scholarly journals RNA Processing of Nitrogenase Transcripts in the Cyanobacterium Anabaena variabilis

2010 ◽  
Vol 192 (13) ◽  
pp. 3311-3320 ◽  
Author(s):  
Justin L. Ungerer ◽  
Brenda S. Pratte ◽  
Teresa Thiel
Keyword(s):  
Transcription Start Site ◽  
Transcription Initiation ◽  
De Novo ◽  
Start Site ◽  
Transcription Start ◽  
Stem Loop ◽  
Upstream Gene ◽  
Genes Encoding ◽  
Nitrogenase Genes ◽  
Cyanobacterium Anabaena

ABSTRACT Little is known about the regulation of nitrogenase genes in cyanobacteria. Transcription of the nifH1 and vnfH genes, encoding dinitrogenase reductases for the heterocyst-specific Mo-nitrogenase and the alternative V-nitrogenase, respectively, was studied by using a lacZ reporter. Despite evidence for a transcription start site just upstream of nifH1 and vnfH, promoter fragments that included these start sites did not drive the transcription of lacZ and, for nifH1, did not drive the expression of nifHDK1. Further analysis using larger regions upstream of nifH1 indicated that a promoter within nifU1 and a promoter upstream of nifB1 both contributed to expression of nifHDK1, with the nifB1 promoter contributing to most of the expression. Similarly, while the region upstream of vnfH, containing the putative transcription start site, did not drive expression of lacZ, the region that included the promoter for the upstream gene, ava4055, did. Characterization of the previously reported nifH1 and vnfH transcriptional start sites by 5′RACE (5′ rapid amplification of cDNA ends) revealed that these 5′ ends resulted from processing of larger transcripts rather than by de novo transcription initiation. The 5′ positions of both the vnfH and nifH1 transcripts lie at the base of a stem-loop structure that may serve to stabilize the nifHDK1 and vnfH specific transcripts compared to the transcripts for other genes in the operons providing the proper stoichiometry for the Nif proteins for nitrogenase synthesis.

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).

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.

scholarly journals The UP Element Is Necessary but Not Sufficient for Growth Rate-Dependent Control of the Escherichia coli guaB Promoter

2008 ◽  
Vol 190 (7) ◽  
pp. 2450-2457 ◽  
Author(s):  
Seyyed I. Husnain ◽  
Mark S. Thomas
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Transcription Start Site ◽  
De Novo ◽  
Start Site ◽  
Transcription Start ◽  
Lower Growth ◽  
Α Subunit ◽  
Rate Dependent ◽  
Up Elements

ABSTRACT The Escherichia coli guaB promoter (P guaB ) regulates the transcription of two genes, guaB and guaA, that are required for de novo synthesis of GMP, a precursor for the synthesis of guanine nucleoside triphosphates. The activity of P guaB is subject to growth rate-dependent control (GRDC). Here we show that the A+T-rich sequence located between positions −59 and −38 relative to the guaB transcription start site stimulates transcription from P guaB ∼8- to 10-fold and, in common with other UP elements, requires the C-terminal domain of the RNA polymerase α subunit for activity. Like the rrnB P1 UP element, the P guaB UP element contains two independently acting subsites located at positions −59 to −47 and −46 to −38 and can stimulate transcription when placed upstream of the lacP1 promoter. We reveal a novel role for the P guaB UP element by demonstrating that it is required for GRDC. The involvement of the UP element in GRDC also requires the participation of sequences located at least 100 bp upstream of the guaB transcription start site. These sequences are required for down-regulation of P guaB activity at lower growth rates.

scholarly journals Ebola Virus VP30-Mediated Transcription Is Regulated by RNA Secondary Structure Formation

2002 ◽  
Vol 76 (17) ◽  
pp. 8532-8539 ◽  
Author(s):  
Michael Weik ◽  
Jens Modrof ◽  
Hans-Dieter Klenk ◽  
Stephan Becker ◽  
Elke Mühlberger
Keyword(s):  
Secondary Structure ◽  
Transcription Start Site ◽  
Ebola Virus ◽  
Loop Structure ◽  
Start Site ◽  
Transcription Activator ◽  
Transcription Start ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Gene Start

ABSTRACT The nucleocapsid protein VP30 of Ebola virus (EBOV), a member of the Filovirus family, is known to act as a transcription activator. By using a reconstituted minigenome system, the role of VP30 during transcription was investigated. We could show that VP30-mediated transcription activation is dependent on formation of a stem-loop structure at the first gene start site. Destruction of this secondary structure led to VP30-independent transcription. Analysis of the transcription products of bicistronic minigenomes with and without the ability to form the secondary structure at the first transcription start signal revealed that transcription initiation at the first gene start site is a prerequisite for transcription of the second gene, independent of the presence of VP30. When the transcription start signal of the second gene was exchanged with the transcription start signal of the first gene, transcription of the second gene also was regulated by VP30, indicating that the stem-loop structure of the first transcription start site acts autonomously and independently of its localization on the RNA genome. Our results suggest that VP30 regulates a very early step of EBOV transcription, most likely by inhibiting pausing of the transcription complex at the RNA structure of the first transcription start site.

MOLECULAR CLONING OF THE HUMAN GENE FOR VON WILLEBRAND FACTOR AND IDENTIFICATION OF THE TRANSCRIPTION INITIATION SITE

1987 ◽  
Author(s):  
Corolyn J Collins ◽  
Richard B Levene ◽  
Christina P Ravera ◽  
Marker J Dombalagian ◽  
David M Livingston ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Transcription Start Site ◽  
Von Willebrand Factor ◽  
Transcription Initiation ◽  
Promoter Region ◽  
Start Site ◽  
Transcription Start ◽  
Von Willebrand's Disease ◽  
Von Willebrand ◽  
Willebrand Factor

Most patients with von Willebrand's disease appear to have a defect affecting the level of expression of the von Willebrand factor (vWf) gene. Thus, an understanding of the pathogenesis of von Willebrand's disease will require an analysis of the structure and function of the vWf gene in normals and in patients. To begin such analyses, we have screened a human genomic cosmid library with probes obtained from vWf cDNA and isolated a colinear segment spanning ≈175 kb in five overlapping clones. This segment extends ≈25 kb upstream and ≈5 kb downstream of the transcription start and stop sites for vWf mRNA, implying the vWf gene has a length of ≈150 kb. Within one of these clones, the vWf transcription initiation sites have been mapped. A portion of the promoter region has been sequenced, revealing a typical TATA box, a downstream CCAAT box, and a perfect downstream repeat of the 8 base pairs containing the major transcription start site. Primer extension analysis suggests that sequences contained within the downstream repeat of the transcription start site may be used as minor initiation sites in endothelial cells. Transfection studies are underway to evaluate the role of sequences within this promoter region in gene regulatory activity. Comparative restriction analyses of cloned and chromosomal DNA segments strongly suggests that no major alterations ocurred during cloning and that there is only one complete copy of the vWf gene in the human haploid genome. Similar analyses of DNA from vWf-expressing endothelial cells and non-expressing white blood cells suggests that no major rearrangements are associated with vWf gene expression. Finally, cross hybridization patterns among seven mammalian species suggests a strong conservation of genomic sequences encoding the plasma portion of vWf, but a lower degree of conservation of sequences encoding the N terminal region of provWf.

scholarly journals Transcription start site profiling uncovers divergent transcription and enhancer-associated RNAs in Drosophila melanogaster

2017 ◽  
Author(s):  
Michael P. Meers ◽  
Karen Adelman ◽  
Robert J. Duronio ◽  
Brian D. Strahl ◽  
Daniel J. McKay ◽  
...  
Keyword(s):  
Transcription Start Site ◽  
Transcription Initiation ◽  
Local Density ◽  
Global Analysis ◽  
Regulatory Elements ◽  
Start Site ◽  
Transcription Start ◽  
Vast Number ◽  
Animal Development ◽  
Divergent Transcription

AbstractBackgroundHigh-resolution transcription start site (TSS) mapping in D. melanogaster embryos and cell lines has revealed a rich and detailed landscape of both cis- and trans-regulatory elements and factors. However, TSS profiling has not been investigated in an orthogonal in vivo setting. Here, we present a comprehensive dataset that links TSS dynamics with nucleosome occupancy and gene expression in the wandering third instar larva, a developmental stage characterized by large-scale shifts in transcriptional programs in preparation for metamorphosis.ResultsThe data recapitulate major regulatory classes of TSSs, based on peak width, promoter-proximal polymerase pausing, and cis-regulatory element density. We confirm the paucity of divergent transcription units in D. melanogaster, but also identify notable exceptions. Furthermore, we identify thousands of novel initiation events occurring at unannotated TSSs that can be classified into functional categories by their local density of histone modifications. Interestingly, a sub-class of these unannotated TSSs overlaps with functionally validated enhancer elements, consistent with a regulatory role for “enhancer RNAs” (eRNAs) in defining transcriptional programs that are important for animal development.ConclusionsHigh-depth TSS mapping is a powerful strategy for identifying and characterizing low-abundance and/or low-stability RNAs. Global analysis of transcription initiation patterns in a developing organism reveals a vast number of novel initiation events that identify potential eRNAs as well as other non-coding transcripts critical for animal development.

The Mauriceville plasmid of Neurospora spp. uses novel mechanisms for initiating reverse transcription in vivo

1994 ◽  
Vol 14 (5) ◽  
pp. 3094-3107
Author(s):  
J C Kennell ◽  
H Wang ◽  
A M Lambowitz
Keyword(s):  
Reverse Transcriptase ◽  
Transcription Start Site ◽  
Reverse Transcription ◽  
De Novo ◽  
Minus Strand ◽  
Start Site ◽  
Cdna Synthesis ◽  
Transcription Start

The Mauriceville plasmid and the closely related Varkud plasmid of Neurospora spp. are retroelements that propagate in mitochondria. Replication appears to occur by a novel mechanism in which a monomer-length plasmid transcript having a 3' tRNA-like structure ending in CCA is reverse transcribed to give a full-length minus-strand cDNA beginning at or near the 3' end of the RNA. Here, we show that the plasmids are transcribed in vitro by the Neurospora mitochondrial RNA polymerase, with the major in vitro transcription start site approximately 260 bp upstream of the 5' end of the plasmid transcript. The location of the transcription start site suggests that the monomer-length transcripts are generated by transcription around the plasmid combined with a site-specific RNA cleavage after the 3'-CCA sequence. The 5' ends of minus-strand cDNAs in ribonucleoprotein particles were analyzed to obtain insight into the mechanism of initiation of reverse transcription in vivo. A major class of minus-strand cDNAs begins opposite C2 of the 3'-CCA sequence, the same site used for de novo initiation of cDNA synthesis by the plasmid reverse transcriptase in vitro. A second class of minus-strand cDNAs begins with putative primer sequences that correspond to cDNA copies of the plasmid or mitochondrial transcripts. These findings are consistent with the possibility that the plasmid reverse transcriptase initiates minus-strand cDNA synthesis in vivo both by de novo initiation and by a novel template-switching mechanism in which the 3' OH of a previously synthesized cDNA is used to prime the synthesis of a new minus-strand cDNA directly at the 3' end of the plasmid transcript.

scholarly journals The Critical cis-Acting Element Required for IMD2 Feedback Regulation by GDP Is a TATA Box Located 202 Nucleotides Upstream of the Transcription Start Site

2003 ◽  
Vol 23 (17) ◽  
pp. 6267-6278 ◽  
Author(s):  
Mafalda Escobar-Henriques ◽  
Bertrand Daignan-Fornier ◽  
Martine A. Collart
Keyword(s):  
Transcription Start Site ◽  
Transcription Initiation ◽  
Feedback Regulation ◽  
Initiation Site ◽  
Nutrient Sensing ◽  
Tata Box ◽  
Start Site ◽  
Sensing Element ◽  
Transcription Start ◽  
Nucleotide Synthesis

ABSTRACT Guanylic nucleotides are essential cellular players, and the critical enzyme in their tightly regulated synthesis in Saccharomyces cerevisiae is encoded by the IMD2 gene. The transcription of IMD2 is subject to general repression by nutrient limitation through the cis nutrient-sensing element. It is also subject to specific feedback regulation by the end products of the guanylic nucleotide synthesis pathway. The critical cis element for this latter mechanism is the guanine response element (GRE), a TATAATA sequence which is located 202 nucleotides upstream of the transcription initiation site and which functions as the IMD2 TATA box. We show that the GRE functions in conjunction with a 52-nucleotide stretch near the transcription start site. This very unusual promoter structure ensures low, basal expression of IMD2 and the recruitment of TFIID to the GRE in response to guanylic nucleotide limitation.

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