The in vitro transcription of a rainbow trout (Salmo gairdnerii) protamine gene. II. Controlled mutation of the cap site region

1985 ◽  
Vol 5 (2) ◽  
pp. 113-120 ◽  
Author(s):  
Jacek M. Jankowski ◽  
Gordon H. Dixon
Keyword(s):  
In Vitro Transcription ◽  
Tata Box ◽  
Fusion Genes ◽  
Complementary Strand ◽  
Coding Region ◽  
Cryptic Promoter ◽  
Cell Lysate ◽  
Multiple Transcripts ◽  
Transcription In Vitro

A series of pJasmids containing new fusion genes in which the trout protamine gene is placed under the control of the complete herpes virus (HSV-I) tk promoter Pvu II-Bgl II fragment (pMg), or a shortened thymidine kinase (tk) promoter in which the region between the TATA box and the cap site is altered by using the Pvu II-Mlu I fragment (pM7), have been constructed. An additional recombinant plasmid was constructed in which the Bgl II-Ava II fragment of the protamine gene containing the entire protamine promoter but missing the protamine coding region was cloned into pBR322 between the Xho II 1666 and Hind III sites (pP5). For in vitro transcription, a HeLa cell lysate system was prepared and the RNA transcription products, after glyoxalation, were electrophoretically analyzed on 5% polyacrylamide gels. In constructing pM8 the DNA sequence between the tk promoter and the cap site was present while in pM7 it was deleted. Similar multiple transcripts were seen in both cases, indicating that the region between the promoter and the cap site has no effect upon transcription in vitro. The multiple transcripts appear to be due to the presence of a cryptic promoter in the complementary strand of the protamine gene. The activity of this cryptic promoter has been confirmed by comparison of the transcription of plasmid pPS, in which the protamine mRNA coding region has been deleted, with a previously described plasmid, p3BRP (Jankowski 3M and Dixon GH (1984) Can. J. Biochem. Cell. Biol. 62, 291–300), containing the intact protamine gene.

Basal and regulated transcription in Archaea

2001 ◽  
Vol 29 (4) ◽  
pp. 392-395 ◽  
Author(s):  
S. D. Bell ◽  
C. P. Magill ◽  
S. P. Jackson
Keyword(s):  
In Vitro Transcription ◽  
Tata Box ◽  
Basal Transcription ◽  
General Transcription Factors ◽  
General Transcription Factor ◽  
Promoter Recognition ◽  
Transcription In Vitro ◽  
Rnap Ii ◽  
Tata Box Binding Protein

The basal transcription machinery of Archaea is fundamentally related to the eucaryal RNA polymerase (RNAP) II apparatus. In addition to a 12-subunit RNAP, Archaea possess two general transcription factors, the activities of which are required for accurate and efficient in vitro transcription. These factors, TBP and TFB, are homologues of the eucaryal TATA-box binding protein and TFIIB respectively. Archaea also possess TFE, a homologue of the eucaryal RNAP II general transcription factor TFIIE. Although not absolutely required for transcription in vitro, TFE nonetheless plays a stimulatory role under conditions where promoter recognition by TBP is sub-optimal. The basal transcription apparatus of Archaea is closely related to that of Eucarya but archaeal transcriptional regulators resemble those of bacteria. The mode of action of two such regulators has been characterized to determine how these ‘bacterial-like’ regulators impinge on the ‘eucaryal-like’ basal machinery.

scholarly journals Regulation of protamine gene expression in an in vitro homologous system.

1996 ◽  
Vol 43 (2) ◽  
pp. 369-377 ◽  
Author(s):  
J M Jankowski ◽  
P D Cannon ◽  
F Van der Hoorn ◽  
L D Wasilewska ◽  
N C Wong ◽  
...  
Keyword(s):  
Gene Expression ◽  
Regulatory Elements ◽  
In Vitro Transcription ◽  
Tata Box ◽  
Transcription System ◽  
Proposed Model ◽  
Binding Factor ◽  
Gc Box ◽  
Responsive Element

An in vitro transcription system from the trout testis nuclei was developed to study trout protamine gene expression. The protamine promoter contains, among others, two regulatory elements: 1) a cAMP-responsive element or CRE element (TGACGTCA) which is present in position 5' to TATA box, and 2) GC box (CCGCCC) which is present in position 3' to TATA box. The removal of the CRE-binding protein by titration (by the addition of appropriate oligonucleotides to the incubation mixture) resulted in a decrease in transcription of the protamine gene. These results were confirmed by experiments in which the pure CRE-binding factor (TPBP1) was used, as well as by those where a stimulatory effect of cAMP on protamine promoter transcription was observed. On the other hand, addition of oligonucleotides containing the GC-box sequence enhanced the protamine gene transcription indicating that the protein (Sp1 like) which binds to this sequence acts as a repressor of protamine gene expression. These results confirm the previously proposed model which suggested that the GC box played a role in negative regulation of the protamine gene expression. Involvement of some other factors in this process was also discussed.

scholarly journals Revisiting T7 RNA polymerase transcription in vitro with the Broccoli RNA aptamer as a simplified real-time fluorescent reporter

2020 ◽  
pp. jbc.RA120.014553
Author(s):  
Zachary J Kartje ◽  
Helen I Janis ◽  
Shaoni Mukhopadhyay ◽  
Keith T Gagnon
Keyword(s):  
Rna Polymerase ◽  
Real Time ◽  
High Throughput Screening ◽  
In Vitro Transcription ◽  
T7 Rna Polymerase ◽  
Rna Aptamer ◽  
Reaction Conditions ◽  
Fluorescent Reporter ◽  
Transcription In Vitro

Methods for rapid and high-throughput screening of transcription in vitro to examine reaction conditions, enzyme mutants, promoter variants, and small molecule modulators can be extremely valuable tools. However, these techniques may be difficult to establish or inaccessible to many researchers. To develop a straightforward and cost-effective platform for assessing transcription in vitro, we used the “Broccoli” RNA aptamer as a direct, real-time fluorescent transcript readout. To demonstrate the utility of our approach, we screened the effect of common reaction conditions and components on bacteriophage T7 RNA polymerase (RNAP) activity using a common quantitative PCR instrument for fluorescence detection. Several essential conditions for in vitro transcription by T7 RNAP were confirmed with this assay, including the importance of enzyme and substrate concentrations, co-variation of magnesium and nucleoside triphosphates, and the effects of several typical additives. When we used this method to assess all possible point mutants of a canonical T7 RNAP promoter, our results coincided well with previous reports. This approach should translate well to a broad variety of bacteriophage in vitro transcription systems and provides a platform for developing fluorescence-based readouts of more complex transcription systems in vitro.

scholarly journals In vitro synthesis and processing of a maize chloroplast transcript encoded by the ribulose 1,5-bisphosphate carboxylase large subunit gene.

1985 ◽  
Vol 5 (10) ◽  
pp. 2733-2745 ◽  
Author(s):  
L Hanley-Bowdoin ◽  
E M Orozco ◽  
N H Chua
Keyword(s):  
Transcription Initiation ◽  
Large Subunit ◽  
Plastid Dna ◽  
Subunit Gene ◽  
Coding Region ◽  
Protein Coding ◽  
Bisphosphate Carboxylase ◽  
Transcription In Vitro ◽  
Maize Chloroplast

The large subunit gene (rbcL) of ribulose 1,5-bisphosphate carboxylase was transcribed in vitro by using maize and pea chloroplast extracts and a cloned plastid DNA template containing 172 base pairs (bp) of the maize rbcL protein-coding region and 791 bp of upstream sequences. Three major in vitro RNA species were synthesized which correspond to in vivo maize rbcL RNAs with 5' termini positioned 300, 100 to 105, and 63 nucleotides upstream of the protein-coding region. A deletion of 109 bp, including the "-300" 5' end (the 5' end at position -300), depressed all rbcL transcription in vitro. A plasmid DNA containing this 109-bp fragment was sufficient to direct correct transcription initiation in vitro. A cloned template, containing 191 bp of plastid DNA which includes the -105 and -63 rbcL termini, did not support transcription in vitro. Exogenously added -300 RNA could be converted to the -63 transcript by maize chloroplast extract. These results established that the -300 RNA is the primary maize rbcL transcript, the -63 RNA is a processed form of the -300 transcript, and synthesis of the -105 RNA is dependent on the -300 region. The promoter for the maize rbcL gene is located within the 109 bp flanking the -300 site. Mutagenesis of the 109-bp chloroplast sequence 11 bp upstream of the -300 transcription initiation site reduced rbcL promoter activity in vitro.

scholarly journals Prolonged exposure to methylglyoxal causes disruption of vascular KATPchannel by mRNA instability

2012 ◽  
Vol 303 (10) ◽  
pp. C1045-C1054 ◽  
Author(s):  
Yang Yang ◽  
Shanshan Li ◽  
Anuhya S. Konduru ◽  
Shuang Zhang ◽  
Timothy C. Trower ◽  
...  
Keyword(s):  
Prolonged Exposure ◽  
In Vitro Transcription ◽  
Untranslated Regions ◽  
Luciferase Reporter ◽  
Coding Region ◽  
Intermediary Metabolites ◽  
Vascular Walls ◽  
Reactive Carbonyl Species ◽  
Reporter Assays

Diabetes mellitus is characterized by hyperglycemia and excessive production of intermediary metabolites including methylglyoxal (MGO), a reactive carbonyl species that can lead to cell injuries. Interacting with proteins, lipids, and DNA, excessive MGO can cause dysfunction of various tissues, especially the vascular walls where diabetic complications often take place. However, the potential vascular targets of excessive MGO remain to be fully understood. Here we show that the vascular Kir6.1/SUR2B isoform of ATP-sensitive K+(KATP) channels is likely to be disrupted with an exposure to submillimolar MGO. Up to 90% of the Kir6.1/SUR2B currents were suppressed by 1 mM MGO with a time constant of ∼2 h. Consistently, MGO treatment caused a vast reduction of both Kir6.1 and SUR2B mRNAs endogenously expressed in the A10 vascular smooth muscle cells. In the presence of the transcriptional inhibitor actinomycin-D, MGO remained to lower the Kir6.1 and SUR2B mRNAs to the same degree as MGO alone, suggesting that the MGO effect is likely to compromise the mRNA stability. Luciferase reporter assays indicated that the 3′-untranslated regions (UTRs) of the Kir6.1 but not SUR2 mRNA were targeted by MGO. In contrast, the SUR2B mRNAs obtained with in vitro transcription were disrupted by MGO directly, while the Kir6.1 transcripts were unaffected. Consistent with these results, the constriction of mesenteric arterial rings was markedly augmented with an exposure to 1 mM MGO for 2 h, and such an MGO effect was totally eliminated in the presence of glibenclamide. These results therefore suggest that acting on the 3′-UTR of Kir6.1 and the coding region of SUR2B, MGO causes instability of Kir6.1 and SUR2B mRNAs, disruption of vascular KATPchannels, and impairment of arterial function.

scholarly journals Even-skipped Represses Transcription by Binding TATA Binding Protein and Blocking the TFIID-TATA Box Interaction

1998 ◽  
Vol 18 (7) ◽  
pp. 3771-3781 ◽  
Author(s):  
Chi Li ◽  
James L. Manley
Keyword(s):  
Rna Polymerase Ii ◽  
Binding Protein ◽  
Direct Interaction ◽  
In Vitro Transcription ◽  
Tata Binding Protein ◽  
Tata Box ◽  
Homeodomain Protein ◽  
Necessary And Sufficient

ABSTRACT The Drosophila homeodomain protein Even-skipped (Eve) is a transcriptional repressor, and previous studies have suggested that it functions by interfering with the basal transcription machinery. Here we describe experiments indicating that the mechanism of Eve repression involves a direct interaction with the TATA binding protein (TBP) that blocks binding of TBP-TFIID to the promoter. We first compared Eve activities in in vitro transcription systems reconstituted with either all the general transcription factors or only TBP, TFIIB, TFIIF30, and RNA polymerase II. In each case, equivalent and very efficient levels of repression were observed, indicating that no factors other than those in the minimal system are required for repression. We then show that Eve can function efficiently when its recognition sites are far from the promoter and that the same regions of Eve required for repression in vivo are necessary and sufficient for in vitro repression. This includes, in addition to an Ala-Pro-rich region, residues within the homeodomain. Using GAL4-Eve fusion proteins, we demonstrate that the homeodomain plays a role in repression in addition to DNA binding, which is to facilitate interaction with TBP. Single-round transcription experiments indicate that Eve must function prior to TBP binding to the promoter, suggesting a mechanism whereby Eve represses by competing with the TATA box for TBP binding. Consistent with this, excess TATA box-containing oligonucleotide is shown to specifically and efficiently disrupt the TBP-Eve interaction. Importantly, we show that Eve binds directly to TFIID and that this interaction can also be disrupted by the TATA oligonucleotide. We conclude that Eve represses transcription via a direct interaction with TBP that blocks TFIID binding to the promoter.

scholarly journals Characterization of a surrogate TATA box promoter that regulates in vitro transcription of the simian virus 40 major late gene.

1985 ◽  
Vol 5 (3) ◽  
pp. 591-594 ◽  
Author(s):  
A Nandi ◽  
G Das ◽  
N P Salzman
Keyword(s):  
Simian Virus 40 ◽  
In Vitro Transcription ◽  
Simian Virus ◽  
Tata Box ◽  
Wild Type Virus ◽  
Base Substitution ◽  
Nucleotide Position ◽  
Start Site ◽  
Additional Base

The presence of a surrogate TATA box sequence located ca. 30 nucleotides upstream of the major late RNA start site at nucleotide position (np) 325 (Brady et al., Cell 31:625-633, 1982) has been confirmed, and its structural specificity has been determined by the generation of additional base substitution mutations at the KpnI restriction site (np 294) in cloned simian virus 40 DNA. Two mutants generated new RNA initiation sites upstream of the np 325 start site and continued to utilize the authentic start site, but with decreased efficiency. The replacement of either one or both cytosines by thymines at np 298 and np 299 specifically enhanced in vitro transcription from the np 325 start site by 430 and 800%, respectively. This enhancement was due to conversion of the simian virus 40 late promoter present in the wild-type virus to a sequence that is similar to the TATA box present in the simian virus 40 early promoter.

scholarly journals In Vitro Transcriptional Studies of thebkd Operon of Pseudomonas putida:l-Branched-Chain Amino Acids and d-Leucine Are the Inducers

1999 ◽  
Vol 181 (9) ◽  
pp. 2889-2894 ◽  
Author(s):  
Kunapuli T. Madhusudhan ◽  
Jinhe Luo ◽  
John R. Sokatch
Keyword(s):  
Amino Acids ◽  
Transcriptional Activation ◽  
In Vitro Transcription ◽  
Branched Chain Amino Acids ◽  
Multienzyme Complex ◽  
Keto Acids ◽  
Transcription In Vitro ◽  
Branched Chain ◽  
Hydroxyl Radical Footprinting

ABSTRACT BkdR is the transcriptional activator of the bkdoperon, which encodes the four proteins of the branched-chain keto acid dehydrogenase multienzyme complex of Pseudomonas putida. In this study, hydroxyl radical footprinting revealed that BkdR bound to only one face of DNA over the same region identified in DNase I protection assays. Deletions of even a few bases in the 5′ region of the BkdR-binding site greatly reduced transcription, confirming that the entire protected region is necessary for transcription. In vitro transcription of the bkd operon was obtained by using a vector containing the bkdR-bkdA1 intergenic region plus the putative ρ-independent terminator of the bkdoperon. Substrate DNA, BkdR, and any of thel-branched-chain amino acids or d-leucine was required for transcription. Branched-chain keto acids,d-valine, and d-isoleucine did not promote transcription. Therefore, the l-branched-chain amino acids and d-leucine are the inducers of the bkdoperon. The concentration of l-valine required for half-maximal transcription was 2.8 mM, which is similar to that needed to cause half-maximal proteolysis due to a conformational change in BkdR. A model for transcriptional activation of the bkdoperon by BkdR during enzyme induction which incorporates these results is presented.

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