed mutagenesis studies span short regions surrounding the initiation sites and bear no obvious sequence homology to any nuclear or procaryotic promoters. In vitro transcription analyses also revealed several minor initiation sites that are characterized by a pyrimidine-rich region followed by a purine-rich region, a feature that is shared by the two major promoters. In this paper, we report a new class of minor promoters in human mitochondrial DNA. These minor promoters were localized to the same duplex DNA sequences that direct major transcriptional events, but they had transcriptional polarity opposite to that of the major promoters. Furthermore, nucleotide changes that affected the major form of transcription similarly affected transcription in the opposite direction. For one of these minor promoters, a corresponding in vivo RNA species initiating from the same site was identified. These observations indicate that the major transcriptional promoters in human mitochondria can function bidirectionally both in vivo and in vitro.
17
Chang, D. D., J. E. Hixson, and D. A. Clayton. "Minor transcription initiation events indicate that both human mitochondrial promoters function bidirectionally." Molecular and Cellular Biology 6, no. 1 (January 1986): 294–301. http://dx.doi.org/10.1128/mcb.6.1.294-301.1986.
Full textAbstract:
Human mitochondrial DNA is transcribed from two distinct, strand-specific promoters located in the displacement loop region of the genome. The transcriptional control sequences identified by deletion mapping and site-directed mutagenesis studies span short regions surrounding the initiation sites and bear no obvious sequence homology to any nuclear or procaryotic promoters. In vitro transcription analyses also revealed several minor initiation sites that are characterized by a pyrimidine-rich region followed by a purine-rich region, a feature that is shared by the two major promoters. In this paper, we report a new class of minor promoters in human mitochondrial DNA. These minor promoters were localized to the same duplex DNA sequences that direct major transcriptional events, but they had transcriptional polarity opposite to that of the major promoters. Furthermore, nucleotide changes that affected the major form of transcription similarly affected transcription in the opposite direction. For one of these minor promoters, a corresponding in vivo RNA species initiating from the same site was identified. These observations indicate that the major transcriptional promoters in human mitochondria can function bidirectionally both in vivo and in vitro.
18
Kassavetis, G. A., and E. P. Geiduschek. "Transcription factor TFIIIB and transcription by RNA polymerase III." Biochemical Society Transactions 34, no. 6 (October 25, 2006): 1082–87. http://dx.doi.org/10.1042/bst0341082.
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pol (RNA polymerase) III is charged with the task of transcribing nuclear genes encoding diverse small structural and catalytic RNAs. We present a brief review of the current understanding of several aspects of the pol III transcription apparatus. The focus is on yeast and, more specifically, on Saccharomyces cerevisiae; preponderant attention is given to the TFs (transcription initiation factors) and especially to TFIIIB, which is the core pol III initiation factor by virtue of its role in recruiting pol III to the transcriptional start site and its essential roles in forming the transcription-ready open promoter complex. Certain relatively recent developments are also selected for brief comment: (i) the genome-wide analysis of occupancy of pol III-transcribed genes (and other loci) by the transcription apparatus and the location of pol III transcription in the cell; (ii) progress toward a mechanistic and molecular understanding of the regulation of transcription by pol III in yeast; and (iii) recent experiments identifying a high mobility group protein as a fidelity factor that assures selection of the precise transcriptional start site at certain pol III promoters.
19
Fujikawa, K., S. Imai, F. Sakane, and H. Kanoh. "Isolation and characterization of the human diacylglycerol kinase gene." Biochemical Journal 294, no. 2 (September 1, 1993): 443–49. http://dx.doi.org/10.1042/bj2940443.
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The 80 kDa diacylglycerol kinase (DGK) is abundantly expressed in oligodendrocytes and lymphocytes but not to a detectable extent in other cells such as neurons and hepatocytes. As an initial attempt to delineate the mechanism of the transcriptional control of the DGK gene, we have cloned from a human genomic library a 22 kb genomic fragment. The genomic clone consists of the 5′-flanking region and 17 exons coding for approx. 53% of the total exons of human DGK, including those encoding EF-hand and zinc-finger regions. The translation initiation site is located in the second exon. S1 nuclease mapping and primer extension analysis of the human DGK mRNA identified a major transcription initiation site (position +1) at 264 bp upstream from the initiator ATG. In the 5′-flanking sequence we detected a single GC box at -35 but no canonical TATA and CAAT sequences. However, the sequence starting from the cap site (AGTTCCTGCCA) is very similar to the initiator element that specifies the transcription initiation site of some housekeeping genes. In addition, the 5′-upstream region contains several putative cis-elements. Jurkat and HepG2 cells were transfected with various 5′-deletion mutants of the upstream region fused to the structural gene of chloramphenicol acetyltransferase (CAT). The CAT assay revealed that among constructs containing up to 3.4 kb of the 5′-flanking region, a fragment of 263 bp from the transcription initiation site contains a basic promoter that is active in both types of cells. Moreover, the region between -263 and -850 contains a negative element that is active in HepG2 but not in Jurkat cells. This negative element may, at least in part, be responsible for the cell type-specific expression of the DGK gene.
20
Kollmar, R., and P. J. Farnham. "Site-Specific Initiation of Transcription by RNA Polymerase II." Experimental Biology and Medicine 203, no. 2 (June 1, 1993): 127–39. http://dx.doi.org/10.3181/00379727-203-43583.
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Fujiwara, Haruhiko, and Hajime Ishikawa. "Structure of theBombyx morirDNA: initiation site for its transcription." Nucleic Acids Research 15, no. 3 (1987): 1245–58. http://dx.doi.org/10.1093/nar/15.3.1245.
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Agapov, Aleksei, Daria Esyunina, Danil Pupov, and Andrey Kulbachinskiy. "Regulation of transcription initiation by Gfh factors from Deinococcus radiodurans." Biochemical Journal 473, no. 23 (November 25, 2016): 4493–505. http://dx.doi.org/10.1042/bcj20160659.
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Transcription factors of the Gre family bind within the secondary channel of bacterial RNA polymerase (RNAP) directly modulating its catalytic activities. Universally conserved Gre factors activate RNA cleavage by RNAP, by chelating catalytic metal ions in the RNAP active site, and facilitate both promoter escape and transcription elongation. Gfh factors are Deinococcus/Thermus-specific homologues of Gre factors whose transcription functions remain poorly understood. Recently, we found that Gfh1 and Gfh2 proteins from Deinococcus radiodurans dramatically stimulate RNAP pausing during transcription elongation in the presence of Mn2+, but not Mg2+, ions. In contrast, we show that Gfh1 and Gfh2 moderately inhibit transcription initiation in the presence of either Mg2+ or Mn2+ ions. By using a molecular beacon assay, we demonstrate that Gfh1 and Gfh2 do not significantly change promoter complex stability or the rate of promoter escape by D. radiodurans RNAP. At the same time, Gfh factors significantly increase the apparent KM value for the 5′-initiating nucleotide, without having major effects on the affinity of metal ions for the RNAP active site. Similar inhibitory effects of Gfh factors are observed for transcription initiation on promoters recognized by the principal and an alternative σ factor. In summary, our data suggest that D. radiodurans Gfh factors impair the binding of initiating substrates independently of the metal ions bound in the RNAP active site, but have only mild overall effects on transcription initiation. Thus the mechanisms of modulation of RNAP activity by these factors are different for various steps of transcription.
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Xiao, Gaoping, Jianxin He, and Laurence G. Rahme. "Mutation analysis of the Pseudomonas aeruginosa mvfR and pqsABCDE gene promoters demonstrates complex quorum-sensing circuitry." Microbiology 152, no. 6 (June 1, 2006): 1679–86. http://dx.doi.org/10.1099/mic.0.28605-0.
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The LysR-type transcriptional regulator MvfR (PqsR) (multiple virulence factor regulator) plays a critical role in Pseudomonas aeruginosa pathogenicity via the transcriptional regulation of multiple quorum-sensing (QS)-regulated virulence factors. LasR activates full mvfR transcription, and MvfR subsequently activates pqsA–E expression. This study identifies and characterizes the key cis-regulatory elements through which mvfR and pqsA–E transcription is regulated in the highly virulent P. aeruginosa strain PA14. Deletion and site-directed mutagenesis indicate that: (1) LasR activates mvfR transcription by binding to a las/rhl box, CTAACAAAAGACATAG, centred at −513 bp upstream of the MvfR translational start site; and (2) RhlR represses pqsA transcription by binding to a las/rhl box, CTGTGAGATTTGGGAG, centred at −311 bp upstream of the pqsA transcriptional initiation site. Furthermore, it is shown that MvfR activates pqsA–E transcription by binding to a LysR box, TTCGGACTCCGAA, centred at −45 bp relative to the pqsA transcriptional initiation site, demonstrating that this LysR box has a critical role in the physical interaction between the MvfR protein and the pqsA promoter. These results provide new insights into the regulatory relationships between LasR and mvfR, and between MvfR/RhlR and the pqs operon, and elucidate further the complex regulation of the P. aeruginosa QS circuitry.
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Wang, Kevin L. C., and Jonathan R. Warner. "Positive and Negative Autoregulation ofREB1 Transcription in Saccharomyces cerevisiae." Molecular and Cellular Biology 18, no. 7 (July 1, 1998): 4368–76. http://dx.doi.org/10.1128/mcb.18.7.4368.
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ABSTRACT Reb1p is a DNA binding protein of Saccharomyces cerevisiae that has been implicated in the activation of transcription by polymerase (Pol) II, in the termination of transcription by Pol I, and in the organization of nucleosomes. Studies of the transcriptional control of the REB1 gene have led us to identify three Reb1p binding sites in the 5′ region of the its gene, termed A, B, and C, at positions −110, −80, and +30 with respect to transcription initiation. In vitro, Reb1p binds to the three sites with the relative affinity of A ≥ C > B. Kinetic parameters suggest that when both A and C sites are present on the same DNA molecule, the C site may recruit Reb1p for the A site. In vivo the A and B sites each contribute to the transcription activity ofREB1 in roughly additive fashion. Mutation of both A and B sites abolishes transcription. On the other hand, the C site is a negative element, reducing transcription by 40%. In cells overexpressing Reb1p, the C site reduces transcription by more than 80%. This effect can be transposed to another transcription unit, demonstrating that the effect of Reb1p binding at the C site does not depend on interaction with upstream Reb1p molecules. Relocation of the C site to a position 105 bp downstream of the transcription initiation site abolishes its effect, suggesting that it does not act as a conventional attenuator of transcription. We conclude that binding of Reb1p at the C site hinders formation of the initiation complex. This arrangement of Reb1p binding sites provides a positive and negative mechanism to autoregulate the expression of REB1. Such an arrangement could serve to dampen the inevitable fluctuation in Rep1p levels caused by the intermittent presence of its mRNA within an individual cell.
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Nam, S. C., and C. W. Kang. "Transcription initiation site selection and abortive initiation cycling of phage SP6 RNA polymerase." Journal of Biological Chemistry 263, no. 34 (December 1988): 18123–27. http://dx.doi.org/10.1016/s0021-9258(19)81332-7.
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Howcroft, T. Kevin, Aparna Raval, Jocelyn D. Weissman, Anne Gegonne, and Dinah S. Singer. "Distinct Transcriptional Pathways Regulate Basal and Activated Major Histocompatibility Complex Class I Expression." Molecular and Cellular Biology 23, no. 10 (May 15, 2003): 3377–91. http://dx.doi.org/10.1128/mcb.23.10.3377-3391.2003.
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ABSTRACT Transcription of major histocompatibility complex (MHC) class I genes is regulated by both tissue-specific (basal) and hormone/cytokine (activated) mechanisms. Although promoter-proximal regulatory elements have been characterized extensively, the role of the core promoter in mediating regulation has been largely undefined. We report here that the class I core promoter consists of distinct elements that are differentially utilized in basal and activated transcription pathways. These pathways recruit distinct transcription factor complexes to the core promoter elements and target distinct transcription initiation sites. Class I transcription initiates at four major sites within the core promoter and is clustered in two distinct regions: “upstream” (−14 and −18) and “downstream” (+12 and +1). Basal transcription initiates predominantly from the upstream start site region and is completely dependent upon the general transcription factor TAF1 (TAFII250). Activated transcription initiates predominantly from the downstream region and is TAF1 (TAFII250) independent. USF1 augments transcription initiating through the upstream start sites and is dependent on TAF1 (TAFII250), a finding consistent with its role in regulating basal class I transcription. In contrast, transcription activated by the interferon mediator CIITA is independent of TAF1 (TAFII250) and focuses initiation on the downstream start sites. Thus, basal and activated transcriptions of an MHC class I gene target distinct core promoter domains, nucleate distinct transcription initiation complexes and initiate at distinct sites within the promoter. We propose that transcription initiation at the core promoter is a dynamic process in which the mechanisms of core promoter function differ depending on the cellular environment.
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Shung, Chia-Yi, Janet Sunter, Shyam S. Sirasanagandla, and Garry Sunter. "Distinct Viral Sequence Elements Are Necessary for Expression of Tomato golden mosaic virus Complementary Sense Transcripts That Direct AL2 and AL3 Gene Expression." Molecular Plant-Microbe Interactions® 19, no. 12 (December 2006): 1394–405. http://dx.doi.org/10.1094/mpmi-19-1394.
Full textAbstract:
Transient expression studies using Nicotiana benthamiana protoplasts and plants have identified sequences important for transcription of complementary sense RNAs derived from Tomato golden mosaic virus (TGMV) DNA component A that direct expression of AL2 and AL3. Transcription of two complementary sense RNAs, initiating at nucleotides 1,935 (AL1935) and 1,629 (AL1629), is directed by unique sequences located upstream of each transcription initiation site. One element is located between 28 and 124 nucleotides (nt) upstream of the AL1935 transcription start site, which differs from a second element located 150 nt downstream, between 129 and 184 nt upstream of the AL1629 transcription start site. Transcription initiation at nucleotide 1,935 is lower than that at nucleotide 1,629 as determined by run-on transcription assays, and the resulting transcript is only capable of expressing AL3. The transcript initiating at nucleotide 1,629 is capable of directing expression of both AL2 and AL3, although expression of AL3 is up to fourfold greater than that for AL2. Nuclear factors purified from tobacco suspension cells bind to sequences upstream of both AL1935 and AL1629, correlating with the ability of these sequences to direct gene expression. Thus, in tobacco, regulatory sequences direct transcription of two unique TGMV messenger RNAs that differentially express AL2 and AL3.
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Lofquist, A. K., A. D. Garcia, and S. J. Sharp. "A discrete region centered 22 base pairs upstream of the initiation site modulates transcription of Drosophila tRNAAsn genes." Molecular and Cellular Biology 8, no. 10 (October 1988): 4441–49. http://dx.doi.org/10.1128/mcb.8.10.4441.
Full textAbstract:
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 tRNA5Asn 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.
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Lofquist, A. K., A. D. Garcia, and S. J. Sharp. "A discrete region centered 22 base pairs upstream of the initiation site modulates transcription of Drosophila tRNAAsn genes." Molecular and Cellular Biology 8, no. 10 (October 1988): 4441–49. http://dx.doi.org/10.1128/mcb.8.10.4441-4449.1988.
Full textAbstract:
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 tRNA5Asn 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.
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Skalenko, Kyle S., Lingting Li, Yuanchao Zhang, Irina O. Vvedenskaya, Jared T. Winkelman, Alexander L. Cope, Deanne M. Taylor, et al. "Promoter-sequence determinants and structural basis of primer-dependent transcription initiation in Escherichia coli." Proceedings of the National Academy of Sciences 118, no. 27 (June 29, 2021): e2106388118. http://dx.doi.org/10.1073/pnas.2106388118.
Full textAbstract:
Chemical modifications of RNA 5′-ends enable “epitranscriptomic” regulation, influencing multiple aspects of RNA fate. In transcription initiation, a large inventory of substrates compete with nucleoside triphosphates for use as initiating entities, providing an ab initio mechanism for altering the RNA 5′-end. In Escherichia coli cells, RNAs with a 5′-end hydroxyl are generated by use of dinucleotide RNAs as primers for transcription initiation, “primer-dependent initiation.” Here, we use massively systematic transcript end readout (MASTER) to detect and quantify RNA 5′-ends generated by primer-dependent initiation for ∼410 (∼1,000,000) promoter sequences in E. coli. The results show primer-dependent initiation in E. coli involves any of the 16 possible dinucleotide primers and depends on promoter sequences in, upstream, and downstream of the primer binding site. The results yield a consensus sequence for primer-dependent initiation, YTSS−2NTSS−1NTSSWTSS+1, where TSS is the transcription start site, NTSS−1NTSS is the primer binding site, Y is pyrimidine, and W is A or T. Biochemical and structure-determination studies show that the base pair (nontemplate-strand base:template-strand base) immediately upstream of the primer binding site (Y:RTSS−2, where R is purine) exerts its effect through the base on the DNA template strand (RTSS−2) through interchain base stacking with the RNA primer. Results from analysis of a large set of natural, chromosomally encoded E. coli promoters support the conclusions from MASTER. Our findings provide a mechanistic and structural description of how TSS-region sequence hard-codes not only the TSS position but also the potential for epitranscriptomic regulation through primer-dependent transcription initiation.
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Li, W. Z., and F. Sherman. "Two types of TATA elements for the CYC1 gene of the yeast Saccharomyces cerevisiae." Molecular and Cellular Biology 11, no. 2 (February 1991): 666–76. http://dx.doi.org/10.1128/mcb.11.2.666.
Full textAbstract:
Functional TATA elements in the 5' untranslated region of the CYC1 gene in the yeast Saccharomyces cerevisiae have been defined by transcriptional analysis of site-directed mutations. Five sites previously suggested to contain functional TATA elements were altered individually and in all possible combinations. The results indicated that only two elements are required for transcription at the normal level and the normal start sites. The two functional TATA elements are located at sites -178 and -123, where the A of the ATG start codon is assigned nucleotide position +1. They direct initiation within windows encompassing -70 to -46 and -46 to -28, respectively. Only when both of the upstream TATA sites were rendered nonfunctional were the third and fourth downstream TATA-like sequences activated, as indicated by the presence of low levels of transcription starting at -28. The two upstream functional TATA elements differed in sequence. The sequence of the most 5' one at site 1, denoted beta-type, was ATATATATAT, whereas that of the second one at site 2, denoted alpha-type, was TATATAAAA. The following rearrangements of the beta-type and alpha-type elements at two sites (1 and 2) were examined: site1 beta-site2 alpha; site 1 alpha-site 2 beta; site1 alpha-site2 alpha; and site1 beta-site2 beta. When different types were at different sites (site1 beta-site2 alpha and site1 alpha-site2 beta), both were used equally. In contrast, when the same type was present at both sites (site1 alpha-site2 alpha and site1 beta-site2 beta), only the upstream element was used. We suggest that the two TATA elements are recognized by different factors of the transcription apparatus.
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Li, W. Z., and F. Sherman. "Two types of TATA elements for the CYC1 gene of the yeast Saccharomyces cerevisiae." Molecular and Cellular Biology 11, no. 2 (February 1991): 666–76. http://dx.doi.org/10.1128/mcb.11.2.666-676.1991.
Full textAbstract:
Functional TATA elements in the 5' untranslated region of the CYC1 gene in the yeast Saccharomyces cerevisiae have been defined by transcriptional analysis of site-directed mutations. Five sites previously suggested to contain functional TATA elements were altered individually and in all possible combinations. The results indicated that only two elements are required for transcription at the normal level and the normal start sites. The two functional TATA elements are located at sites -178 and -123, where the A of the ATG start codon is assigned nucleotide position +1. They direct initiation within windows encompassing -70 to -46 and -46 to -28, respectively. Only when both of the upstream TATA sites were rendered nonfunctional were the third and fourth downstream TATA-like sequences activated, as indicated by the presence of low levels of transcription starting at -28. The two upstream functional TATA elements differed in sequence. The sequence of the most 5' one at site 1, denoted beta-type, was ATATATATAT, whereas that of the second one at site 2, denoted alpha-type, was TATATAAAA. The following rearrangements of the beta-type and alpha-type elements at two sites (1 and 2) were examined: site1 beta-site2 alpha; site 1 alpha-site 2 beta; site1 alpha-site2 alpha; and site1 beta-site2 beta. When different types were at different sites (site1 beta-site2 alpha and site1 alpha-site2 beta), both were used equally. In contrast, when the same type was present at both sites (site1 alpha-site2 alpha and site1 beta-site2 beta), only the upstream element was used. We suggest that the two TATA elements are recognized by different factors of the transcription apparatus.
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Gumina, Richard J., Nancy E. Kirschbaum, Kim Piotrowski, and Peter J. Newman. "Characterization of the Human Platelet/Endothelial Cell Adhesion Molecule-1 Promoter: Identification of a GATA-2 Binding Element Required for Optimal Transcriptional Activity." Blood 89, no. 4 (February 15, 1997): 1260–69. http://dx.doi.org/10.1182/blood.v89.4.1260.
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Abstract Platelet/endothelial cell adhesion molecule-1 (PECAM-1) is a 130-kD member of the Ig gene superfamily that is expressed on platelets, endothelial cells, and certain leukocyte subsets. To examine the factors controlling vascular-specific expression of PECAM-1, we cloned the 5′-flanking region of the PECAM-1 gene and analyzed its transcriptional activity. 5′-Rapid amplification of cDNA ends (5′-RACE) analysis showed that transcription initiation occurred at several closely spaced nearby sites originating approximately 204 bp upstream from the translation start site. Analysis of the sequence immediately upstream from the transcription initiation site (TIS) showed no canonical TATA or CAAT elements, however an initiator element commonly found in TATA-less promoters encompassed the TIS. 5′-serially truncated PECAM-1 promoter segments cloned in front of a luciferase reporter drove transcription in both a lineage- and orientation-specific manner. Putative cis-acting control elements present within a 300-bp core promoter included two ets sites, an Sp1 site, tandem E-box domains, two GATA-associated sites (CACCC), an AP-2 binding site, and a GATA element at −24. Mutational analysis showed that optimal transcriptional activity required the GATA sequence at position −24, and gel-shift assays further showed that the GATA-2 transcription factor, but not GATA-1, bound to this region of the PECAM-1 promoter. Understanding the cis- and trans-acting factors that regulate the tissue-specific expression of PECAM-1 should increase our understanding of the mechanisms by which vascular-specific gene expression is achieved.
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García-Escudero, Ramón, and Eladio Viñuela. "Structure of African Swine Fever Virus Late Promoters: Requirement of a TATA Sequence at the Initiation Region." Journal of Virology 74, no. 17 (September 1, 2000): 8176–82. http://dx.doi.org/10.1128/jvi.74.17.8176-8182.2000.
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ABSTRACT A number of mutations, including deletions, linker scan substitutions, and point mutations, were performed in the promoter of the late African swine fever virus (ASFV) gene coding for the capsid protein p72. The consequences of the mutations in terms of promoter activity were analyzed by luciferase assays using plasmids transfected into infected cells. The results showed that the promoter function is contained between nucleotides −36 and +5 relative to the transcription initiation site. Moreover, two major essential regions for promoter activity, centered at positions −13 and +3, were located along the 41-bp sequence, the latter mapping in the transcription start site. Sequence alignment with other ASFV late promoters showed homology in the region of transcriptional initiation, where the presence of the sequence TATA was observed in most of the promoters. Substitution of these four residues in three other late viral promoters strongly reduced their respective activities. These results show thatcis-acting control elements of ASFV p72 gene transcription are restricted to a short sequence of about 40 bp and suggest that transcription of late genes is initiated around a TATA sequence that would function as an initiator element.
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Schilling, L. J., and P. J. Farnham. "Identification of a new promoter upstream of the murine dihydrofolate reductase gene." Molecular and Cellular Biology 9, no. 10 (October 1989): 4568–70. http://dx.doi.org/10.1128/mcb.9.10.4568.
Full textAbstract:
In vitro reactions identified a transcription initiation site located 740 nucleotides upstream of the dihydrofolate reductase translational start. Transcription from this site proceeded in the direction opposite to that of dihydrofolate reductase mRNA. Deletion mapping indicated that this new promoter can be separated from the dihydrofolate reductase promoter and that separation increased transcription at -740. Transcripts that initiate at -740 were also detected in cellular RNA, indicating that this is a bona fide transcription initiation site in vivo.
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Schilling, L. J., and P. J. Farnham. "Identification of a new promoter upstream of the murine dihydrofolate reductase gene." Molecular and Cellular Biology 9, no. 10 (October 1989): 4568–70. http://dx.doi.org/10.1128/mcb.9.10.4568-4570.1989.
Full textAbstract:
In vitro reactions identified a transcription initiation site located 740 nucleotides upstream of the dihydrofolate reductase translational start. Transcription from this site proceeded in the direction opposite to that of dihydrofolate reductase mRNA. Deletion mapping indicated that this new promoter can be separated from the dihydrofolate reductase promoter and that separation increased transcription at -740. Transcripts that initiate at -740 were also detected in cellular RNA, indicating that this is a bona fide transcription initiation site in vivo.
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Walker, Kimberly A., and Robert Osuna. "Factors Affecting Start Site Selection at the Escherichia coli fis Promoter." Journal of Bacteriology 184, no. 17 (September 1, 2002): 4783–91. http://dx.doi.org/10.1128/jb.184.17.4783-4791.2002.
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ABSTRACT Transcription initiation with CTP is an uncommon feature among Escherichia coli σ70 promoters. The fis promoter (fis P), which is subject to growth phase-dependent regulation, is among the few that predominantly initiate transcription with CTP. Mutations in this promoter that cause a switch from utilization of CTP to either ATP or GTP as the initiation nucleotide drastically alter its growth phase regulation pattern, suggesting that the choice of the primary initiating nucleotide can significantly affect its regulation. To better understand what factors influence this choice in fis P, we made use of a series of promoter mutations that altered the nucleotide or position used for initiation. Examination of these promoters indicates that start site selection is determined by a combination of factors that include preference for a nucleotide distance from the −10 region (8 > 7 > 9 ≫ 6 ≫ 10 > 11), initiation nucleotide preference (A = G ≫ CTP ≥ UTP), the DNA sequence surrounding the initiation region, the position of the −35 region, and changes in the intracellular nucleoside triphosphate pools. We describe the effects that each of these factors has on start site selection in the fis P and discuss the interplay between position and nucleotide preference in this important process.
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Healy, A. M., T. L. Helser, and R. S. Zitomer. "Sequences required for transcriptional initiation of the Saccharomyces cerevisiae CYC7 genes." Molecular and Cellular Biology 7, no. 10 (October 1987): 3785–91. http://dx.doi.org/10.1128/mcb.7.10.3785.
Full textAbstract:
A series of BAL 31 deletions were constructed in the upstream region of the Saccharomyces cerevisiae CYC7 gene to determine sequences required for transcriptional initiation. These deletions identified the TATA box as an alternating A-T sequence at -160 and the initiation sequences as well as the spatial relationship between them. The TATA box was necessary for wild-type levels of expression of the CYC7 gene. Decreasing the distance between the TATA sequence and the initiation site did not alter gene expression, but the site of transcription was shifted 3'-ward. In most cases, transcription initiated at a number of sites, the 5'-most of which was the first suitable site greater than 45 base pairs 3' of the TATA sequence, suggesting a spatial relationship between these sequences. Consensus sequences previously proposed for initiation sites were evaluated with respect to the start sites identified in this study as well as the start sites of other yeast genes.
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Healy, A. M., T. L. Helser, and R. S. Zitomer. "Sequences required for transcriptional initiation of the Saccharomyces cerevisiae CYC7 genes." Molecular and Cellular Biology 7, no. 10 (October 1987): 3785–91. http://dx.doi.org/10.1128/mcb.7.10.3785-3791.1987.
Full textAbstract:
A series of BAL 31 deletions were constructed in the upstream region of the Saccharomyces cerevisiae CYC7 gene to determine sequences required for transcriptional initiation. These deletions identified the TATA box as an alternating A-T sequence at -160 and the initiation sequences as well as the spatial relationship between them. The TATA box was necessary for wild-type levels of expression of the CYC7 gene. Decreasing the distance between the TATA sequence and the initiation site did not alter gene expression, but the site of transcription was shifted 3'-ward. In most cases, transcription initiated at a number of sites, the 5'-most of which was the first suitable site greater than 45 base pairs 3' of the TATA sequence, suggesting a spatial relationship between these sequences. Consensus sequences previously proposed for initiation sites were evaluated with respect to the start sites identified in this study as well as the start sites of other yeast genes.
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Lu, Fang, Jing Zhou, Andreas Wiedmer, Kevin Madden, Yan Yuan, and Paul M. Lieberman. "Chromatin Remodeling of the Kaposi's Sarcoma-Associated Herpesvirus ORF50 Promoter Correlates with Reactivation from Latency." Journal of Virology 77, no. 21 (November 1, 2003): 11425–35. http://dx.doi.org/10.1128/jvi.77.21.11425-11435.2003.
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ABSTRACT The switch from latent to lytic infection of Kaposi's sarcoma-associated herpesvirus is initiated by the immediate early transcriptional activator protein Rta/open reading frame 50 (ORF50). We examined the transcriptional regulation of the ORF50 core promoter in response to lytic cycle stimulation. We show that the ORF50 promoter is highly responsive to sodium butyrate (NaB) and trichostatin A (TSA), two chemicals known to inhibit histone deacetylases. The NaB and TSA responsive element was mapped to a 70-bp minimal promoter containing an essential GC box that binds Sp1/Sp3 in vitro and in vivo. Micrococcal nuclease mapping studies revealed that a nucleosome is positioned over the transcriptional initiation and the Sp1/3 binding sites. Stimulation with NaB or TSA increased histone acetylation and restriction enzyme accessibility of the ORF50 promoter transcription initiation site. Chromatin immunoprecipitation assay was used to demonstrate that the ORF50 promoter is associated with several different histone deacetylase proteins (including HDAC1, 5, and 7) in latently infected cells. NaB treatment led to the rapid association of Ini1/Snf5, a component of the Swi/Snf family of chromatin remodeling proteins, with the ORF50 promoter. Ectopic expression of the CREB-binding protein (CBP) histone acetyltransferase (HAT) stimulated plasmid-based ORF50 transcription in a HAT-dependent manner, suggesting that CBP recruitment to the ORF50 promoter can be an initiating event for transcription and viral reactivation. Together, these results suggest that remodeling of a stably positioned nucleosome at the transcriptional initiation site of ORF50 is a regulatory step in the transition from latent to lytic infection.
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Yu, K., and R. T. Elder. "Some of the signals for 3'-end formation in transcription of the Saccharomyces cerevisiae Ty-D15 element are immediately downstream of the initiation site." Molecular and Cellular Biology 9, no. 6 (June 1989): 2431–44. http://dx.doi.org/10.1128/mcb.9.6.2431.
Full textAbstract:
Fragments from the Ty-D15 element of Saccharomyces cerevisiae were assayed for the ability to direct 3'-end formation for RNA initiated by the GAL1 promoter. The delta, the direct repeat at each end of the element, was capable of forming 3' ends at two sites, an inefficient upstream site and an efficient downstream site near the end of the delta. Different sequences were required for 3'-end formation at these sites. For the efficient site, all transcripts had 3' ends in the delta and no downstream transcription was detected, which suggested that these sequences terminate transcription. Surprisingly, the delta region downstream of the initiation site for Ty RNA comprised part of this major site and terminated more than 50% of the transcripts that read into it. Sequences necessary for the efficient site were localized to two small regions. Both regions were upstream of the 3' end and contained similarities to a tripartite consensus sequence that has been proposed as a terminator element. Sequences near the position of the 3' end could also affect termination; a short G + C-rich sequence inserted just downstream changed an efficient terminator to an inefficient one. Initiation in the delta had no effect on the efficiency or positions or termination in that delta. A new initiation site was seen when the same delta terminated transcription, but transcriptional interference did not occur, since the amount of initiation was not decreased.
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Yu, K., and R. T. Elder. "Some of the signals for 3'-end formation in transcription of the Saccharomyces cerevisiae Ty-D15 element are immediately downstream of the initiation site." Molecular and Cellular Biology 9, no. 6 (June 1989): 2431–44. http://dx.doi.org/10.1128/mcb.9.6.2431-2444.1989.
Full textAbstract:
Fragments from the Ty-D15 element of Saccharomyces cerevisiae were assayed for the ability to direct 3'-end formation for RNA initiated by the GAL1 promoter. The delta, the direct repeat at each end of the element, was capable of forming 3' ends at two sites, an inefficient upstream site and an efficient downstream site near the end of the delta. Different sequences were required for 3'-end formation at these sites. For the efficient site, all transcripts had 3' ends in the delta and no downstream transcription was detected, which suggested that these sequences terminate transcription. Surprisingly, the delta region downstream of the initiation site for Ty RNA comprised part of this major site and terminated more than 50% of the transcripts that read into it. Sequences necessary for the efficient site were localized to two small regions. Both regions were upstream of the 3' end and contained similarities to a tripartite consensus sequence that has been proposed as a terminator element. Sequences near the position of the 3' end could also affect termination; a short G + C-rich sequence inserted just downstream changed an efficient terminator to an inefficient one. Initiation in the delta had no effect on the efficiency or positions or termination in that delta. A new initiation site was seen when the same delta terminated transcription, but transcriptional interference did not occur, since the amount of initiation was not decreased.
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Lace, Michael J., Yasushi Yamakawa, Masato Ushikai, James R. Anson, Thomas H. Haugen, and Lubomir P. Turek. "Cellular factor YY1 downregulates the human papillomavirus 16 E6/E7 promoter, P97, in vivo and in vitro from a negative element overlapping the transcription-initiation site." Journal of General Virology 90, no. 10 (October 1, 2009): 2402–12. http://dx.doi.org/10.1099/vir.0.012708-0.
Full textAbstract:
Cellular factors that bind to cis sequences in the human papillomavirus 16 (HPV-16) upstream regulatory region (URR) positively and negatively regulate the viral E6 and E7 oncogene promoter, P97. DNase I footprinting has revealed the binding of cellular proteins to two previously undetected cis elements overlapping and 3′ of the transcription-initiation site of the P97 promoter. Mutations within homologous motifs found in both of these cis elements abolished their negative function in vivo and the binding of the same cellular complex in vitro. This factor was identified as YY1 by complex mobility and binding specificity in comparison with vaccinia virus-expressed, purified recombinant YY1 protein and by antigenic reactivity with YY1 antisera. Cis mutations in the ‘initiator’ YY1 site activated the P97 promoter in vivo and in vitro. P97 was also activated threefold in vitro by depletion of endogenous YY1 with wild-type, but not mutant, YY1 oligonucleotides from the IgH kappa E3′ enhancer. Furthermore, increasing concentrations of exogenous, purified recombinant YY1 repressed wild-type P97 transcript levels by up to threefold, but did not influence the P97 promoter mutated in the ‘initiator’ YY1 site. Thus, the promoter-proximal YY1 site was not necessary for correct transcription initiation at the P97 promoter, but was found to be required for downregulation of P97 transcription in vivo and in vitro. In contrast to other viral and cellular promoters, where YY1 is thought to function as a positive transcription-‘initiator’ factor, HPV-16 P97 transcription is downregulated by YY1 from a critical motif overlapping the transcription start site.
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Gong, X., C. A. Radebaugh, G. K. Geiss, M. N. Simon, and M. R. Paule. "Site-directed photo-cross-linking of rRNA transcription initiation complexes." Molecular and Cellular Biology 15, no. 9 (September 1995): 4956–63. http://dx.doi.org/10.1128/mcb.15.9.4956.
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Site-specific photo-cross-linking of the rRNA committed transcription complex was carried out by using 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUMP-derivatized promoter DNA. Putative TAFIs of 145, 99, 96, and 91 kDa, as well as TATA-binding protein (TBP), were found to specifically photo-cross-link to different positions along the promoter. These had been identified as potential subunits of the fundamental transcription initiation factor TIF-IB (also known as SL1, factor D, and TFID) from Acanthamoeba castellanii by purification to apparent homogeneity. No other polypeptides attributable to the rRNA architectural transcription factor UBF were identified, suggesting that this protein is not part of the committed complex. Scanning transmission electron microscopy of the complexes was used to estimate the mass of the complex and the contour length of the DNA in the complex. This showed that a single molecule of TIF-IB is in each committed complex and that the DNA is not looped around the protein, as would be expected if UBF were in the complex. A circular permutation analysis of DNA bending resulting from TIF-IB binding revealed a 45 +/- 3.1 degrees (n = 14) bend centered 23 bp upstream of the transcription initiation site. This degree of bending and the position of the bend relative to the site of TBP photo-cross-linking are consistent with earlier data showing that the TBP TATA box-binding domain is not utilized in the assembly of the rRNA committed complex (C. A. Radebaugh, J. L. Mathews, G. K. Geiss, F. Liu, J. Wong, E. Bateman, S. Camier, A. Sentenac, and M. R. Paule, Mol. Cell. Biol. 14:597-605, 1994).
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Nymark-McMahon, M. Henrietta, Nadejda S. Beliakova-Bethell, Jean-Luc Darlix, Stuart F. J. Le Grice, and Suzanne B. Sandmeyer. "Ty3 Integrase Is Required for Initiation of Reverse Transcription." Journal of Virology 76, no. 6 (March 15, 2002): 2804–16. http://dx.doi.org/10.1128/jvi.76.6.2804-2816.2002.
Full textAbstract:
ABSTRACT The integrase (IN) encoded by the Saccharomyces cerevisiae retroviruslike element Ty3 has features found in retrovirus IN proteins including the catalytic triad, an amino-terminal zinc-binding motif, and a nuclear localization sequence. Mutations in the amino- and carboxyl-terminal domains of Ty3 IN cause reduced accumulation of full-length cDNA in the viruslike particles. We show that the reduction in cDNA is accompanied by reduced amounts of early intermediates such as minus-strand, strong-stop DNA. Expression of a capsid (CA)-IN fusion protein (CA-IN) complemented catalytic site and nuclear localization mutants, but not DNA mutants. However, expression of a fusion of CA, reverse transcriptase (RT), and IN (CA-RT-IN) complemented transposition of catalytic site and nuclear localization signal mutants, increased the amount of cDNA in some of the mutants, and complemented transposition of several mutants to low frequencies. Expression of a CA-RT-IN protein with a Ty3 IN catalytic site mutation did not complement transposition of either a Ty3 catalytic site mutant or a nuclear localization mutant but did increase the amount of cDNA in several mutants and complement at least one of the cDNA mutants for transposition. These in vivo data support a model in which independent IN domains can contribute to reverse transcription and integration. We conclude that during reverse transcription, the Ty3 IN domain interacts closely with the polymerase domain and may even constitute a domain within a heterodimeric RT. These studies also suggest that during integration the IN catalytic site and at least portions of the IN carboxyl-terminal domain act in cis.
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Farnham, P. J., and A. L. Means. "Sequences downstream of the transcription initiation site modulate the activity of the murine dihydrofolate reductase promoter." Molecular and Cellular Biology 10, no. 4 (April 1990): 1390–98. http://dx.doi.org/10.1128/mcb.10.4.1390.
Full textAbstract:
The murine dihydrofolate reductase gene is regulated by a bidirectional promoter that lacks a TATA box. To identify the DNA sequences required for dihydrofolate reductase transcription, the activities of various templates were determined by in vitro transcription analysis. Our data indicate that sequences both upstream and downstream of the transcription initiation site modulate the activity of the dihydrofolate reductase promoter. We have focused on two regions downstream of the transcription initiation site that are important in determining the overall efficiency of the promoter. Region 1, which included exon 1 and part of intron 1, could stimulate transcription when placed in either orientation in the normal downstream position and when inserted upstream of the transcription start site. This region could also stimulate transcription in trans when the enhancer was physically separate from the promoter. Deletion of region 2, spanning 46 nucleotides of the 5' untranslated region, reduced transcriptional activity by fivefold. DNase I footprinting reactions identified protein-binding sites in both downstream stimulatory regions. Protein bound to two sites in region 1, both of which contain an inverted CCAAT box. The protein-binding site in the 5' untranslated region has extensive homology to binding sites in promoters that both lack (simian virus 40 late) and contain (adenovirus type 2 major late promoter and c-myc) TATA boxes.
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Farnham, P. J., and A. L. Means. "Sequences downstream of the transcription initiation site modulate the activity of the murine dihydrofolate reductase promoter." Molecular and Cellular Biology 10, no. 4 (April 1990): 1390–98. http://dx.doi.org/10.1128/mcb.10.4.1390-1398.1990.
Full textAbstract:
The murine dihydrofolate reductase gene is regulated by a bidirectional promoter that lacks a TATA box. To identify the DNA sequences required for dihydrofolate reductase transcription, the activities of various templates were determined by in vitro transcription analysis. Our data indicate that sequences both upstream and downstream of the transcription initiation site modulate the activity of the dihydrofolate reductase promoter. We have focused on two regions downstream of the transcription initiation site that are important in determining the overall efficiency of the promoter. Region 1, which included exon 1 and part of intron 1, could stimulate transcription when placed in either orientation in the normal downstream position and when inserted upstream of the transcription start site. This region could also stimulate transcription in trans when the enhancer was physically separate from the promoter. Deletion of region 2, spanning 46 nucleotides of the 5' untranslated region, reduced transcriptional activity by fivefold. DNase I footprinting reactions identified protein-binding sites in both downstream stimulatory regions. Protein bound to two sites in region 1, both of which contain an inverted CCAAT box. The protein-binding site in the 5' untranslated region has extensive homology to binding sites in promoters that both lack (simian virus 40 late) and contain (adenovirus type 2 major late promoter and c-myc) TATA boxes.
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Wilmańska, Dorota, Malgorzata Czyz, Kazimierz Studzian, Mariola K. Piestrzeniewicz, and Marek Gniazdowski. "Effects of Anticancer Drugs on Transcription in vitro." Zeitschrift für Naturforschung C 56, no. 9-10 (October 1, 2001): 886–91. http://dx.doi.org/10.1515/znc-2001-9-1034.
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AbstractThe effects of DNA interacting drugs on: (1) total RNA synthesis catalyzed by E.coli and T7 RNA polymerase; (2) synthesis of the initiating dinucleotide (pppApU) by E .coli RNA polymerase (“abortive initiation“); (3) elongation of RNA chains synthesized by T7 RNA polymerase on pT7-7 plasmid DNA bearing T7 RNA polymerase promoter ϕ 10 with human Cu/Zn superoxide dismutase coding sequence, (4) interaction of transcription factor Sp1 and its binding site were studied. Intercalating ligands which form quickly dissociating complexes with DNA (anthracyclines, proflavine, ethidium bromide) are compared with the slowly dissociating drug of d(G · C ) specificity (actinomycin D), the non-intercalating, d(A · T ) specific pyrrole antibiotics (netropsin and distamycin A) and covalently binding to DNA 1-nitroacridine derivative (nitracrine). The obtained results indicate that rapidly dissociating ligands, proflavine and ethidium bromide, inhibit total RNA synthesis in vitro and the abortive initiation to a similar extent while they do not induce discrete elongation stops of RNA polymerase. Actinomycin D and nitracrine exhibit a high inhibitory effect on total RNA synthesis and induce stops of RNA polymerase while not affecting abortive initiation. Pyrrole antibiotics primarily inhibit the initiation, while no elongation stops are induced. Actinomycin D inhibits complex formation between nuclear proteins and the Sp1 binding site. Netropsin, ethidium bromide, proflavine and other intercalating acridines do not affect Sp1 binding. The results indicate that the effects primarily depend on sequence specificity and secondarily on the dissociation rate of ligands from their complexes with DNA.
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MacDougald, O. A., and D. B. Jump. "Identification of functional cis-acting elements within the rat liver S14 promoter." Biochemical Journal 280, no. 3 (December 15, 1991): 761–67. http://dx.doi.org/10.1042/bj2800761.
Full textAbstract:
The structure of DNAase I hypersensitive site 1 (Hss-1), located adjacent to the 5′ end of the rat liver S14 gene, is regulated by tissue-specific factors, and its formation correlates with the transcriptional activation of the S14 gene. We propose that tissue-specific trans-acting factors interacting with key cis-linked elements within this site function in the initiation of S14 gene transcription. To examine this hypothesis we used DNAase I footprint, gel shift and in vitro transcriptional analyses to identify cis-linked elements that function in the control of S14 gene transcription. Binding of rat liver nuclear proteins to the S14 promoter (from -8 to -464 bp) produced four DNAase I footprints (designated A-D). Gel shift studies showed that DNA-protein binding was tissue- and sequence-specific, differentially heat-sensitive, and abolished by proteinase K. The function of the four cis-acting elements was assessed by using an in vitro transcription initiation assay in which the S14 promoter was fused to a reporter gene (G-free cassette). Deletion studies showed that nuclear factors binding to regions A (-48 to -63 bp), B (-88 to -113 bp) and D (-286 to -310 bp) enhanced the rate of initiation of transcription, while proteins binding to region C (-227 to -244 bp) suppressed the rate of initiation of transcription. Based on oligonucleotide competition studies, we suggest that hepatic NF-1 (or a related protein) binding to the A region enhances the rate of initiation of S14 gene transcription. Since trans-acting factors interacting with regions B and D are found in liver but not in spleen or kidney, we suggest that the proteins interacting with these regions may be involved in the tissue-specific augmentation of S14 gene transcription.
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Liu, Yujie, Michael R. Nonnemacher, Aikaterini Alexaki, Vanessa Pirrone, Anupam Banerjee, Luna Li, Evelyn Kilareski, and Brian Wigdahl. "Functional Studies of CCAAT/Enhancer Binding Protein Site Located Downstream of the Transcriptional Start Site." Clinical Medicine Insights: Pathology 10 (January 1, 2017): 117955571769455. http://dx.doi.org/10.1177/1179555717694556.
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Previous studies have identified a CCAAT/enhancer binding protein (C/EBP) site located downstream of the transcriptional start site (DS3). The role of the DS3 element with respect to HIV-1 transactivation by Tat and viral replication has not been characterized. We have demonstrated that DS3 was a functional C/EBPβ binding site and mutation of this site to the C/EBP knockout DS3-9C variant showed lower HIV-1 long terminal repeat (LTR) transactivation by C/EBPβ. However, it was able to exhibit similar or even higher transcription levels by Tat compared to the parental LTR. C/EBPβ and Tat together further enhanced the transcription level of the parental LAI-LTR and DS3-9C LTR, with higher levels in the DS3-9C LTR. HIV molecular clone viruses carrying the DS3-9C variant LTR demonstrated a decreased replication capacity and delayed rate of replication. These results suggest that DS3 plays a role in virus transcriptional initiation and provides new insight into C/EBP regulation of HIV-1.