scholarly journals Multiple control elements in the TRP1 promoter of Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (12) ◽  
pp. 4251-4258 ◽  
Author(s):  
S Kim ◽  
J Mellor ◽  
A J Kingsman ◽  
S M Kingsman
Keyword(s):  
Positive Element ◽  
Northern Blotting ◽  
Tata Box ◽  
Base Pairs ◽  
Multiple Control ◽  
Negative Element ◽  
Consensus Sequences ◽  
Positive Elements ◽  
The Difference

The TRP1 promoter generates two groups of mRNAs, transcript I and transcript II. The difference in size between the largest and smallest mRNAs is about 200 base pairs. A series of one-sided and internal deletions were constructed in vitro throughout the TRP1 promoter, and the effect of each deletion on transcription was assessed by Northern blotting. We showed that 395 base pairs of the TRP1 promoter were sufficient for the normal transcription of all RNAs and that the promoter contained two control domains. The control domain for transcript I consisted of one positive element and one negative element, while the control domain for transcript II contained two positive elements. The negative element, mapped between -293 and -318, expression of transcript I. Two regions of transcript I. Two regions (-280 to -236 and -235 to -209) were required for accurate initiation of transcript I. Each region contained sequences homologous to known consensus sequences of the TATA box.

Multiple control elements in the TRP1 promoter of Saccharomyces cerevisiae

1986 ◽  
Vol 6 (12) ◽  
pp. 4251-4258
Author(s):  
S Kim ◽  
J Mellor ◽  
A J Kingsman ◽  
S M Kingsman
Keyword(s):  
Positive Element ◽  
Northern Blotting ◽  
Tata Box ◽  
Base Pairs ◽  
Multiple Control ◽  
Negative Element ◽  
Consensus Sequences ◽  
Positive Elements ◽  
The Difference

The TRP1 promoter generates two groups of mRNAs, transcript I and transcript II. The difference in size between the largest and smallest mRNAs is about 200 base pairs. A series of one-sided and internal deletions were constructed in vitro throughout the TRP1 promoter, and the effect of each deletion on transcription was assessed by Northern blotting. We showed that 395 base pairs of the TRP1 promoter were sufficient for the normal transcription of all RNAs and that the promoter contained two control domains. The control domain for transcript I consisted of one positive element and one negative element, while the control domain for transcript II contained two positive elements. The negative element, mapped between -293 and -318, expression of transcript I. Two regions of transcript I. Two regions (-280 to -236 and -235 to -209) were required for accurate initiation of transcript I. Each region contained sequences homologous to known consensus sequences of the TATA box.

Overlapping positive and negative regulatory elements determine lens-specific activity of the delta 1-crystallin enhancer

1993 ◽  
Vol 13 (9) ◽  
pp. 5206-5215 ◽  
Author(s):  
Y Kamachi ◽  
H Kondoh
Keyword(s):  
Mutational Analysis ◽  
Specific Activity ◽  
Regulatory Elements ◽  
Positive Element ◽  
Specific Expression ◽  
Enhancer Activity ◽  
Negative Element ◽  
Positive Elements ◽  
Lens Cells ◽  
Specific Regulation

Lens-specific expression of the delta 1-crystallin gene is governed by an enhancer in the third intron, and the 30-bp-long DC5 fragment was found to be responsible for eliciting the lens-specific activity. Mutational analysis of the DC5 fragment identified two contiguous, interdependent positive elements and a negative element which overlaps the 3'-located positive element. Previously identified ubiquitous factors delta EF1 bound to the negative element and repressed the enhancer activity in nonlens cells. Mutation and cotransfection analyses indicated the existence of an activator which counteracts the action of delta EF1 in lens cells, probably through binding site competition. We also found a group of nuclear factors, collectively called delta EF2, which bound to the 5'-located positive element. delta EF2a and -b were the major species in lens cells, whereas delta EF2c and -d predominated in nonlens cells. These delta EF2 proteins probably cooperate with factors bound to the 3'-located element in activation in lens cells and repression in nonlens cells. delta EF2 proteins also bound to a promoter sequence of the gamma F-crystallin gene, suggesting that delta EF2 proteins are involved in lens-specific regulation of various crystallin classes.

scholarly journals Sequences required for transcriptional initiation of the Saccharomyces cerevisiae CYC7 genes.

1987 ◽  
Vol 7 (10) ◽  
pp. 3785-3791 ◽  
Author(s):  
A M Healy ◽  
T L Helser ◽  
R S Zitomer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Spatial Relationship ◽  
Initiation Site ◽  
Tata Box ◽  
Upstream Region ◽  
Base Pairs ◽  
Transcriptional Initiation ◽  
Consensus Sequences ◽  
Tata Sequence ◽  
Yeast Genes

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.

The rat albumin promoter is composed of six distinct positive elements within 130 nucleotides

1989 ◽  
Vol 9 (11) ◽  
pp. 4750-4758
Author(s):  
P Herbomel ◽  
A Rollier ◽  
F Tronche ◽  
M O Ott ◽  
M Yaniv ◽  
...  
Keyword(s):  
Regulatory Elements ◽  
Negative Regulation ◽  
Base Pairs ◽  
Tissue Specific ◽  
Positive Elements ◽  
Specific Factors ◽  
Ccaat Box ◽  
Rat Hepatoma

No fewer than six different positive regulatory elements concentrated within 130 base pairs constitute the rat albumin promoter, which drives highly tissue specific transcription in rat hepatoma cells in culture. Inactivation of each element led to a decrease in transcriptional efficiency: from upstream to downstream, 3- to 4-fold for distal elements III and II, 15-fold for distal element I, and 50-fold for the CCAAT box and the proximal element (PE). Three of these elements, distal elements III and II and, more crucially, the PE, were found to be involved in the tissue-specific character of transcription, with an additional negative regulation possibly superimposed at the level of the PE. Finally, our mapping of these regulatory elements in vivo entirely coincided with footprint data obtained in vitro, thereby allowing the tentative assignment of specific factors to the effects observed in vivo.

scholarly journals A Novel Upstream RNA Polymerase III Promoter Element Becomes Essential When the Chromatin Structure of the Yeast U6 RNA Gene Is Altered

2001 ◽  
Vol 21 (19) ◽  
pp. 6429-6439 ◽  
Author(s):  
Michael P. Martin ◽  
Valerie L. Gerlach ◽  
David A. Brow
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Point Mutations ◽  
Tata Box ◽  
Initiation Factor ◽  
Base Pairs ◽  
Polymerase Iii ◽  
U6 Rna

ABSTRACT The Saccharomyces cerevisiae U6 RNA gene,SNR6, possesses upstream sequences that allow productive binding in vitro of the RNA polymerase III (Pol III) transcription initiation factor IIIB (TFIIIB) in the absence of TFIIIC or other assembly factors. TFIIIC-independent transcription ofSNR6 in vitro is highly sensitive to point mutations in a consensus TATA box at position −30. In contrast, the TATA box is dispensable for SNR6 transcription in vivo, apparently because TFIIIC bound to the intragenic A block and downstream B block can recruit TFIIIB via protein-protein interactions. A mutant allele ofSNR6 with decreased spacing between the A and B blocks,snr6-Δ42, exhibits increased dependence on the upstream sequences in vivo. Unexpectedly, we find that in vivo expression of snr6-Δ42 is much more sensitive to mutations in a (dT-dA)7 tract between the TATA box and transcription start site than to mutations in the TATA box itself. Inversion of single base pairs in the center of the dT-dA tract nearly abolishes transcription of snr6-Δ42, yet inversion of all 7 base pairs has little effect on expression, indicating that the dA-dT tract is relatively orientation independent. Although it is within the TFIIIB footprint, point mutations in the dT-dA tract do not inhibit TFIIIB binding or TFIIIC-independent transcription ofSNR6 in vitro. In the absence of the chromatin architectural protein Nhp6, dT-dA tract mutations are lethal even when A-to-B block spacing is wild type. We conclude that the (dT-dA)7 tract and Nhp6 cooperate to direct productive transcription complex assembly on SNR6 in vivo.

scholarly journals Overlapping positive and negative regulatory elements determine lens-specific activity of the delta 1-crystallin enhancer.

1993 ◽  
Vol 13 (9) ◽  
pp. 5206-5215 ◽  
Author(s):  
Y Kamachi ◽  
H Kondoh
Keyword(s):  
Mutational Analysis ◽  
Specific Activity ◽  
Regulatory Elements ◽  
Positive Element ◽  
Specific Expression ◽  
Enhancer Activity ◽  
Negative Element ◽  
Positive Elements ◽  
Lens Cells ◽  
Specific Regulation

Lens-specific expression of the delta 1-crystallin gene is governed by an enhancer in the third intron, and the 30-bp-long DC5 fragment was found to be responsible for eliciting the lens-specific activity. Mutational analysis of the DC5 fragment identified two contiguous, interdependent positive elements and a negative element which overlaps the 3'-located positive element. Previously identified ubiquitous factors delta EF1 bound to the negative element and repressed the enhancer activity in nonlens cells. Mutation and cotransfection analyses indicated the existence of an activator which counteracts the action of delta EF1 in lens cells, probably through binding site competition. We also found a group of nuclear factors, collectively called delta EF2, which bound to the 5'-located positive element. delta EF2a and -b were the major species in lens cells, whereas delta EF2c and -d predominated in nonlens cells. These delta EF2 proteins probably cooperate with factors bound to the 3'-located element in activation in lens cells and repression in nonlens cells. delta EF2 proteins also bound to a promoter sequence of the gamma F-crystallin gene, suggesting that delta EF2 proteins are involved in lens-specific regulation of various crystallin classes.

scholarly journals Simian virus 40 major late promoter: a novel tripartite structure that includes intragenic sequences.

1988 ◽  
Vol 8 (5) ◽  
pp. 2021-2033 ◽  
Author(s):  
D E Ayer ◽  
W S Dynan
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Point Mutations ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Transcription Unit ◽  
Tata Box ◽  
Base Pairs

Unlike most genes transcribed by RNA polymerase II, the simian virus 40 late transcription unit does not have a TATA box. To determine what sequences are required for initiation at the major late mRNA cap site of simian virus 40, clustered point mutations were constructed and tested for transcriptional activity in vitro and in vivo. Three promoter elements were defined. The first is centered 31 base pairs upstream of the cap site in a position normally reserved for a TATA box. The second is at the cap site. The third occupies a novel position centered 28 base pairs downstream of the cap site within a protein-coding sequence. The ability of RNA polymerase II to recognize this promoter suggests that there is greater variation in promoter architecture than had been believed previously.

scholarly journals DNA sequences required for specific and efficient initiation of transcription at the polyoma virus early promoter.

1982 ◽  
Vol 2 (7) ◽  
pp. 737-751 ◽  
Author(s):  
P Jat ◽  
U Novak ◽  
A Cowie ◽  
C Tyndall ◽  
R Kamen
Keyword(s):  
Dna Sequences ◽  
In Vitro Transcription ◽  
Polyoma Virus ◽  
Tata Box ◽  
Deletion Mutants ◽  
Base Pairs ◽  
Transcription Analysis ◽  
In Vivo Expression

The 5'-flanking DNA sequences involved in the specific and efficient transcription of the polyoma virus early region have been investigated. Sequence requirements for efficient in vivo expression differed from those in vitro. Deletion of DNA located between 200 and 400 base pairs before the principal cap sites severely inhibited in vivo expression as measured by transformation ability, but did not affect in vitro transcription. Viable deletion mutants which lack the principal cap sites and the "TATA" box were very poor templates for in vitro transcription. Analysis of other deletion mutants in vitro demonstrated that no specific sequences more than 46 base pairs before the cap sites were important. Removal of the TATA box reduced in vitro transcriptional efficiency but did not alter the initiation sites. The synthesis of transcripts with abnormal 5' termini did not occur in vitro until sequence between the TATA box and the normal cap sites was also deleted. We further observed a nonspecific requirement for 90 to 100 base pairs of DNA 5' to the cap site for optimal transcription of DNA fragments in vitro.

scholarly journals Elements in the 3' untranslated region of procyclin mRNA regulate expression in insect forms of Trypanosoma brucei by modulating RNA stability and translation.

1997 ◽  
Vol 17 (8) ◽  
pp. 4372-4380 ◽  
Author(s):  
A Furger ◽  
N Schürch ◽  
U Kurath ◽  
I Roditi
Keyword(s):  
Steady State ◽  
Trypanosoma Brucei ◽  
Posttranscriptional Regulation ◽  
Untranslated Region ◽  
Rna Stability ◽  
State Level ◽  
Positive Element ◽  
Systematic Analysis ◽  
Negative Element ◽  
Positive Elements

Procyclins are the major surface glycoproteins of insect forms of Trypanosoma brucei. We have previously shown that a conserved 16-mer in the 3' untranslated region (UTR) of procyclin transcripts functions as a positive element in procyclic-form trypanosomes. A systematic analysis of the entire 297-base 3' UTR has now revealed additional elements which are involved in posttranscriptional regulation: a positive element which requires the first 40 bases of the 3' UTR and at least one negative element between nucleotides 101 and 173 (the LII domain). Deletion of either positive element resulted in a >8-fold reduction in the amount of protein but only an approximately 2-fold decrease in the steady-state level of mRNA, suggesting that regulation also occurred at the level of translation. In contrast, deletion of LII caused a threefold increase in the steady-state levels of both the mRNA and protein. LII-16-mer double deletions also gave high levels of expression, suggesting that the 16-mer functions as an antirepressor of the negative element rather than as an independent activator. All three elements have an effect on RNA turnover. When either positive element was deleted, the half-life (t(1/2)) of the mRNA was reduced from approximately 50 min (the t(1/2) of the wild-type 3' UTR) to < 15 min, whereas removal of the LII element resulted in an increased t(1/2) of approximately 100 min. We present a model of posttranscriptional regulation in which the negative domain is counteracted by two positive elements which shield it from nucleases and/or translational repressors.

Transcription of a trout protamine gene in vitro: the effects of alteration of promoters

1984 ◽  
Vol 62 (5) ◽  
pp. 291-300 ◽  
Author(s):  
Jacek M. Jankowski ◽  
Gordon H. Dixon
Keyword(s):  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Tata Box ◽  
Base Pairs ◽  
S1 Nuclease ◽  
Promoter Sequences ◽  
Transcription System ◽  
Nuclease Digestion ◽  
Simplex Virus

An in vitro approach has been used to study trout protamine gene expression using various recombinant plasmids containing trout protamine genes as templates in the HeLa cell lysate transcription system. The specific RNA transcript which is protected against S1 nuclease digestion by hybridization to the protamine gene sequence is α-amanitin sensitive (1 μg/mL), showing that RNA polymerase II is involved. The sizes of transcripts from templates linearized with Bam HI, Rsa I, and Hpa II (all downstream from the putative TATA box) are consistent with those predicted from the known sequence of the protamine gene. Digestion at an Alu I site only 14 base pairs (bp) upstream from TATA box has no effect on the accuracy of transcription in vitro; however, cutting at an Ava II site 9 bp downstream from the TATA box (reading from the first T) abolishes transcription. Chimeric plasmids, in which a herpes simplex virus (HSV-1) thymidine kinase (tk) promoter is tandemly inserted upstream from the trout protamine DNA sequences or as a replacement of the natural protamine promoter, were constructed. Use of these plasmids allowed an examination in a single assay of eight different putative promoter sequences (TATAAAA, TATAAA, TACAAA, TATATA, TATTTAA, CATATTA, TATATTAT, and TATTTAT) that are localized in either the protamine or the tk genes. The canonical TATAAAA promoter (the natural protamine promoter) was the strongest one and, in its presence, none of the others were used significantly for transcription. However, when this promoter was removed the weaker promoters were able to promote transcription.

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