scholarly journals The in vitro transcription-translation of DNA and RNA templates by extracts of Rhodopseudomonas sphaeroides. Optimization and comparison of template specificity with Escherichia coli extracts and in vivo synthesis.

1982 ◽  
Vol 257 (24) ◽  
pp. 15110-15121
Author(s):  
J Chory ◽  
S Kaplan
Keyword(s):  
Escherichia Coli ◽  
In Vitro Transcription ◽  
Rhodopseudomonas Sphaeroides ◽  
Dna And Rna ◽  
Template Specificity ◽  
In Vivo Synthesis

scholarly journals Cyclic AMP Receptor Protein and RhaR Synergistically Activate Transcription from the l-Rhamnose-Responsive rhaSR Promoter in Escherichia coli

2005 ◽  
Vol 187 (19) ◽  
pp. 6708-6718 ◽  
Author(s):  
Jason R. Wickstrum ◽  
Thomas J. Santangelo ◽  
Susan M. Egan
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
In Vitro Transcription ◽  
Dnase I ◽  
Receptor Protein ◽  
Transcription Activator ◽  
Transcription Start ◽  
Dnase I Footprinting

ABSTRACT The Escherichia coli rhaSR operon encodes two AraC family transcription activator proteins, RhaS and RhaR, which regulate expression of the l-rhamnose catabolic regulon in response to l-rhamnose availability. RhaR positively regulates rhaSR in response to l-rhamnose, and RhaR activation can be enhanced by the cyclic AMP (cAMP) receptor protein (CRP) protein. CRP is a well-studied global transcription regulator that binds to DNA as a dimer and activates transcription in the presence of cAMP. We investigated the mechanism of CRP activation at rhaSR both alone and in combination with RhaR in vivo and in vitro. Base pair substitutions at potential CRP binding sites in the rhaSR-rhaBAD intergenic region demonstrate that CRP site 3, centered at position −111.5 relative to the rhaSR transcription start site, is required for the majority of the CRP-dependent activation of rhaSR. DNase I footprinting confirms that CRP binds to site 3; CRP binding to the other potential CRP sites at rhaSR was not detected. We show that, at least in vitro, CRP is capable of both RhaR-dependent and RhaR-independent activation of rhaSR from a total of three transcription start sites. In vitro transcription assays indicate that the carboxy-terminal domain of the alpha subunit (α-CTD) of RNA polymerase is at least partially dispensable for RhaR-dependent activation but that the α-CTD is required for CRP activation of rhaSR. Although CRP requires the presence of RhaR for efficient in vivo activation of rhaSR, DNase I footprinting assays indicated that cooperative binding between RhaR and CRP does not make a significant contribution to the mechanism of CRP activation at rhaSR. It therefore appears that CRP activates transcription from rhaSR as it would at simple class I promoters, albeit from a relatively distant position.

scholarly journals Nitrogen Regulation of the codBA (Cytosine Deaminase) Operon from Escherichia coli by the Nitrogen Assimilation Control Protein, NAC

2003 ◽  
Vol 185 (9) ◽  
pp. 2920-2926 ◽  
Author(s):  
Wilson B. Muse ◽  
Christopher J. Rosario ◽  
Robert A. Bender
Keyword(s):  
Escherichia Coli ◽  
Nitrogen Assimilation ◽  
Cytosine Deaminase ◽  
In Vitro Transcription ◽  
Dependent Manner ◽  
E Coli ◽  
Different Response ◽  
Control Protein

ABSTRACT Transcription of the cytosine deaminase (codBA) operon of Escherichia coli is regulated by nitrogen, with about three times more codBA expression in cells grown in nitrogen-limiting medium than in nitrogen-excess medium. β-Galactosidase expression from codBp-lacZ operon fusions showed that the nitrogen assimilation control protein NAC was necessary for this regulation. In vitro transcription from the codBA promoter with purified RNA polymerase was stimulated by the addition of purified NAC, confirming that no other factors are required. Gel mobility shifts and DNase I footprints showed that NAC binds to a site centered at position −59 relative to the start site of transcription and that mutants that cannot bind NAC there cannot activate transcription. When a longer promoter region (positions −120 to +67) was used, a double footprint was seen with a second 26-bp footprint separated from the first by a hypersensitive site. When a shorter fragment was used (positions −83 to +67), only the primary footprint was seen. Nevertheless, both the shorter and longer fragments showed NAC-mediated regulation in vivo. Cytosine deaminase expression in Klebsiella pneumoniae was also regulated by nitrogen in a NAC-dependent manner. K. pneumoniae differs from E. coli in having two cytosine deaminase genes, an intervening open reading frame between the codB and codA orthologs, and a different response to hypoxanthine which increased cod expression in K. pneumoniae but decreased it in E. coli.

Attenuation regulation in the thr operon of Escherichia coli K-12: molecular cloning and transcription of the controlling region

1982 ◽  
Vol 152 (1) ◽  
pp. 363-371
Author(s):  
S P Lynn ◽  
J F Gardner ◽  
W S Reznikoff
Keyword(s):  
Escherichia Coli ◽  
Transcription Termination ◽  
Regulatory Region ◽  
In Vitro Transcription ◽  
Regulatory Mutation ◽  
Rna Transcripts ◽  
In Vitro Transcripts ◽  
K 12

Recombinant plasmids were constructed which carry defined regions of the threonine (thr) operon regulatory region of Escherichia coli. In vitro transcription experiments utilizing plasmid or restriction fragment templates showed that two major RNA transcripts, which differ in length by one to a few bases, are transcribed from this region. The approximate length of the transcripts is 150 to 170 bases, and the site(s) of termination is near or within the thr attenuator. The efficiency of termination at the thr operon attenuator in vitro is approximately 90%. A regulatory mutation, thr79-20, which is a G-C insertion in the attenuator, reduces the frequency of transcription termination to 75%. In addition, in vivo RNA transcripts were identified which hybridize to the thr operon regulatory region. These transcripts appeared to be identical to the two major in vitro transcripts as judged by their mobilities on 8% polyacrylamide-8 M urea gels. This result indicates that the thr operon regulatory region is transcribed in vivo and that termination occurs near or within the thr attenuator.

scholarly journals An Antisense RNA-Mediated Transcriptional Attenuation Mechanism Functions in Escherichia coli

2002 ◽  
Vol 184 (10) ◽  
pp. 2740-2747 ◽  
Author(s):  
Sabine Brantl ◽  
E. Gerhart H. Wagner
Keyword(s):  
Escherichia Coli ◽  
Antisense Rna ◽  
In Vitro Transcription ◽  
Control Mode ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Antisense Rnas ◽  
Replication Control

ABSTRACT Antisense RNA-mediated transcriptional attenuation is a regulatory mechanism operating in the replication control of two groups of plasmids in gram-positive bacteria, the pT181 group and the inc18 family, represented by pIP501. In contrast, this control mechanism has so far not been identified in gram-negative bacteria or their plasmids. In this work we asked whether such a mechanism can be supported by Escherichia coli. The core replication control regions of plasmids pT181 and pIP501 were transferred into this heterologous host. In vivo lacZ reporter gene assays showed that the antisense RNAs of these plasmids can inhibit lacZ expression and that most of this effect can be accounted for by reduced mRNA readthrough. Northern analyses confirmed that the ratio of attenuated to readthrough target RNA was increased in the presence of the cognate antisense RNA, as expected for this mechanism. Similarly, both antisense RNAs induced premature termination of their cognate target RNAs in an E. coli in vitro transcription system, whereas the noncognate antisense RNAs had no effect. Thus, this report shows that antisense RNA-mediated transcriptional attenuation is supported by at least one gram-negative host, although the data indicate that inhibitory efficiencies are lower than those for, e.g., Bacillus subtilis. Possible explanations for the apparent absence of this control mode in plasmids of gram-negative bacteria are discussed.

scholarly journals RNA Polymerase α and ς70 Subunits Participate in Transcription of the Escherichia coli uhpT Promoter

1999 ◽  
Vol 181 (23) ◽  
pp. 7266-7273 ◽  
Author(s):  
Igor N. Olekhnovich ◽  
Robert J. Kadner
Keyword(s):  
Escherichia Coli ◽  
Transcription Factors ◽  
Rna Polymerase ◽  
Affect Regulation ◽  
Response Regulator ◽  
In Vitro Transcription ◽  
Receptor Protein ◽  
Bacterial Gene

ABSTRACT Fundamental questions in bacterial gene regulation concern how multiple regulatory proteins interact with the transcription apparatus at a single promoter and what are the roles of protein contacts with RNA polymerase and changes in DNA conformation. Transcription of theEscherichia coli uhpT gene, encoding the inducible sugar phosphate transporter, is dependent on the response regulator UhpA and is stimulated by the cyclic AMP receptor protein (CAP). UhpA binds to multiple sites in the uhpT promoter between positions −80 and −32 upstream of the transcription start site, and CAP binds to a single site centered at position −103.5. The role in uhpTtranscription of portions of RNA polymerase Eς70holoenzyme which affect regulation at other promoters was examined by using series of alanine substitutions throughout the C-terminal domains of RpoA (residues 255 to 329) and of RpoD (residues 570 to 613). Alanine substitutions that affected in vivo expression of auhpT-lacZ transcriptional fusion were tested for their effect on in vitro transcription activity by using reconstituted holoenzymes. Consistent with the binding of UhpA near the −35 region, residues K593 and K599 in the C-terminal region of RpoD were necessary for efficient uhpT expression in response to UhpA alone. Their requirement was overcome when CAP was also present. In addition, residues R265, G296, and S299 in the DNA-binding surface of the C-terminal domain of RpoA (αCTD) were important for uhpTtranscription even in the presence of CAP. Substitutions at several other positions had effects in cells but not during in vitro transcription with saturating levels of the transcription factors. Two DNase-hypersensitive sites near the upstream end of the UhpA-binding region were seen in the presence of all three transcription factors. Their appearance required functional αCTD but not the presence of upstream DNA. These results suggest that both transcription activators depend on or interact with different subunits of RNA polymerase, although their role in formation of proper DNA geometry may also be crucial.

Безопасность соединений из группы терпенов ментанового ряда in vitro и in vivo

2020 ◽  
Vol 83 (4) ◽  
pp. 24-30
Author(s):  
Ирина Владимировна Акулина ◽  
Светлана Ивановна Павлова ◽  
Ирина Семеновна Степаненко ◽  
Назира Сунагатовна Карамова ◽  
Александр Владиславович Сергеев ◽  
...  
Keyword(s):  
Escherichia Coli

Проведено токсикологическое исследование соединений с антибактериальными свойствами из группы терпенов ментанового ряда в условиях in vitro и in vivo: лимонена (B34), его производного (+)-1,2-оксида лимонена (B60) и серосодержащего монотерпенового соединения 2-(1’-гидрокси-4’-изопренил-1’-метилциклогексил-2’-тио)метилэтаноата (B65). В условиях in vitro (культура опухолевых клеток HeLa) изучаемые монотерпены в диапазоне концентраций 2 – 200 мкг/мл обладали цитотоксичностью. Ингибирующая концентрация (ИК50) для B34 составила 231 (167 – 295) мкг/мл, для B60 – 181 (105 – 257) мкг/мл, ИК50 B65 – 229 (150 – 308) мкг/мл. Исследование генотоксичности показало, что B34 и B65 в диапазоне концентраций 50 – 1000 мкг/мл не индуцируют SOS мутагенез в клетках Escherichia coli PQ37, тогда как B60 в концентрациях 500 и 1000 мкг/мл проявляет генотоксичность. In vivo в остром эксперименте на беспородных мышах установлена низкая токсичность B34 и его производных при различных путях введения. Наименьший показатель острой токсичности имеет B65, в связи с чем дополнительно на крысах проведено изучение его хронической токсичности. Ежедневное внутрижелудочное введение B65 в разовых дозах, составляющих 1/10 и 1/20 ЛД50 (1000 мг/кг и 500 мг/кг), в течение 1 мес не вызывало гибели животных, значимых нарушений общего состояния, изменения динамики массы тела, морфопатологических изменений. Внутрижелудочное введение B65 крысам в высокой токсической дозе 2000 мг/кг (1/5 ЛД50) в течение месяца вызывает патоморфологические изменения структуры печени.

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