Combination of in vitro capping and ribonuclease protection improves the detection of transcription start sites in chloroplasts

1992 ◽  
Vol 19 (2) ◽  
pp. 309-311 ◽  
Author(s):  
Antonio Vera ◽  
Masahiro Sugiura
Keyword(s):  
Transcription Start ◽  
Transcription Start Sites ◽  
In Vitro Capping ◽  
Ribonuclease Protection

scholarly journals Bromo- and Extraterminal Domain Chromatin Regulators Serve as Cofactors for Murine Leukemia Virus Integration

2013 ◽  
Vol 87 (23) ◽  
pp. 12721-12736 ◽  
Author(s):  
Saumya Shree Gupta ◽  
Tobias Maetzig ◽  
Goedele N. Maertens ◽  
Azar Sharif ◽  
Michael Rothe ◽  
...  
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Retroviral Vectors ◽  
Transcription Start ◽  
Preintegration Complex ◽  
Transcription Start Sites ◽  
Bet Inhibitors ◽  
Murine Leukemia

Retroviral integrase (IN) proteins catalyze the permanent integration of proviral genomes into host DNA with the help of cellular cofactors. Lens epithelium-derived growth factor (LEDGF) is a cofactor for lentiviruses, including human immunodeficiency virus type 1 (HIV-1), and targets lentiviral integration toward active transcription units in the host genome. In contrast to lentiviruses, murine leukemia virus (MLV), a gammaretrovirus, tends to integrate near transcription start sites. Here, we show that the bromodomain and extraterminal domain (BET) proteins BRD2, BRD3, and BRD4 interact with gammaretroviral INs and stimulate the catalytic activity of MLV INin vitro. We mapped the interaction site to a characteristic structural feature within the BET protein extraterminal (ET) domain and to three amino acids in MLV IN. The ET domains of different BET proteins stimulate MLV integrationin vitroand, in the case of BRD2, alsoin vivo. Furthermore, two small-molecule BET inhibitors, JQ1 and I-BET, decrease MLV integration and shift it away from transcription start sites. Our data suggest that BET proteins might act as chromatin-bound acceptors for the MLV preintegration complex. These results could pave a way to redirecting MLV DNA integration as a basis for creating safer retroviral vectors.

scholarly journals Organization of the human β-1,2-N-acetylglucosaminyltransferase I gene (MGAT1), which controls complex and hybrid N-glycan synthesis

1997 ◽  
Vol 321 (2) ◽  
pp. 465-474 ◽  
Author(s):  
Betty YIP ◽  
Shi-Hao CHEN ◽  
Hans MULDER ◽  
Jo W. M. HÖPPENER ◽  
Harry SCHACHTER
Keyword(s):  
Genomic Dna ◽  
Transient Transfection ◽  
Transcription Start ◽  
Exon 2 ◽  
Transcription Start Sites ◽  
Exon 1 ◽  
Dna Fragment ◽  
Flanking Region ◽  
Ribonuclease Protection ◽  
I Gene

UDP-GlcNAc:α-3-d-mannoside α-1,2-N-acetylglucosaminyltransferase I (EC 2.4.1.101; GlcNAc-T I) is a medial-Golgi enzyme which catalyses the first step in the conversion of oligomannose-type to N-acetyl-lactosamine- and hybrid-type N-glycans and is essential for normal embryogenesis in the mouse. Previous work indicated the presence of at least two exons in the human GlcNAc-T I gene MGAT1, exon 2 containing part of the 5ƀ untranslated region and the complete coding and 3ƀ untranslated regions, and exon 1 with the remainder of the 5ƀ untranslated region. We now report the cloning and sequencing of a human genomic DNA fragment containing exon 1, which is between 5.6 and 15 kb upstream of exon 2. Transient transfection, ribonuclease protection and reverse transcriptase-mediated PCR indicated the absence of transcription start sites in intron 1 between exons 1 and 2. Northern analysis, ribonuclease protection, primer extension analysis and rapid amplification of 5ƀ-cDNA ends showed that there are multiple transcription start sites for exon 1 compatible with the expression by several human cell lines and tissues of two transcripts, a broad band ranging in size from 2.7 to 3.0 kb and a sharper band at 3.1 kb. The 5ƀ flanking region of exon 1 has a GC content of 81% and has no canonical TATA or CCAAT boxes but contains potential binding sites for transcription factors Sp1, GC-binding factor and epidermal growth factor receptor-specific transcription factor. Chloramphenicol acetyltransferase (CAT) expression was observed on transient transfection into HeLa cells of a fusion construct containing the gene for CAT and a genomic DNA fragment from the 5ƀ flanking region of exon 1. It is concluded that MGAT1 is a typical housekeeping gene although there is, in addition, tissue-specific expression of the larger 3.1 kb transcript.

scholarly journals ExsA Recruits RNA Polymerase to an Extended −10 Promoter by Contacting Region 4.2 of Sigma-70

2010 ◽  
Vol 192 (14) ◽  
pp. 3597-3607 ◽  
Author(s):  
Christopher A. Vakulskas ◽  
Evan D. Brutinel ◽  
Timothy L. Yahr
Keyword(s):  
Rna Polymerase ◽  
Binding Sites ◽  
Type Iii Secretion ◽  
Transcription Initiation ◽  
Additional Contribution ◽  
Transcription Start ◽  
Transcription Start Sites ◽  
Collective Data ◽  
Sigma 70

ABSTRACT ExsA is a member of the AraC family of transcriptional activators and is required for expression of the Pseudomonas aeruginosa type III secretion system (T3SS). ExsA-dependent promoters consist of two binding sites for monomeric ExsA located approximately 50 bp upstream of the transcription start sites. Binding to both sites is required for recruitment of σ70-RNA polymerase (RNAP) to the promoter. ExsA-dependent promoters also contain putative −35 hexamers that closely match the σ70 consensus but are atypically spaced 21 or 22 bp from the −10 hexamer. Because several nucleotides located within the putative −35 region are required for ExsA binding, it is unclear whether the putative −35 region makes an additional contribution to transcription initiation. In the present study we demonstrate that the putative −35 hexamer is dispensable for ExsA-independent transcription from the P exsC promoter and that deletion of σ70 region 4.2, which contacts the −35 hexamer, has no effect on ExsA-independent transcription from P exsC . Region 4.2 of σ70, however, is required for ExsA-dependent activation of the P exsC and P exsD promoters. Genetic data suggest that ExsA directly contacts region 4.2 of σ70, and several amino acids were found to contribute to the interaction. In vitro transcription assays demonstrate that an extended −10 element located in the P exsC promoter is important for overall promoter activity. Our collective data suggest a model in which ExsA compensates for the lack of a −35 hexamer by interacting with region 4.2 of σ70 to recruit RNAP to the promoter.

scholarly journals DNA-guided establishment of canonical nucleosome patterns in a eukaryotic genome

2014 ◽  
Author(s):  
Leslie Y Beh ◽  
Noam Kaplan ◽  
Manuel M Muller ◽  
Tom W Muir ◽  
Laura F Landweber
Keyword(s):  
Tetrahymena Thermophila ◽  
Gc Content ◽  
Transcription Start ◽  
Transcription Start Sites ◽  
Genome Wide ◽  
Nucleosome Formation ◽  
Eukaryotic Chromatin ◽  
Nucleosome Array

A conserved hallmark of eukaryotic chromatin architecture is the distinctive array of well-positioned nucleosomes downstream of transcription start sites (TSS). Recent studies indicate that trans-acting factors establish this stereotypical array. Here, we present the first genome-wide in vitro and in vivo nucleosome maps for the ciliate Tetrahymena thermophila. In contrast with previous studies in yeast, we find that the stereotypical nucleosome array is preserved in the in vitro reconstituted map, which is governed only by the DNA sequence preferences of nucleosomes. Remarkably, this average in vitro pattern arises from the presence of subsets of nucleosomes, rather than the whole array, in individual Tetrahymena genes. Variation in GC content contributes to the positioning of these sequence-directed nucleosomes, and affects codon usage and amino acid composition in genes. We propose that these ‘seed’ nucleosomes may aid the AT-rich Tetrahymena genome – which is intrinsically unfavorable for nucleosome formation – in establishing nucleosome arrays in vivo in concert with trans-acting factors, while minimizing changes to the coding sequences they are embedded within.

scholarly journals Extended Region of Nodulation Genes in Rhizobium meliloti 1021. II. Nucleotide Sequence, Transcription Start Sites and Protein Products

Genetics ◽  
1987 ◽  
Vol 117 (2) ◽  
pp. 191-201
Author(s):  
Robert F Fisher ◽  
Jean A Swanson ◽  
John T Mulligan ◽  
Sharon R Long
Keyword(s):  
Binding Site ◽  
Rhizobium Meliloti ◽  
Open Reading Frames ◽  
Translation System ◽  
Transcription Start ◽  
Nod Genes ◽  
Transcription Start Sites ◽  
Conserved Sequence ◽  
Reading Frames

ABSTRACT We have established the DNA sequence and analyzed the transcription and translation products of a series of putative nodulation (nod) genes in Rhizobium meliloti strain 1021. Four loci have been designated nodF, nodE, nodG and nodH. The correlation of transposon insertion positions with phenotypes and open reading frames was confirmed by sequencing the insertion junctions of the transposons. The protein products of these nod genes were visualized by in vitro expression of cloned DNA segments in a R. meliloti transcription-translation system. In addition, the sequence for nodG was substantiated by creating translational fusions in all three reading frames at several points in the sequence; the resulting fusions were expressed in vitro in both E. coli and R. meliloti transcription-translation systems. A DNA segment bearing several open reading frames downstream of nodG corresponds to the putative nod gene mutated in strain nod-216. The transcription start sites of nodF and nodH were mapped by primer extension of RNA from cells induced with the plant flavone, luteolin. Initiation of transcription occurs approximately 25 bp downstream from the conserved sequence designated the "nod box," suggesting that this conserved sequence acts as an upstream regulator of inducible nod gene expression. Its distance from the transcription start site is more suggestive of an activator binding site rather than an RNA polymerase binding site.

scholarly journals Transcriptional start site heterogeneity modulates the structure and function of the HIV-1 genome

2016 ◽  
Vol 113 (47) ◽  
pp. 13378-13383 ◽  
Author(s):  
Siarhei Kharytonchyk ◽  
Sarah Monti ◽  
Philip J. Smaldino ◽  
Verna Van ◽  
Nicholas C. Bolden ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Transcriptional Start Site ◽  
Structure And Function ◽  
Transcription Start ◽  
Transcription Start Sites ◽  
Hiv Type 1 ◽  
And Function ◽  
Hiv 1

The promoter in HIV type 1 (HIV-1) proviral DNA contains three sequential guanosines at the U3–R boundary that have been proposed to function as sites for transcription initiation. Here we show that all three sites are used in cells infected with HIV-1 and that viral RNAs containing a single 5′ capped guanosine (Cap1G) are specifically selected for packaging in virions, consistent with a recent report [Masuda et al. (2015)Sci Rep5:17680]. In addition, we now show that transcripts that begin with two or three capped guanosines (Cap2G orCap3G) are enriched on polysomes, indicating that RNAs synthesized from different transcription start sites have different functions in viral replication. Because genomes are selected for packaging as dimers, we examined the in vitro monomer–dimer equilibrium properties ofCap1G,Cap2G, andCap3G 5′-leader RNAs in the NL4-3 strain of HIV-1. Strikingly, under physiological-like ionic conditions in which theCap1G 5′-leader RNA adopts a dimeric structure, theCap2G andCap3G 5′-leader RNAs exist predominantly as monomers. Mutagenesis studies designed to probe for base-pairing interactions suggest that the additional guanosines of the 2G and 3G RNAs remodel the base of the PolyA hairpin, resulting in enhanced sequestration of dimer-promoting residues and stabilization of the monomer. Our studies suggest a mechanism through which the structure, function, and fate of the viral genome can be modulated by the transcriptionally controlled presence or absence of a single 5′ guanosine.

scholarly journals Nascent RNA sequencing identifies a widespread sigma70-dependent pausing regulated by Gre factors in bacteria

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zhe Sun ◽  
Alexander V. Yakhnin ◽  
Peter C. FitzGerald ◽  
Carl E. Mclntosh ◽  
Mikhail Kashlev
Keyword(s):  
Environmental Cues ◽  
Start Site ◽  
Transcription Start ◽  
Genome Wide Analysis ◽  
Genome Wide ◽  
Eukaryotic Gene Expression ◽  
Eukaryotic Gene ◽  
Vitro Analysis ◽  
In Vitro Analysis

AbstractPromoter-proximal pausing regulates eukaryotic gene expression and serves as checkpoints to assemble elongation/splicing machinery. Little is known how broadly this type of pausing regulates transcription in bacteria. We apply nascent elongating transcript sequencing combined with RNase I footprinting for genome-wide analysis of σ70-dependent transcription pauses in Escherichia coli. Retention of σ70 induces strong backtracked pauses at a 10−20-bp distance from many promoters. The pauses in the 10−15-bp register of the promoter are dictated by the canonical −10 element, 6−7 nt spacer and “YR+1Y” motif centered at the transcription start site. The promoters for the pauses in the 16−20-bp register contain an additional −10-like sequence recognized by σ70. Our in vitro analysis reveals that DNA scrunching is involved in these pauses relieved by Gre cleavage factors. The genes coding for transcription factors are enriched in these pauses, suggesting that σ70 and Gre proteins regulate transcription in response to changing environmental cues.

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