Mechanism of regulation of transcription initiation by ppGpp. I. Effects of ppGpp on transcription initiation in vivo and in vitro

2001 ◽  
Vol 305 (4) ◽  
pp. 673-688 ◽  
Author(s):  
Melanie M. Barker ◽  
Tamas Gaal ◽  
Cathleen A. Josaitis ◽  
Richard L. Gourse
Keyword(s):  
Transcription Initiation ◽  
Regulation Of Transcription

scholarly journals Stimulation of the λ p R promoter by Escherichia coli SeqA protein requires downstream GATC sequences and involves late stages of transcription initiation

Microbiology ◽  
2006 ◽  
Vol 152 (10) ◽  
pp. 2985-2992 ◽  
Author(s):  
Robert Łyżeń ◽  
Grzegorz Wȩgrzyn ◽  
Alicja Wȩgrzyn ◽  
Agnieszka Szalewska-Pałasz
Keyword(s):  
Escherichia Coli ◽  
Transcription Initiation ◽  
Negative Regulator ◽  
Regulation Of Transcription ◽  
Chromosomal Dna ◽  
Transcription Activator ◽  
Transcription Analysis ◽  
Stimulation Of

Escherichia coli SeqA protein is a major negative regulator of chromosomal DNA replication acting by sequestration, and thus inactivation, of newly formed oriC regions. However, other activities of this protein have been discovered recently, one of which is regulation of transcription. SeqA has been demonstrated to be a specific transcription factor acting at bacteriophage λ promoters p I, p aQ and p R. While SeqA-mediated stimulation of p I and p aQ occurs by facilitating functions of another transcription activator protein, cII, a mechanism for stimulation of p R remains largely unknown. Here, it has been demonstrated that two GATC sequences, located 82 and 105 bp downstream of the p R transcription start site, are necessary for this stimulation both in vivo and in vitro. SeqA-mediated activation of p R was as effective on a linear DNA template as on a supercoiled one, indicating that alterations in DNA topology are not likely to facilitate the SeqA effect. In vitro transcription analysis demonstrated that the most important regulatory effect of SeqA in p R transcription occurs after open complex formation, namely during promoter clearance. SeqA did not influence the appearance and level of abortive transcripts or the pausing during transcription elongation. Interestingly, SeqA is one of few known prokaryotic transcription factors which bind downstream of the regulated promoter and still act as transcription activators.

scholarly journals Novel regulation of transcription initiation of the peptide IX gene of adenovirus 2.

1989 ◽  
Vol 9 (10) ◽  
pp. 4265-4271 ◽  
Author(s):  
T Matsui
Keyword(s):  
Transcriptional Repression ◽  
Transcription Initiation ◽  
Nuclear Extract ◽  
Negative Control ◽  
Tata Box ◽  
Regulation Of Transcription ◽  
Direct Initiation ◽  
Upstream Element

cis-Acting elements involved in transcription of the peptide IX (pIX) gene of adenovirus 2 were identified by using in vivo transient expression assays and two in vitro transcription systems. Deletion of either the sequence between positions -45 and -70 or the TATA box abolished the initiation of pIX gene transcription in vivo and transcription with HeLa cell nuclear extracts in vitro. These results initially suggested the presence of a positive factor acting on the upstream element. However, when proteins in the nuclear extract were fractionated by column chromatography and analyzed by reconstitution of transcription in vitro, it was found that a certain fraction could direct TATA box-dependent transcription initiation even in the absence of the upstream element. Furthermore, activity inhibiting TATA box-dependent transcription was found in the nuclear extract. In contrast, inhibition of TATA box-dependent transcription was suppressed by deletion of a downstream sequence between positions +33 and +122. These results indicate that the TATA box of the pIX gene by itself has the ability to direct initiation of constitutive transcription but that the function of this element is under negative control by a repressor acting on a downstream sequence. Thus, the upstream element of the pIX gene appears to have a novel function: suppression of the transcriptional repression exerted by a downstream sequence, leading to a net transcription activation. Possible mechanisms for transcription initiation of pIX DNA are discussed.

scholarly journals Direct Regulation of Mitochondrial RNA Synthesis by Thyroid Hormone

1999 ◽  
Vol 19 (1) ◽  
pp. 657-670 ◽  
Author(s):  
José A. Enríquez ◽  
Patricio Fernández-Silva ◽  
Nuria Garrido-Pérez ◽  
Manuel J. López-Pérez ◽  
Acisclo Pérez-Martos ◽  
...  
Keyword(s):  
Thyroid Hormone ◽  
Transcription Initiation ◽  
Dimethyl Sulfate ◽  
Nuclear Genes ◽  
Regulation Of Transcription ◽  
Mitochondrial Rna ◽  
Mitochondrial Transcription ◽  
In Organello

ABSTRACT We have analyzed the influence of in vivo treatment and in vitro addition of thyroid hormone on in organello mitochondrial DNA (mtDNA) transcription and, in parallel, on the in organello footprinting patterns at the mtDNA regions involved in the regulation of transcription. We found that thyroid hormone modulates mitochondrial RNA levels and the mRNA/rRNA ratio by influencing the transcriptional rate. In addition, we found conspicuous differences between the mtDNA dimethyl sulfate footprinting patterns of mitochondria derived from euthyroid and hypothyroid rats at the transcription initiation sites but not at the mitochondrial transcription termination factor (mTERF) binding region. Furthermore, direct addition of thyroid hormone to the incubation medium of mitochondria isolated from hypothyroid rats restored the mRNA/rRNA ratio found in euthyroid rats as well as the mtDNA footprinting patterns at the transcription initiation area. Therefore, we conclude that the regulatory effect of thyroid hormone on mitochondrial transcription is partially exerted by a direct influence of the hormone on the mitochondrial transcription machinery. Particularly, the influence on the mRNA/rRNA ratio is achieved by selective modulation of the alternative H-strand transcription initiation sites and does not require the previous activation of nuclear genes. These results provide the first functional demonstration that regulatory signals, such as thyroid hormone, that modify the expression of nuclear genes can also act as primary signals for the transcriptional apparatus of mitochondria.

Novel regulation of transcription initiation of the peptide IX gene of adenovirus 2

1989 ◽  
Vol 9 (10) ◽  
pp. 4265-4271
Author(s):  
T Matsui
Keyword(s):  
Transcriptional Repression ◽  
Transcription Initiation ◽  
Nuclear Extract ◽  
Negative Control ◽  
Tata Box ◽  
Regulation Of Transcription ◽  
Direct Initiation ◽  
Upstream Element

cis-Acting elements involved in transcription of the peptide IX (pIX) gene of adenovirus 2 were identified by using in vivo transient expression assays and two in vitro transcription systems. Deletion of either the sequence between positions -45 and -70 or the TATA box abolished the initiation of pIX gene transcription in vivo and transcription with HeLa cell nuclear extracts in vitro. These results initially suggested the presence of a positive factor acting on the upstream element. However, when proteins in the nuclear extract were fractionated by column chromatography and analyzed by reconstitution of transcription in vitro, it was found that a certain fraction could direct TATA box-dependent transcription initiation even in the absence of the upstream element. Furthermore, activity inhibiting TATA box-dependent transcription was found in the nuclear extract. In contrast, inhibition of TATA box-dependent transcription was suppressed by deletion of a downstream sequence between positions +33 and +122. These results indicate that the TATA box of the pIX gene by itself has the ability to direct initiation of constitutive transcription but that the function of this element is under negative control by a repressor acting on a downstream sequence. Thus, the upstream element of the pIX gene appears to have a novel function: suppression of the transcriptional repression exerted by a downstream sequence, leading to a net transcription activation. Possible mechanisms for transcription initiation of pIX DNA are discussed.

High resolution mapping of nucleic acid containing complexes in vitro and in situ

1996 ◽  
Vol 54 ◽  
pp. 48-49
Author(s):  
D. P. Bazett-Jones ◽  
M. J. Hendzel
Keyword(s):  
Nucleic Acid ◽  
High Resolution ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Heavy Atom ◽  
Regulation Of Transcription ◽  
High Exposure ◽  
Resolution Mapping ◽  
Tata Sequence

Structural analysis of combinations of nucleosomes and transcription factors on promoter and enhancer elements is necessary in order to understand the molecular mechanisms responsible for the regulation of transcription initiation. Such complexes are often not amenable to study by high resolution crystallographic techniques. We have been applying electron spectroscopic imaging (ESI) to specific problems in molecular biology related to transcription regulation. There are several advantages that this technique offers in studies of nucleoprotein complexes. First, an intermediate level of spatial resolution can be achieved because heavy atom contrast agents are not necessary. Second, mass and stoichiometric relationships of protein and nucleic acid can be estimated by phosphorus detection, an element in much higher proportions in nucleic acid than protein. Third, wrapping or bending of the DNA by the protein constituents can be observed by phosphorus mapping of the complexes. Even when ESI is used with high exposure of electrons to the specimen, important macromolecular information may be provided. For example, an image of the TATA binding protein (TBP) bound to DNA is shown in the Figure (top panel). It can be seen that the protein distorts the DNA away from itself and much of its mass sits off the DNA helix axis. Moreover, phosphorus and mass estimates demonstrate whether one or two TBP molecules interact with this particular promoter TATA sequence.

scholarly journals Interactions between RNA polymerase and the core recognition element are a determinant of transcription start site selection

2016 ◽  
Vol 113 (21) ◽  
pp. E2899-E2905 ◽  
Author(s):  
Irina O. Vvedenskaya ◽  
Hanif Vahedian-Movahed ◽  
Yuanchao Zhang ◽  
Deanne M. Taylor ◽  
Richard H. Ebright ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Transcription Start Site ◽  
Transcription Initiation ◽  
Specific Protein ◽  
Start Site ◽  
Transcription Start ◽  
Transcription Bubble ◽  
Recognition Element

During transcription initiation, RNA polymerase (RNAP) holoenzyme unwinds ∼13 bp of promoter DNA, forming an RNAP-promoter open complex (RPo) containing a single-stranded transcription bubble, and selects a template-strand nucleotide to serve as the transcription start site (TSS). In RPo, RNAP core enzyme makes sequence-specific protein–DNA interactions with the downstream part of the nontemplate strand of the transcription bubble (“core recognition element,” CRE). Here, we investigated whether sequence-specific RNAP–CRE interactions affect TSS selection. To do this, we used two next-generation sequencing-based approaches to compare the TSS profile of WT RNAP to that of an RNAP derivative defective in sequence-specific RNAP–CRE interactions. First, using massively systematic transcript end readout, MASTER, we assessed effects of RNAP–CRE interactions on TSS selection in vitro and in vivo for a library of 47 (∼16,000) consensus promoters containing different TSS region sequences, and we observed that the TSS profile of the RNAP derivative defective in RNAP–CRE interactions differed from that of WT RNAP, in a manner that correlated with the presence of consensus CRE sequences in the TSS region. Second, using 5′ merodiploid native-elongating-transcript sequencing, 5′ mNET-seq, we assessed effects of RNAP–CRE interactions at natural promoters in Escherichia coli, and we identified 39 promoters at which RNAP–CRE interactions determine TSS selection. Our findings establish RNAP–CRE interactions are a functional determinant of TSS selection. We propose that RNAP–CRE interactions modulate the position of the downstream end of the transcription bubble in RPo, and thereby modulate TSS selection, which involves transcription bubble expansion or transcription bubble contraction (scrunching or antiscrunching).

Analysis of Saccharomyces cerevisiae his3 transcription in vitro: biochemical support for multiple mechanisms of transcription

1990 ◽  
Vol 10 (6) ◽  
pp. 2832-2839
Author(s):  
A S Ponticelli ◽  
K Struhl
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Initiation Site ◽  
Activator Proteins ◽  
Nuclear Extracts ◽  
Transcription In Vitro

The promoter region of the Saccharomyces cerevisiae his3 gene contains two TATA elements, TC and TR, that direct transcription initiation to two sites designated +1 and +13. On the basis of differences between their nucleotide sequences and their responsiveness to upstream promoter elements, it has previously been proposed that TC and TR promote transcription by different molecular mechanisms. To begin a study of his3 transcription in vitro, we used S. cerevisiae nuclear extracts together with various DNA templates and transcriptional activator proteins that have been characterized in vivo. We demonstrated accurate transcription initiation in vitro at the sites used in vivo, transcriptional activation by GCN4, and activation by a GAL4 derivative on various gal-his3 hybrid promoters. In all cases, transcription stimulation was dependent on the presence of an acidic activation region in the activator protein. In addition, analysis of promoters containing a variety of TR derivatives indicated that the level of transcription in vitro was directly related to the level achieved in vivo. The results demonstrated that the in vitro system accurately reproduced all known aspects of in vivo his3 transcription that depend on the TR element. However, in striking contrast to his3 transcription in vivo, transcription in vitro yielded approximately 20 times more of the +13 transcript than the +1 transcript. This result was not due to inability of the +1 initiation site to be efficiently utilized in vitro, but rather it reflects the lack of TC function in vitro. The results support the idea that TC and TR mediate transcription from the wild-type promoter by distinct mechanisms.

Mechanism of initiator-mediated transcription: evidence for a functional interaction between the TATA-binding protein and DNA in the absence of a specific recognition sequence

1993 ◽  
Vol 13 (7) ◽  
pp. 3841-3849
Author(s):  
B Zenzie-Gregory ◽  
A Khachi ◽  
I P Garraway ◽  
S T Smale
Keyword(s):  
Transcription Initiation ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Upstream Region ◽  
Promoter Strength ◽  
Recognition Sequence ◽  
Specific Recognition ◽  
Rate Limiting

Promoters containing Sp1 binding sites and an initiator element but lacking a TATA box direct high levels of accurate transcription initiation by using a mechanism that requires the TATA-binding protein (TBP). We have begun to address the role of TBP during transcription from Sp1-initiator promoters by varying the nucleotide sequence between -14 and -33 relative to the start site. With each of several promoters containing different upstream sequences, we detected accurate transcription both in vitro and in vivo, but the promoter strengths varied widely, particularly with the in vitro assay. The variable promoter activities correlated with, but were not proportional to, the abilities of the upstream sequences to function as TATA boxes, as assessed by multiple criteria. These results confirm that accurate transcription can proceed in the presence of an initiator, regardless of the sequence present in the -30 region. However, the results reveal a role for this upstream region, most consistent with a model in which initiator-mediated transcription requires binding of TBP to the upstream DNA in the absence of a specific recognition sequence. Moreover, in vivo it appears that the promoter strength is modulated less severely by altering the -30 sequence, consistent with a previous suggestion that TBP is not rate limiting in vivo for TATA-less promoters. Taken together, these results suggest that variations in the structure of a core promoter might alter the rate-limiting step for transcription initiation and thereby alter the potential modes of transcriptional regulation, without severely changing the pathway used to assemble a functional preinitiation complex.

scholarly journals Interaction between Acetylated MyoD and the Bromodomain of CBP and/or p300

2001 ◽  
Vol 21 (16) ◽  
pp. 5312-5320 ◽  
Author(s):  
Anna Polesskaya ◽  
Irina Naguibneva ◽  
Arnaud Duquet ◽  
Eyal Bengal ◽  
Philippe Robin ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Protein Function ◽  
Live Cells ◽  
Regulation Of Transcription ◽  
Muscle Creatine Kinase ◽  
Nonhistone Protein ◽  
Myogenic Factor ◽  
First Time

ABSTRACT Acetylation is emerging as a posttranslational modification of nuclear proteins that is essential to the regulation of transcription and that modifies transcription factor affinity for binding sites on DNA, stability, and/or nuclear localization. Here, we present both in vitro and in vivo evidence that acetylation increases the affinity of myogenic factor MyoD for acetyltransferases CBP and p300. In myogenic cells, the fraction of endogenous MyoD that is acetylated was found associated with CBP or p300. In vitro, the interaction between MyoD and CBP was more resistant to high salt concentrations and was detected with lower doses of MyoD when MyoD was acetylated. Interestingly, an analysis of CBP mutants revealed that the interaction with acetylated MyoD involves the bromodomain of CBP. In live cells, MyoD mutants that cannot be acetylated did not associate with CBP or p300 and were strongly impaired in their ability to cooperate with CBP for transcriptional activation of a muscle creatine kinase-luciferase construct. Taken together, our data suggest a new mechanism for activation of protein function by acetylation and demonstrate for the first time an acetylation-dependent interaction between the bromodomain of CBP and a nonhistone protein.

Sign in / Sign up

Export Citation Format

Share Document