scholarly journals Chromatin Remodeling around Nucleosome-Free Regions Leads to Repression of Noncoding RNA Transcription

2010 ◽  
Vol 30 (21) ◽  
pp. 5110-5122 ◽  
Author(s):  
Adam N. Yadon ◽  
Daniel Van de Mark ◽  
Ryan Basom ◽  
Jeffrey Delrow ◽  
Iestyn Whitehouse ◽  
...  
Keyword(s):  
Chromatin Remodeling ◽  
Transcription Initiation ◽  
Noncoding Rna ◽  
Open Reading Frames ◽  
Yeast Genome ◽  
Transcription Start Sites ◽  
Transcriptional Regulatory ◽  
First Time ◽  
Reading Frames

ABSTRACT Nucleosome-free regions (NFRs) at the 5′ and 3′ ends of genes are general sites of transcription initiation for mRNA and noncoding RNA (ncRNA). The presence of NFRs within transcriptional regulatory regions and the conserved location of transcription start sites at NFRs strongly suggest that the regulation of NFRs profoundly affects transcription initiation. To date, multiple factors are known to facilitate transcription initiation by positively regulating the formation and/or size of NFRs in vivo. However, mechanisms to repress transcription by negatively regulating the size of NFRs have not been identified. We identified four distinct classes of NFRs located at the 5′ and 3′ ends of genes, within open reading frames (ORFs), and far from ORFs. The ATP-dependent chromatin-remodeling enzyme Isw2 was found enriched at all classes of NFRs. Analysis of RNA levels also demonstrated Isw2 is required to repress ncRNA transcription from many of these NFRs. Thus, by the systematic annotation of NFRs across the yeast genome and analysis of ncRNA transcription, we established, for the first time, a mechanism by which NFR size is negatively regulated to repress ncRNA transcription from NFRs. Finally, we provide evidence suggesting that one biological consequence of repression of ncRNA, by Isw2 or by the exosome, is prevention of transcriptional interference of mRNA.

scholarly journals Independent regulation of H-NS-mediated silencing of the bgl operon at two levels: upstream by BglJ and LeuO and downstream by DnaKJ

Microbiology ◽  
2005 ◽  
Vol 151 (10) ◽  
pp. 3349-3359 ◽  
Author(s):  
S. Madhusudan ◽  
Andreas Paukner ◽  
Yvonne Klingen ◽  
Karin Schnetz
Keyword(s):  
Escherichia Coli ◽  
Transcription Initiation ◽  
Promoter Activity ◽  
Transposon Mutagenesis ◽  
Open Reading Frames ◽  
Wild Type ◽  
Coding Region ◽  
First Time ◽  
Insertion Mutations ◽  
Reading Frames

Silencing of the Escherichia coli bgl operon by the histone-like nucleoid-structuring protein H-NS occurs at two levels. Binding of H-NS upstream of the promoter represses transcription initiation, whilst binding within the coding region is also proposed to repress transcription elongation. The latter, downstream level of repression is counteracted by the protease Lon and, thus, silencing of the bgl operon is more effective in lon mutants. Transposon-mutagenesis screens for suppression of this lon phenotype on bgl were performed and insertion mutations disrupting rpoS and crl were obtained, as well as mutations mapping upstream of the open reading frames of bglJ, leuO and dnaK. In rpoS and crl mutants, bgl promoter activity is known to be higher. Likewise, as shown here, bgl promoter activity is increased in the bglJ and leuO mutants, which express BglJ and LeuO constitutively. However, BglJ and LeuO have no impact on downstream repression. A dnaKJ mutant was isolated for the first time in the context of the bgl operon. The mutant expresses lower levels of DnaK than the wild-type. Interestingly, in this dnaKJ : : miniTn10 mutant, downstream repression of bgl by H-NS is less effective, whilst upstream repression by H-NS remains unaffected. Together, the data show that the two levels of bgl silencing by H-NS are regulated independently.

scholarly journals Violaxanthin conversion by recombinant diatom and plant de-epoxidases, expressed in Escherichia coli – comparative analysis

2019 ◽  
Author(s):  
Monika Olchawa-Pajor ◽  
Monika Bojko ◽  
Wojciech Strzałka ◽  
Kazimierz Strzałka ◽  
Dariusz Latowski
Keyword(s):  
Escherichia Coli ◽  
Open Reading Frames ◽  
Amino Acid Residues ◽  
Metal Affinity ◽  
Comparable Activity ◽  
First Time ◽  
Kinetics Of ◽  
Reading Frames

The purpose of this research was to obtain recombinant violaxanthin de-epoxidases (VDEs) from two species. The first one was VDE of Arabidopsis thaliana (L.) Heynh. (WT Columbia strain) (AtVDE) which in vivo catalyzes conversion of violaxanthin (Vx) to zeaxanthin (Zx) via anteraxanthin (Ax). The second one was VDE of Phaeodactylum tricornutum Bohlin, 1897 (CCAP 1055/1 strain) (PtVDE) which is responsible for de-epoxidation of diadinoxanthin (Ddx) to diatoxanthin (Dtx). As the first step of our experiments, open reading frames coding for studied enzymes were amplified and subsequently cloned into pET-15b plasmid. For recombinant proteins production Escherichia coli Origami b strain was used. The molecular weight of the produced enzymes were estimated approximately at 45kDa and 50kDa for AtVDE and PtVDE, respectively. Both enzymes, purified under native conditions by immobilized metal affinity chromatography, displayed comparable activity in assay mixture and converted up to 90% Vx in 10 min in two steps enzymatic de-epoxidation, irrespective of enzyme origin. No statistically significant differences were observed when kinetics of the reactions catalyzed by these enzymes were compared. Putative role of selected amino-acid residues of AtVDE and PtVDE was also considered. The significance of the first time obtained recombinant PtVDE as a useful tool in various comparative investigations of de-epoxidation reactions in main types of xanthophyll cycles existing in nature are also indicated.

scholarly journals Nucleosome Remodeling and Transcriptional Repression Are Distinct Functions of Isw1 in Saccharomyces cerevisiae

2009 ◽  
Vol 29 (9) ◽  
pp. 2419-2430 ◽  
Author(s):  
Marina Pinskaya ◽  
Anitha Nair ◽  
David Clynes ◽  
Antonin Morillon ◽  
Jane Mellor
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromatin Remodeling ◽  
Transcriptional Repression ◽  
Transcription Initiation ◽  
Copy Number ◽  
Regulatory Proteins ◽  
Nucleosome Remodeling ◽  
Transcriptional Regulatory ◽  
Sant Domain

ABSTRACT The SANT domain is a nucleosome recognition module found in transcriptional regulatory proteins, including chromatin-modifying enzymes. It shows high functional degeneracy between species, varying in sequence and copy number. Here, we investigate functions in vivo associated with two SANT motifs, SANT and SLIDE, in the Saccharomyces cerevisiae Isw1 chromatin-remodeling ATPase. We show that differences in the primary structures of the SANT and SLIDE domains in yeast and Drosophila melanogaster reflect their different functions. In yeast, the SLIDE domain is required for histone interactions, while this is a function of the SANT domain in flies. In yeast, both motifs are required for optimal association with chromatin and for formation of the Isw1b complex (Isw1, Ioc2, and Ioc4). Moreover, nucleosome remodeling at the MET16 locus is defective in strains lacking the SANT or SLIDE domain. In contrast, the SANT domain is dispensable for the interaction between Isw1 and Ioc3 in the Isw1a complex. We show that, although defective in nucleosome remodeling, Isw1 lacking the SANT domain is able to repress transcription initiation at the MET16 promoter. Thus, chromatin remodeling and transcriptional repression are distinct activities of Isw1.

scholarly journals Bias between the Left and Right Inverted Repeats during IS911 Targeted Insertion

2008 ◽  
Vol 190 (18) ◽  
pp. 6111-6118 ◽  
Author(s):  
P. Rousseau ◽  
C. Loot ◽  
C. Turlan ◽  
S. Nolivos ◽  
M. Chandler
Keyword(s):  
Insertion Sequence ◽  
Regulatory Protein ◽  
Open Reading Frames ◽  
Inverted Repeats ◽  
Functional Differences ◽  
Left And Right ◽  
Ordered Assembly ◽  
Overlapping Open Reading Frames ◽  
Reading Frames

ABSTRACT IS911 is a bacterial insertion sequence composed of two consecutive overlapping open reading frames (ORFs [orfA and orfB]) encoding the transposase (OrfAB) as well as a regulatory protein (OrfA). These ORFs are bordered by terminal left and right inverted repeats (IRL and IRR, respectively) with several differences in nucleotide sequence. IS911 transposition is asymmetric: each end is cleaved on one strand to generate a free 3′-OH, which is then used as the nucleophile in attacking the opposite insertion sequence (IS) end to generate a free IS circle. This will be inserted into a new target site. We show here that the ends exhibit functional differences which, in vivo, may favor the use of one compared to the other during transposition. Electromobility shift assays showed that a truncated form of the transposase [OrfAB(1-149)] exhibits higher affinity for IRR than for IRL. While there was no detectable difference in IR activities during the early steps of transposition, IRR was more efficient during the final insertion steps. We show here that the differential activities between the two IRs correlate with the different affinities of OrfAB(1-149) for the IRs during assembly of the nucleoprotein complexes leading to transposition. We conclude that the two inverted repeats are not equivalent during IS911 transposition and that this asymmetry may intervene to determine the ordered assembly of the different protein-DNA complexes involved in the reaction.

scholarly journals Genomic Sequence of Rhesus Cytomegalovirus 180.92: Insights into the Coding Potential of Rhesus Cytomegalovirus

2006 ◽  
Vol 80 (8) ◽  
pp. 4179-4182 ◽  
Author(s):  
Pierre Rivailler ◽  
Amitinder Kaur ◽  
R. Paul Johnson ◽  
Fred Wang
Keyword(s):  
Genomic Sequence ◽  
Open Reading Frames ◽  
Rhesus Cytomegalovirus ◽  
Human Cmv ◽  
Coding Capacity ◽  
Coding Potential ◽  
Reading Frames ◽  
Genetic Rearrangements

ABSTRACT A pathogenic isolate of rhesus cytomegalovirus (rhCMV 180.92) was cloned, sequenced, and annotated. Comparisons with the published rhCMV 68.1 genome revealed 8 open reading frames (ORFs) in isolate 180.92 that are absent in 68.1, 10 ORFs in 68.1 that are absent in 180.92, and 34 additional ORFs that were not previously annotated. Most of the differences appear to be due to genetic rearrangements in both isolates from a region that is frequently altered in human CMV (hCMV) during in vitro passage. These results indicate that the rhCMV ORF repertoire is larger than previously recognized. Like hCMV, understanding of the complete coding capacity of rhCMV is complicated by genomic instability and may require comparisons with additional isolates in vitro and in vivo.

The first and fourth upstream open reading frames in GCN4 mRNA have similar initiation efficiencies but respond differently in translational control to change in length and sequence

1988 ◽  
Vol 8 (12) ◽  
pp. 5439-5447
Author(s):  
P P Mueller ◽  
B M Jackson ◽  
P F Miller ◽  
A G Hinnebusch
Keyword(s):  
Translational Control ◽  
Normal Growth ◽  
Open Reading Frames ◽  
Regulatory Function ◽  
Coding Sequences ◽  
Lacz Fusions ◽  
Upstream Open Reading Frames ◽  
Reading Frames ◽  
Starvation Conditions

The third and fourth AUG codons in GCN4 mRNA efficiently repress translation of the GCN4-coding sequences under normal growth conditions. The first AUG codon is approximately 30-fold less inhibitory and is required under amino acid starvation conditions to override the repressing effects of AUG codons 3 and 4. lacZ fusions constructed to functional, elongated versions of the first and fourth upstream open reading frames (URFs) were used to show that AUG codons 1 and 4 function similarly as efficient translational start sites in vivo, raising the possibility that steps following initiation distinguish the regulatory properties of URFs 1 and 4. In accord with this idea, we observed different consequences of changing the length and termination site of URF1 versus changing those of URFs 3 and 4. The latter were lengthened considerably, with little or no effect on regulation. In fact, the function of URFs 3 and 4 was partially reconstituted with a completely heterologous URF. By contrast, certain mutations that lengthen URF1 impaired its positive regulatory function nearly as much as removing its AUG codon did. The same mutations also made URF1 a much more inhibitory element when it was present alone in the mRNA leader. These results strongly suggest that URFs 1 and 4 both function in regulation as translated coding sequences. To account for the phenotypes of the URF1 mutations, we suggest the most ribosomes normally translate URF1 and that the mutations reduce the number of ribosomes that are able to complete URF1 translation and resume scanning downstream. This effect would impair URF1 positive regulatory function if ribosomes must first translate URF1 in order to overcome the strong translational block at the 3'-proximal URFs. Because URF1-lacZ fusions were translated at the same rate under repressing and derepressing conditions, it appears that modulating initiation at URF1 is not the means that is used to restrict the regulatory consequences of URF1 translation to starvation conditions.

E1−E4+ Adenoviral Gene Transfer Vectors Function as a “Pro-Life” Signal to Promote Survival of Primary Human Endothelial Cells

Blood ◽  
1999 ◽  
Vol 93 (9) ◽  
pp. 2936-2944 ◽  
Author(s):  
Ramachandran Ramalingam ◽  
Shahin Rafii ◽  
Stefan Worgall ◽  
Douglas E. Brough ◽  
Ronald G. Crystal
Keyword(s):  
Endothelial Cells ◽  
Open Reading Frames ◽  
Adenoviral Gene Transfer ◽  
Vector Genome ◽  
Transfer Vectors ◽  
Reading Frames ◽  
Human Endothelium ◽  
Pro Life

Abstract Although endothelial cells are quiescent and long-lived in vivo, when they are removed from blood vessels and cultured in vitro they die within days to weeks. In studies of the interaction of E1−E4+ replication–deficient adenovirus (Ad) vectors and human endothelium, the cells remained quiescent and were viable for prolonged periods. Evaluation of these cultures showed that E1−E4+ Ad vectors provide an “antiapoptotic” signal that, in association with an increase in the ratio of Bcl2 to Bax levels, induces the endothelial cells to enter a state of “suspended animation,” remaining viable for at least 30 days, even in the absence of serum and growth factors. Although the mechanisms initiating these events are unclear, the antiapoptoic signal requires the presence of E4 genes in the vector genome, suggesting that one or more E4 open reading frames of subgroup C Ad initiate a “pro-life” program that modifies cultured endothelial cells to survive for prolonged periods.

scholarly journals Characterization of Endogenous Retroviruses in Sheep

2003 ◽  
Vol 77 (20) ◽  
pp. 11268-11273 ◽  
Author(s):  
Nikolai Klymiuk ◽  
Mathias Müller ◽  
Gottfried Brem ◽  
Bernhard Aigner
Keyword(s):  
Endogenous Retrovirus ◽  
Ovis Aries ◽  
Endogenous Retroviruses ◽  
Open Reading Frames ◽  
In Vivo Experiments ◽  
Pregnant Sheep ◽  
Reading Frames

ABSTRACT Endogenous retrovirus (ERV) sequences have been found in all mammals. In vitro and in vivo experiments revealed ERV activation and cross-species infection in several species. Sheep (Ovis aries) are used for various biotechnological purposes; however, they have not yet been comprehensively screened for ERV sequences. Therefore, the aim of the study was to classify the ERV sequences in the ovine genome (OERV) by analyzing the retroviral pro-pol sequences. Three OERV β families and nine OERV γ families were revealed. Novel open reading frames (ORF) in the amplified proviral fragment were found in one OERV β family and two OERV γ families. Hybrid OERV produced by putative recombination events were not detected. Quantitative analysis of the OERV sequences in the ovine genome revealed no relevant variations in the endogenous retroviral loads of different breeds. Expression analysis of different tissues from fetal and pregnant sheep detected mRNA from both gammaretrovirus families, showing ORF fragments. Thus, the release of retroviruses from sheep cells cannot be excluded.

scholarly journals Accurate detection of short and long active ORFs using Ribo-seq data

2019 ◽  
Vol 36 (7) ◽  
pp. 2053-2059 ◽  
Author(s):  
Saket Choudhary ◽  
Wenzheng Li ◽  
Andrew D. Smith
Keyword(s):  
Caenorhabditis Elegans ◽  
Deep Sequencing ◽  
Open Reading Frames ◽  
Supplementary Information ◽  
Supplementary Data ◽  
Accurate Detection ◽  
Short Orfs ◽  
Coding Potential ◽  
Reading Frames

Abstract Motivation Ribo-seq, a technique for deep-sequencing ribosome-protected mRNA fragments, has enabled transcriptome-wide monitoring of translation in vivo. It has opened avenues for re-evaluating the coding potential of open reading frames (ORFs), including many short ORFs that were previously presumed to be non-translating. However, the detection of translating ORFs, specifically short ORFs, from Ribo-seq data, remains challenging due to its high heterogeneity and noise. Results We present ribotricer, a method for detecting actively translating ORFs by directly leveraging the three-nucleotide periodicity of Ribo-seq data. Ribotricer demonstrates higher accuracy and robustness compared with other methods at detecting actively translating ORFs including short ORFs on multiple published datasets across species inclusive of Arabidopsis, Caenorhabditis elegans, Drosophila, human, mouse, rat, yeast and zebrafish. Availability and implementation Ribotricer is available at https://github.com/smithlabcode/ribotricer. All analysis scripts and results are available at https://github.com/smithlabcode/ribotricer-results. Supplementary information Supplementary data are available at Bioinformatics online.

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