scholarly journals Transcription of the juvenile hormone esterase gene under the control of both an initiator and AT-rich motif

1998 ◽  
Vol 335 (1) ◽  
pp. 79-84 ◽  
Author(s):  
Grace JONES ◽  
Maria MANCZAK ◽  
Douglas SCHELLING ◽  
Helen TURNER ◽  
Davy JONES
Keyword(s):  
Core Promoter ◽  
Tata Box ◽  
Binding Motif ◽  
Start Site ◽  
Wild Type ◽  
Transcription Start ◽  
Juvenile Hormone Esterase ◽  
A Cell ◽  
Esterase Gene ◽  
Wild Type Promoter

The binding of transcription factors to the core promoter of the juvenile hormone esterase gene was functionally characterized using both a cell-free in vitrotranscription functional assay and a cell transfection assay. A core JHE promoter (-61 to +28 bp relative to transcription start site) supported faithful transcription from the in vivo transcription start site. The nuclear extracts from the Sf9 insect cell line that provided transcription from that template also bound to that template as a probe in gel-mobility shift assays. Deletion or transversion of the initiator-binding motif (-1 to +4 bp) abolished detectable transcription either in vitro or in transfected cells. An AT-rich motif (ATATAT; -28 to -23 bp) serves another transcription factor-binding site. Mutation of the AT-rich motif to a canonical TATA-box preserved transcription, while either its deletion or complete transversion abolished or significantly reduced detectable transcriptional activity. These results indicate that, under these conditions, the functional operation of this core promoter approaches that of a composite promoter in which both the TATA- and initiator-binding protein complexes are necessary, even for basal transcription. On the other hand, these debilitating mutations to either the TATA box or initiator motif did not prevent the ability of the corresponding gel-shift competitive probes to compete with the wild-type promoter for binding by the transcription factors. Even a double transversion of both the AT-rich motif and the initiator-binding motif was able to competitively displace the protein complex that bound to the labelled wild-type probe. These data strongly indicate the presence of (an) additional core-promoter-associated transcription factor(s) (that is not the ‘downstream element ’) that contact(s) the AT-binding complex and/or initiator-binding factor with sufficient avidity to remove them from binding to the competing wild-type promoter sequence.

scholarly journals Core Promoter-Dependent TFIIB Conformation and a Role for TFIIB Conformation in Transcription Start Site Selection

2002 ◽  
Vol 22 (19) ◽  
pp. 6697-6705 ◽  
Author(s):  
Jennifer A. Fairley ◽  
Rachel Evans ◽  
Nicola A. Hawkes ◽  
Stefan G. E. Roberts
Keyword(s):  
Transcription Start Site ◽  
Site Selection ◽  
Transcription Initiation ◽  
Core Promoter ◽  
Initiation Site ◽  
Start Site ◽  
Wild Type ◽  
Transcription Start ◽  
General Transcription Factor ◽  
Selection Of

ABSTRACT The general transcription factor TFIIB plays a central role in the selection of the transcription initiation site. The mechanisms involved are not clear, however. In this study, we analyze core promoter features that are responsible for the susceptibility to mutations in TFIIB and cause a shift in the transcription start site. We show that TFIIB can modulate both the 5′ and 3′ parameters of transcription start site selection in a manner dependent upon the sequence of the initiator. Mutations in TFIIB that cause aberrant transcription start site selection concentrate in a region that plays a pivotal role in modulating TFIIB conformation. Using epitope-specific antibody probes, we show that a TFIIB mutant that causes aberrant transcription start site selection assembles at the promoter in a conformation different from that for wild-type TFIIB. In addition, we uncover a core promoter-dependent effect on TFIIB conformation and provide evidence for novel sequence-specific TFIIB promoter contacts.

Saturation Mutagenesis of the TATA Box and Upstream Activator Sequence in the Haloarchaeal bop Gene Promoter

1999 ◽  
Vol 181 (8) ◽  
pp. 2513-2518 ◽  
Author(s):  
Nitin S. Baliga ◽  
Shiladitya DasSarma
Keyword(s):  
Transcription Start Site ◽  
Direct Evidence ◽  
Consensus Sequence ◽  
Gene Promoter ◽  
Tata Box ◽  
Saturation Mutagenesis ◽  
Start Site ◽  
Wild Type ◽  
Transcription Start ◽  
Transcriptional Terminator

ABSTRACT Degenerate oligonucleotides were used to randomize 21 bp of the 53-bp minimal bop promoter in three 7-bp segments, including the putative TATA box and the upstream activator sequence (UAS). The mutagenized bop promoter and the wild-type structural gene and transcriptional terminator were inserted into a shuttle plasmid capable of replication in the halophilic archaeonHalobacterium sp. strain S9. Active promoters were isolated by screening transformants of an orange (Pum− bop) Halobacterium mutant for purple (Pum+ bop +) colonies on agar plates and analyzed for bop mRNA and/or bacteriorhodopsin content. Sequence analysis yielded the consensus sequence 5′-tyT(T/a)Ta-3′, corresponding to the promoter TATA box element 30 to 25 bp 5′ of the transcription start site. A putative UAS, 5′-ACCcnactagTTnG-3′, located 52 to 39 bp 5′ of the transcription start site was found to be conserved in active promoters. This study provides direct evidence for the requirement of the TATA box and UAS for bop promoter activity.

scholarly journals Regulation of the juvenile hormone esterase gene by a composite core promoter

2000 ◽  
Vol 346 (1) ◽  
pp. 233-240 ◽  
Author(s):  
Grace JONES ◽  
Yan Xia CHU ◽  
Douglas SCHELLING ◽  
Davy JONES
Keyword(s):  
Juvenile Hormone ◽  
Mutational Analysis ◽  
Core Promoter ◽  
Tata Box ◽  
Transient Transfection Assay ◽  
Core Element ◽  
Juvenile Hormone Esterase ◽  
Transcription In Vitro ◽  
Esterase Gene

Transcription from the core promoter of the juvenile hormone esterase gene (-61 to +28) requires the presence of both an AT-rich motif (TATA box) and an initiator motif for any transcription to occur, when assayed by either transcription in vitro with lepidopteran Sf9 nuclear extracts or by transient-transfection assay in Sf9 cells. Additional gel-shift experiments indicated that at least one additional binding site is essential for transcription to occur. Mutational analysis in the transcription-in vitro and cell-transfection assays demonstrated that a 14-bp region from +13 to +27 relative to the transcription start site is also essential for transcription to occur. Whereas the wild-type core promoter is highly transcriptionally active, inclusion of additional flanking sequences to position -212 reduces that activity approx. 100-fold, and inclusion of the 5ʹ region out to position -500 reduces transcription by 200-fold. The pattern of dependence on both the AT-rich motif and the initiator for detectable transcription, and the high innate activity being repressed by 5ʹ-binding factors, was recapitulated in mosquito C7-10 cells. This study demonstrates that the cellular juvenile hormone esterase gene is organized as a composite core promoter, dependent on both TATA-box and initiator-binding factors, an organization that has been more commonly reported for viral promoters. This highly active composite core promoter is made more complex by the absolute dependence on the presence of a third site shortly downstream from the initiator, which is distinct from the ‘downstream promoter element’ described from some TATA-less genes. The juvenile hormone esterase gene thus appears to be a model of a cellular composite core promoter with a multipartite, indispensible requirement for not just both the TATA box and initiator, but also for at least a third core element as well.

scholarly journals A cis Element between the TATA Box and the Transcription Start Site of the Major Immediate-Early Promoter of Human Cytomegalovirus Determines Efficiency of Viral Replication

2007 ◽  
Vol 82 (2) ◽  
pp. 849-858 ◽  
Author(s):  
Hiroki Isomura ◽  
Mark F. Stinski ◽  
Ayumi Kudoh ◽  
Sanae Nakayama ◽  
Takayuki Murata ◽  
...  
Keyword(s):  
Viral Replication ◽  
Transcription Start Site ◽  
Gene Transcription ◽  
Human Cytomegalovirus ◽  
Recombinant Virus ◽  
Tata Box ◽  
Immediate Early ◽  
Wild Type Virus ◽  
Start Site ◽  
Transcription Start

ABSTRACT The promoter of the major immediate-early (MIE) genes of human cytomegalovirus (HCMV), also referred to as the CMV promoter, possesses a cis-acting element positioned downstream of the TATA box between positions −14 and −1 relative to the transcription start site (+1). We determined the role of the cis-acting element in viral replication by comparing recombinant viruses with the cis-acting element replaced with other sequences. Recombinant virus with the simian CMV counterpart replicated efficiently in human foreskin fibroblasts, as well as wild-type virus. In contrast, replacement with the murine CMV counterpart caused inefficient MIE gene transcription, RNA splicing, MIE and early viral gene expression, and viral DNA replication. To determine which nucleotides in the cis-acting element are required for efficient MIE gene transcription and splicing, we constructed mutations within the cis-acting element in the context of a recombinant virus. While mutations in the cis-acting element have only a minor effect on in vitro transcription, the effects on viral replication are major. The nucleotides at −10 and −9 in the cis-acting element relative to the transcription start site (+1) affect efficient MIE gene transcription and splicing at early times after infection. The cis-acting element also acts as a cis-repression sequence when the viral IE86 protein accumulates in the infected cell. We demonstrate that the cis-acting element has an essential role in viral replication.

scholarly journals Variations in Intracellular Levels of TATA Binding Protein Can Affect Specific Genes by Different Mechanisms

2007 ◽  
Vol 28 (1) ◽  
pp. 83-92 ◽  
Author(s):  
Stephanie D. Bush ◽  
Patricia Richard ◽  
James L. Manley
Keyword(s):  
Binding Protein ◽  
Core Promoter ◽  
Tata Binding Protein ◽  
Tata Box ◽  
Wild Type ◽  
The Core ◽  
Mitotic Delay ◽  
Reduced Activity ◽  
Dt40 Cells ◽  
Repressor Element

ABSTRACT We previously showed that reduced intracellular levels of the TATA binding protein (TBP), brought about by tbp heterozygosity in DT40 cells, resulted in a mitotic delay reflecting reduced expression of the mitotic regulator cdc25B but did not significantly affect overall transcription. Here we extend these findings in several ways. We first provide evidence that the decrease in cdc25B expression reflects reduced activity of the cdc25B core promoter in the heterozygous (TBP-het) cells. Strikingly, mutations in a previously described repressor element that overlaps the TATA box restored promoter activity in TBP-het cells, supporting the idea that the sensitivity of this promoter to TBP levels reflects a competition between TBP and the repressor for DNA binding. To determine whether cells might have mechanisms to compensate for fluctuations in TBP levels, we next examined expression of the two known vertebrate TBP homologues, TLP and TBP2. Significantly, mRNAs encoding both were significantly overexpressed relative to levels observed in wild-type cells. In the case of TLP, this was shown to reflect regulation of the core promoter by both TBP and TLP. Together, our results indicate that variations in TBP levels can affect the transcription of specific promoters in distinct ways, but overall transcription may be buffered by corresponding alterations in the expression of TBP homologues.

scholarly journals Maneuver at the transcription start site: Mot1p and NC2 navigate TFIID/TBP to specific core promoter elements

Epigenetics ◽  
2009 ◽  
Vol 4 (1) ◽  
pp. 1-4 ◽  
Author(s):  
Zhuo Zhou ◽  
I-Ju Lin ◽  
Russell P. Darst ◽  
Jörg Bungert
Keyword(s):  
Transcription Start Site ◽  
Core Promoter ◽  
Start Site ◽  
Transcription Start ◽  
Promoter Elements

scholarly journals Synthetic enhancement of a TFIIB defect by a mutation in SSU72, an essential yeast gene encoding a novel protein that affects transcription start site selection in vivo.

1996 ◽  
Vol 16 (4) ◽  
pp. 1557-1566 ◽  
Author(s):  
Z W Sun ◽  
M Hampsey
Keyword(s):  
Site Selection ◽  
Zinc Binding ◽  
Binding Motif ◽  
Start Site ◽  
Transcription Start ◽  
Gene Encoding ◽  
N Terminus ◽  
Zinc Binding Motif ◽  
Novel Protein

An ssu72 mutant of Saccharomyces cerevisiae was identified as an enhancer of a TFIIB defect (sua7-1) that confers both a cold-sensitive growth defect and a downstream shift in transcription start site selection. The ssu72-1 allele did not affect cold sensitivity but, in combination with sua7-1, created a heat-sensitive phenotype. Moreover, start site selection at the ADH1 gene was dramatically shifted further downstream of the normal sites. Both of these effects could be rescued by either SUA7 or SSU72, thereby defining a functional relationship between the two genes. SSU72 is a single-copy, essential gene encoding a novel protein of 206 amino acids. The ssu72-1 allele is the result of a 30-bp duplication creating a sequence encoding a Cys-X2-Cys-X6-Cys-X2-Cys zinc binding motif near the N terminus of Ssu72p. Mutational analysis demonstrated that the N terminus of Ssu72p is essential for function and that cysteine residues in both the normal and mutant proteins are critical. We discuss the possibility that the potential zinc binding motif of Ssu72 facilitates assembly of the transcription preinitiation complex and that this effect is important for accurate start site selection in vivo.

In vitro transcription of a Drosophila U1 small nuclear RNA gene requires TATA box-binding protein and two proximal cis-acting elements with stringent spacing requirements

1993 ◽  
Vol 13 (9) ◽  
pp. 5918-5927
Author(s):  
Z Zamrod ◽  
C M Tyree ◽  
Y Song ◽  
W E Stumph
Keyword(s):  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Tata Box ◽  
Start Site ◽  
Small Nuclear Rna ◽  
Wild Type ◽  
Nuclear Rna ◽  
Tata Sequence ◽  
Tata Box Binding Protein

Transcription of a Drosophila U1 small nuclear RNA gene was functionally analyzed in cell extracts derived from 0- to 12-h embryos. Two promoter elements essential for efficient initiation of transcription in vitro by RNA polymerase II were identified. The first, termed PSEA, is located between positions -41 and -61 relative to the transcription start site, is crucial for promoter activity, and is the dominant element for specifying the transcription initiation site. PSEA thus appears to be functionally homologous to the proximal sequence element of vertebrate small nuclear RNA genes. The second element, termed PSEB, is located at positions -25 to -32 and is required for an efficient level of transcription initiation because mutation of PSEB, or alteration of the spacing between PSEA and PSEB, severely reduced transcriptional activity relative to that of the wild-type promoter. Although the PSEB sequence does not have any obvious sequence similarity to a TATA box, conversion of PSEB to the canonical TATA sequence dramatically increased the efficiency of the U1 promoter and simultaneously relieved the requirement for the upstream PSEA. Despite these effects, introduction of the TATA sequence into the U1 promoter had no effect on the choice of start site or on the RNA polymerase II specificity of the promoter. Finally, evidence is presented that the TATA box-binding protein is required for transcription from the wild-type U1 promoter as well as from the TATA-containing U1 promoter.

Drosophila OVO regulates ovarian tumor transcription by binding unusually near the transcription start site

Development ◽  
2001 ◽  
Vol 128 (9) ◽  
pp. 1671-1686 ◽  
Author(s):  
J. Lu ◽  
B. Oliver
Keyword(s):  
Transcription Start Site ◽  
Ovarian Tumor ◽  
Core Promoter ◽  
Regulatory Region ◽  
Start Site ◽  
Loss Of Function ◽  
Transcription Start ◽  
Germline Expression ◽  
Promoter Swapping

Evolutionarily conserved ovo loci encode developmentally regulated, sequence-specific, DNA-binding, C(2)H(2)-zinc-finger proteins required in the germline and epidermal cells of flies and mice. The direct targets of OVO activity are not known. Genetic experiments suggest that ovo acts in the same regulatory network as ovarian tumor (otu), but the relative position of these genes in the pathway is controversial. Three OVO-binding sites exist in a compact regulatory region that controls germline expression of the otu gene. Interestingly, the strongest OVO-binding site is very near the otu transcription start, where basal transcriptional complexes must function. Loss-of-function, gain-of-function and promoter swapping constructs demonstrate that OVO binding near the transcription start site is required for OVO-dependent otu transcription in vivo. These data unambiguously identify otu as a direct OVO target gene and raise the tantalizing possibility that an OVO site, at the location normally occupied by basal components, functions as part of a specialized core promoter.

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