scholarly journals The effect of macromolecular crowding on single-round transcription by E. coli RNA polymerase

2017 ◽  
Author(s):  
SangYoon Chung ◽  
Eitan Lerner ◽  
Yan Jin ◽  
Soohong Kim ◽  
Yazan Alhadid ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Transcription Initiation ◽  
Macromolecular Crowding ◽  
Reaction Rates ◽  
Scaled Particle Theory ◽  
Hard Sphere Model ◽  
Enzymatic Reactions ◽  
Crowding Effects ◽  
Cellular Environment

ABSTRACTBiological reactions in the cellular environment differ physicochemically from those performed in dilute buffer solutions due to, in part, slower diffusion of various components in the cellular milieu, increase in their chemical activities, and modulation of their binding affinities and conformational stabilities.In vivotranscription is therefore expected to be strongly influenced by the ‘crowdedness’ of the cell. Previous studies of transcription under macromolecular crowding conditions have focused mainly on multiple cycles of RNAP-Promoter associations, assuming that the association is the rate-determining step of the entire transcription process. However, recent reports demonstrated that late initiation and promoter escape could be the rate-determining steps for some promoter DNA sequences. The investigation of crowding effects on these steps undersingle-roundconditions is therefore crucial for better understanding of transcription initiationin vivo. Here, we have implemented anin vitrotranscription quenched-kinetics single-molecule assay to investigate the dependence of transcription reaction rates on the sizes and concentrations of crowders. Our results demonstrate an expected slowdown of transcription kinetics due to increased viscosity, and an unexpected enhancement in transcription kinetics by large crowding agents (at a given viscosity). More importantly, the enhancement’s dependence on crowder size significantly deviates from hard-sphere model (scaled-particle theory) predictions, commonly used for description of crowding effects. Our findings shed new light on how enzymatic reactions are affected by crowding conditions in the cellular milieu.

Spacer promoters are orientation-dependent activators of pre-rRNA transcription in Drosophila melanogaster

1990 ◽  
Vol 10 (9) ◽  
pp. 4667-4677
Author(s):  
G Grimaldi ◽  
P Fiorentini ◽  
P P Di Nocera
Keyword(s):  
Drosophila Melanogaster ◽  
Dna Sequences ◽  
Transcription Initiation ◽  
Gene Promoter ◽  
Large Set ◽  
Gene Promoters ◽  
Stimulatory Action ◽  
Rrna Transcription ◽  
Tandem Arrays

In Drosophila melanogaster, 240-base-pair (bp) repeats, clustered in tandem arrays within the ribosomal DNA nontranscribed spacer region, include sites of RNA polymerase I-dependent transcription initiation and elements that stimulate the rate of transcription from the downstream precursor rRNA (pre-rRNA) promoter. We have analyzed the in vivo transcriptional activity of a large set of recombinant constructs in which tandem arrays of distinct segments derived from a 240-bp repeat were inserted upstream of the pre-rRNA promoter. The results indicate that activating spacer elements are confined to a region of 70 bp. Enhancing units overlap with spacer promoters, since DNA segments that stimulate transcription at the gene promoter also efficiently drive transcription initiation. The finding that artificial spacer arrays invariably stimulate pre-rRNA transcription initiation in an orientation-dependent fashion suggest that spacer-initiated transcription is involved in the enhancement process. The minimal spacer activating segment includes a perfect copy of a core domain of the gene promoter extending from -24 to +10 flanked by poorly homologous upstream DNA sequences. Spacer and gene promoters are functionally interchangeable as activating units. However, the different combination of DNA elements within the two determines a functional hierarchy, as only the pre-rRNA promoter is responsive to the stimulatory action of upstream units.

scholarly journals Spacer promoters are orientation-dependent activators of pre-rRNA transcription in Drosophila melanogaster.

1990 ◽  
Vol 10 (9) ◽  
pp. 4667-4677 ◽  
Author(s):  
G Grimaldi ◽  
P Fiorentini ◽  
P P Di Nocera
Keyword(s):  
Drosophila Melanogaster ◽  
Dna Sequences ◽  
Transcription Initiation ◽  
Gene Promoter ◽  
Large Set ◽  
Gene Promoters ◽  
Stimulatory Action ◽  
Rrna Transcription ◽  
Tandem Arrays

In Drosophila melanogaster, 240-base-pair (bp) repeats, clustered in tandem arrays within the ribosomal DNA nontranscribed spacer region, include sites of RNA polymerase I-dependent transcription initiation and elements that stimulate the rate of transcription from the downstream precursor rRNA (pre-rRNA) promoter. We have analyzed the in vivo transcriptional activity of a large set of recombinant constructs in which tandem arrays of distinct segments derived from a 240-bp repeat were inserted upstream of the pre-rRNA promoter. The results indicate that activating spacer elements are confined to a region of 70 bp. Enhancing units overlap with spacer promoters, since DNA segments that stimulate transcription at the gene promoter also efficiently drive transcription initiation. The finding that artificial spacer arrays invariably stimulate pre-rRNA transcription initiation in an orientation-dependent fashion suggest that spacer-initiated transcription is involved in the enhancement process. The minimal spacer activating segment includes a perfect copy of a core domain of the gene promoter extending from -24 to +10 flanked by poorly homologous upstream DNA sequences. Spacer and gene promoters are functionally interchangeable as activating units. However, the different combination of DNA elements within the two determines a functional hierarchy, as only the pre-rRNA promoter is responsive to the stimulatory action of upstream units.

scholarly journals Mutational Analysis of Escherichia coli Heat Shock Transcription Factor Sigma 32 Reveals Similarities with Sigma 70 in Recognition of the −35 Promoter Element and Differences in Promoter DNA Melting and −10 Recognition

2005 ◽  
Vol 187 (19) ◽  
pp. 6762-6769 ◽  
Author(s):  
Olga V. Kourennaia ◽  
Laura Tsujikawa ◽  
Pieter L. deHaseth
Keyword(s):  
Escherichia Coli ◽  
Transcription Factor ◽  
Amino Acid ◽  
Heat Shock ◽  
Dna Sequences ◽  
Transcription Initiation ◽  
Amino Acid Sequences ◽  
Single Amino Acid ◽  
Promoter Dna

ABSTRACT Upon the exposure of Escherichia coli to high temperature (heat shock), cellular levels of the transcription factor σ32 rise greatly, resulting in the increased formation of the σ32 holoenzyme, which is capable of transcription initiation at heat shock promoters. Higher levels of heat shock proteins render the cell better able to cope with the effects of higher temperatures. To conduct structure-function studies on σ32 in vivo, we have carried out site-directed mutagenesis and employed a previously developed system involving σ32 expression from one plasmid and a β-galactosidase reporter gene driven by the σ32-dependent groE promoter on another in order to monitor the effects of single amino acid substitutions on σ32 activity. It was found that the recognition of the −35 region involves similar amino acid residues in regions 4.2 of E. coli σ32 and σ70. Three conserved amino acids in region 2.3 of σ32 were found to be only marginally important in determining activity in vivo. Differences between σ32 and σ70 in the effects of mutation in region 2.4 on the activities of the two sigma factors are consistent with the pronounced differences between both the amino acid sequences in this region and the recognized promoter DNA sequences.

scholarly journals Minor transcription initiation events indicate that both human mitochondrial promoters function bidirectionally.

1986 ◽  
Vol 6 (1) ◽  
pp. 294-301 ◽  
Author(s):  
D D Chang ◽  
J E Hixson ◽  
D A Clayton
Keyword(s):  
Mitochondrial Dna ◽  
Dna Sequences ◽  
Transcription Initiation ◽  
Site Directed Mutagenesis ◽  
Deletion Mapping ◽  
Rich Region ◽  
Human Mitochondrial Dna ◽  
Loop Region

Human mitochondrial DNA is transcribed from two distinct, strand-specific promoters located in the displacement loop region of the genome. The transcriptional control sequences identified by deletion mapping and site-directed mutagenesis studies span short regions surrounding the initiation sites and bear no obvious sequence homology to any nuclear or procaryotic promoters. In vitro transcription analyses also revealed several minor initiation sites that are characterized by a pyrimidine-rich region followed by a purine-rich region, a feature that is shared by the two major promoters. In this paper, we report a new class of minor promoters in human mitochondrial DNA. These minor promoters were localized to the same duplex DNA sequences that direct major transcriptional events, but they had transcriptional polarity opposite to that of the major promoters. Furthermore, nucleotide changes that affected the major form of transcription similarly affected transcription in the opposite direction. For one of these minor promoters, a corresponding in vivo RNA species initiating from the same site was identified. These observations indicate that the major transcriptional promoters in human mitochondria can function bidirectionally both in vivo and in vitro.

Minor transcription initiation events indicate that both human mitochondrial promoters function bidirectionally

1986 ◽  
Vol 6 (1) ◽  
pp. 294-301
Author(s):  
D D Chang ◽  
J E Hixson ◽  
D A Clayton
Keyword(s):  
Mitochondrial Dna ◽  
Dna Sequences ◽  
Transcription Initiation ◽  
Site Directed Mutagenesis ◽  
Deletion Mapping ◽  
Rich Region ◽  
Human Mitochondrial Dna ◽  
Loop Region

Human mitochondrial DNA is transcribed from two distinct, strand-specific promoters located in the displacement loop region of the genome. The transcriptional control sequences identified by deletion mapping and site-directed mutagenesis studies span short regions surrounding the initiation sites and bear no obvious sequence homology to any nuclear or procaryotic promoters. In vitro transcription analyses also revealed several minor initiation sites that are characterized by a pyrimidine-rich region followed by a purine-rich region, a feature that is shared by the two major promoters. In this paper, we report a new class of minor promoters in human mitochondrial DNA. These minor promoters were localized to the same duplex DNA sequences that direct major transcriptional events, but they had transcriptional polarity opposite to that of the major promoters. Furthermore, nucleotide changes that affected the major form of transcription similarly affected transcription in the opposite direction. For one of these minor promoters, a corresponding in vivo RNA species initiating from the same site was identified. These observations indicate that the major transcriptional promoters in human mitochondria can function bidirectionally both in vivo and in vitro.

Transcription factor/DNA interactions visualized by electron spectroscopic imaging

1992 ◽  
Vol 50 (1) ◽  
pp. 450-451
Author(s):  
David P. Bazett-Jones ◽  
Mark L. Brown
Keyword(s):  
Transcription Factor ◽  
Dna Sequences ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Phosphorus Content ◽  
Spectroscopic Imaging ◽  
Electron Spectroscopic Imaging ◽  
Dna Backbone ◽  
Two Parameters ◽  
High Level

A multisubunit RNA polymerase enzyme is ultimately responsible for transcription initiation and elongation of RNA, but recognition of the proper start site by the enzyme is regulated by general, temporal and gene-specific trans-factors interacting at promoter and enhancer DNA sequences. To understand the molecular mechanisms which precisely regulate the transcription initiation event, it is crucial to elucidate the structure of the transcription factor/DNA complexes involved. Electron spectroscopic imaging (ESI) provides the opportunity to visualize individual DNA molecules. Enhancement of DNA contrast with ESI is accomplished by imaging with electrons that have interacted with inner shell electrons of phosphorus in the DNA backbone. Phosphorus detection at this intermediately high level of resolution (≈lnm) permits selective imaging of the DNA, to determine whether the protein factors compact, bend or wrap the DNA. Simultaneously, mass analysis and phosphorus content can be measured quantitatively, using adjacent DNA or tobacco mosaic virus (TMV) as mass and phosphorus standards. These two parameters provide stoichiometric information relating the ratios of protein:DNA content.

scholarly journals DNA thermodynamics shape chromosome organization and topology

2013 ◽  
Vol 41 (2) ◽  
pp. 548-553 ◽  
Author(s):  
Andrew A. Travers ◽  
Georgi Muskhelishvili
Keyword(s):  
Dna Sequence ◽  
Dna Sequences ◽  
Chromosome Organization ◽  
Topological Properties ◽  
Genetic Organization ◽  
And Topology ◽  
Dna Translocases

How much information is encoded in the DNA sequence of an organism? We argue that the informational, mechanical and topological properties of DNA are interdependent and act together to specify the primary characteristics of genetic organization and chromatin structures. Superhelicity generated in vivo, in part by the action of DNA translocases, can be transmitted to topologically sensitive regions encoded by less stable DNA sequences.

scholarly journals Adipose and Muscle Cell Co-Culture System: A Novel In Vitro Tool to Mimic the In Vivo Cellular Environment

Biology ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 6
Author(s):  
Palaniselvam Kuppusamy ◽  
Dahye Kim ◽  
Ilavenil Soundharrajan ◽  
Inho Hwang ◽  
Ki Choon Choi
Keyword(s):  
Drug Development ◽  
Culture System ◽  
Cell Types ◽  
Culture Cell ◽  
In Vitro Techniques ◽  
Culture Systems ◽  
Complex Interactions ◽  
Cellular Environment

A co-culture system allows researchers to investigate the complex interactions between two cell types under various environments, such as those that promote differentiation and growth as well as those that mimic healthy and diseased states, in vitro. In this paper, we review the most common co-culture systems for myocytes and adipocytes. The in vitro techniques mimic the in vivo environment and are used to investigate the causal relationships between different cell lines. Here, we briefly discuss mono-culture and co-culture cell systems and their applicability to the study of communication between two or more cell types, including adipocytes and myocytes. Also, we provide details about the different types of co-culture systems and their applicability to the study of metabolic disease, drug development, and the role of secretory factors in cell signaling cascades. Therefore, this review provides details about the co-culture systems used to study the complex interactions between adipose and muscle cells in various environments, such as those that promote cell differentiation and growth and those used for drug development.

scholarly journals Macromolecular Crowding Effects on Reactions of TePixD (Tll0078)†

2010 ◽  
Vol 87 (3) ◽  
pp. 584-589 ◽  
Author(s):  
Tsuguyoshi Toyooka ◽  
Keisuke Tanaka ◽  
Koji Okajima ◽  
Masahiko Ikeuchi ◽  
Satoru Tokutomi ◽  
...  
Keyword(s):  
Macromolecular Crowding ◽  
Crowding Effects

scholarly journals A 5′ Splice Site Enhances the Recruitment of Basal Transcription Initiation Factors In Vivo

2008 ◽  
Vol 29 (2) ◽  
pp. 271-278 ◽  
Author(s):  
Christian Kroun Damgaard ◽  
Søren Kahns ◽  
Søren Lykke-Andersen ◽  
Anders Lade Nielsen ◽  
Torben Heick Jensen ◽  
...  
Keyword(s):  
Splice Site ◽  
Transcription Initiation ◽  
Basal Transcription ◽  
Initiation Factors
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