Accurate transcription of a plant mitochondrial gene in vitro

1991 ◽  
Vol 11 (4) ◽  
pp. 2035-2039
Author(s):  
P J Hanic-Joyce ◽  
M W Gray
Keyword(s):  
Mitochondrial Gene ◽  
Plant Mitochondria ◽  
In Vitro Transcription ◽  
Dna Templates ◽  
In Vitro System ◽  
Transcription System ◽  
Translation Initiation Codon ◽  
Mitochondrial Extract

To investigate transcriptional mechanisms in plant mitochondria, we have developed an accurate and efficient in vitro transcription system consisting of a partially purified wheat mitochondrial extract programmed with cloned DNA templates containing the promoter for the wheat mitochondrial cytochrome oxidase subunit II gene (coxII). Using this system, we localize the coxII promoter to a 372-bp region spanning positions -56 to -427 relative to the coxII translation initiation codon. We show that in vitro transcription of coxII is initiated at position -170, precisely the same site at which transcription is initiated in vivo. Transcription begins within the sequence GTATAGTAAGTA (the initiating nucleotide is underlined), which is similar to the consensus yeast mitochondrial promoter motif, (A/T)TATAAGTA. This is the first in vitro system that faithfully reproduces in vivo transcription of a plant mitochondrial gene.

scholarly journals Accurate transcription of a plant mitochondrial gene in vitro.

1991 ◽  
Vol 11 (4) ◽  
pp. 2035-2039 ◽  
Author(s):  
P J Hanic-Joyce ◽  
M W Gray
Keyword(s):  
Mitochondrial Gene ◽  
Plant Mitochondria ◽  
In Vitro Transcription ◽  
Dna Templates ◽  
In Vitro System ◽  
Transcription System ◽  
Translation Initiation Codon ◽  
Mitochondrial Extract

To investigate transcriptional mechanisms in plant mitochondria, we have developed an accurate and efficient in vitro transcription system consisting of a partially purified wheat mitochondrial extract programmed with cloned DNA templates containing the promoter for the wheat mitochondrial cytochrome oxidase subunit II gene (coxII). Using this system, we localize the coxII promoter to a 372-bp region spanning positions -56 to -427 relative to the coxII translation initiation codon. We show that in vitro transcription of coxII is initiated at position -170, precisely the same site at which transcription is initiated in vivo. Transcription begins within the sequence GTATAGTAAGTA (the initiating nucleotide is underlined), which is similar to the consensus yeast mitochondrial promoter motif, (A/T)TATAAGTA. This is the first in vitro system that faithfully reproduces in vivo transcription of a plant mitochondrial gene.

scholarly journals Activation of yeast polymerase II transcription by herpesvirus VP16 and GAL4 derivatives in vitro.

1989 ◽  
Vol 9 (11) ◽  
pp. 4746-4749 ◽  
Author(s):  
D I Chasman ◽  
J Leatherwood ◽  
M Carey ◽  
M Ptashne ◽  
R D Kornberg
Keyword(s):  
Rna Polymerase Ii ◽  
Fusion Proteins ◽  
In Vitro System ◽  
Transcription System ◽  
Natural Mechanism ◽  
Transcription In Vitro ◽  
Rna Polymerase Ii Transcription ◽  
Fold Stimulation

Fusion proteins known to activate transcription in vivo were tested for the ability to stimulate transcription in vitro in a recently developed Saccharomyces cerevisiae RNA polymerase II transcription system. One fusion protein, whose activation domain was derived from the herpesvirus transcriptional activator VP16, gave more than 100-fold stimulation in the in vitro system. The order of effects of the various proteins was the same for transcription in vitro and in vivo, suggesting that the natural mechanism of activation is preserved in vitro.

scholarly journals In vitro transcription and delimitation of promoter elements of the murine dihydrofolate reductase gene.

1986 ◽  
Vol 6 (7) ◽  
pp. 2392-2401 ◽  
Author(s):  
P J Farnham ◽  
R T Schimke
Keyword(s):  
Dihydrofolate Reductase ◽  
In Vitro Transcription ◽  
S Phase ◽  
Specific Factor ◽  
Or Efficiency ◽  
Transcription System ◽  
Nuclear Extracts ◽  
Reductase Gene

We have developed an in vitro transcription system for the murine dihydrofolate reductase gene. Although transcription in vitro from a linearized template was initiated at the same start sites as in vivo, the correct ratios were more closely approximated when a supercoiled template was used. In addition, whereas the dihydrofolate reductase promoter functions bidirectionally in vivo, the initiation signals directed unidirectional transcription in this in vitro system. The dihydrofolate reductase gene does not have a typical TATA box, but has four GGGCGG hexanucleotides within 300 base pairs 5' of the AUG codon. Deletion analysis suggested that, although sequences surrounding each of the GC boxes could specify initiation approximately 40 to 50 nucleotides downstream, three of the four GC boxes could be removed without changing the accuracy or efficiency of initiation at the major in vivo site. The dihydrofolate reductase promoter initiated transcription very rapidly in vitro, with transcripts visible by 1 min and almost maximal by 2 min at 30 degrees C with no preincubation. Nuclear extracts prepared from cells blocked in the S phase by aphidicolin or from adenovirus-infected cells at 16 h postinfection had enhanced dihydrofolate reductase transcriptional activity. This increased in vitro transcription mimicked the increase in dihydrofolate reductase mRNA seen in S-phase cells and suggested the presence of a cell-cycle-specific factor(s) which stimulated transcription from the dihydrofolate reductase gene.

scholarly journals Analysis of bvgR Expression in Bordetella pertussis

2003 ◽  
Vol 185 (23) ◽  
pp. 6902-6912 ◽  
Author(s):  
Tod J. Merkel ◽  
Philip E. Boucher ◽  
Scott Stibitz ◽  
Vanessa K. Grippe
Keyword(s):  
Bordetella Pertussis ◽  
Transcriptional Activation ◽  
Northern Blot Analysis ◽  
Whooping Cough ◽  
Transmembrane Protein ◽  
In Vitro Transcription ◽  
Environmental Signals ◽  
Transcription System

ABSTRACT Bordetella pertussis, the causative agent of whooping cough, produces a wide array of factors that are associated with its ability to cause disease. The expression and regulation of these virulence factors are dependent upon the bvg locus, which encodes three proteins: BvgA, a 23-kDa cytoplasmic protein; BvgS, a 135-kDa transmembrane protein; and BvgR, a 32-kDa protein. It is hypothesized that BvgS responds to environmental signals and interacts with BvgA, a transcriptional regulator, which upon modification by BvgS binds to specific promoters and activates transcription. An additional class of genes is repressed by the products of the bvg locus. The repression of these genes is dependent upon the third gene, bvgR. Expression of bvgR is dependent upon the function of BvgA and BvgS. This led to the hypothesis that the binding of phosphorylated BvgA to the bvgR promoter activates the expression of bvgR. We undertook an analysis of the transcriptional activation of bvgR expression. We identified the bvgR transcript by Northern blot analysis and identified the start site of transcription by primer extension. We determined that transcriptional activation of the bvgR promoter in an in vitro transcription system requires the addition of phosphorylated BvgA. Additionally, we have identified cis-acting regions that are required for BvgA activation of the bvgR promoter by in vitro footprinting and in vivo deletion and linker scanning analyses. A model of BvgA binding to the bvgR promoter is presented.

scholarly journals A tridecamer DNA sequence supports human mitochondrial RNA 3'-end formation in vitro.

1988 ◽  
Vol 8 (10) ◽  
pp. 4502-4509 ◽  
Author(s):  
T W Christianson ◽  
D A Clayton
Keyword(s):  
Dna Sequence ◽  
Transcription Termination ◽  
In Vitro Transcription ◽  
Mitochondrial Rna ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Transcription System ◽  
Flanking Regions

Vertebrate mitochondrial genomes contain a putative transcription termination site at the boundary between the genes for 16S rRNA and leucyl-tRNA. We have described previously an in vitro transcription system from human cells with the capacity to generate RNA 3' ends with the same map positions as those synthesized in vivo. By assaying the ability of variously truncated templates to support 3'-end formation, we demonstrated that the tridecamer sequence 5'-TGGCAGAGCCCCGG-3', contained entirely within the gene for leucyl-tRNA, is necessary to direct accurate termination. When two tridecamer sequences and their immediate flanking regions were placed in tandem, termination occurred at both promoter-proximal and promoter-distal sites. Furthermore, termination was competitively inhibited, in a concentration-dependent manner, by DNA containing the tridecamer sequence. These results suggest a modest sequence requirement for transcription termination that is contingent on a factor capable of recognizing the presence of the tridecamer DNA sequence.

Binding of the transcription factor EBP-80 mediates the methylation response of an intracisternal A-particle long terminal repeat promoter

1991 ◽  
Vol 11 (1) ◽  
pp. 117-125
Author(s):  
M Falzon ◽  
E L Kuff
Keyword(s):  
Promoter Activity ◽  
In Vitro Transcription ◽  
Site Directed Mutagenesis ◽  
Long Terminal Repeats ◽  
Transcription System ◽  
A Cell ◽  
Induced Changes ◽  
Intracisternal A Particle

Intracisternal A-particle (IAP) expression in mouse cells has been correlated with hypomethylation of HhaI and HpaII sites in proviral long terminal repeats (LTRs). In a previous study, in vitro methylation of three HhaI sites in the U3 region of the LTR from the cloned genomic IAP element, MIA14, was shown to inhibit promoter activity in vivo. In this study, we found by site-directed mutagenesis that the two more downstream HhaI sites within this LTR were responsible for the methylation effects on promoter activity in vivo; methylation of the other (5') HhaI site, which lies within a putative SP1 binding domain, did not affect promoter activity. Methylation of the HhaI sites also inhibited promoter activity of the LTR in a cell-free transcription system. Exonuclease III footprinting demonstrated methylation-induced changes in protein binding over the region encompassing the downstream HhaI site, designated the Enh2 domain. The protein that interacts specifically with this domain, EBP-80, was characterized in a previous study (M. Falzon and E. L. Kuff, J. Biol. Chem. 264:21915-21922, 1989). We show here that the presence of methylcytosine in the HhaI site within the Enh2 domain inhibited binding of EBP-80 in vitro. The methylated MIA14 LTR construct was much less responsive to added EBP-80 in an in vitro transcription system than was the unmethylated construct. These data suggest that CpG methylation within the Enh2 domain may exert its effect on transcription in vivo by altering the interaction between EBP-80 and its cognate DNA sequence.

A tridecamer DNA sequence supports human mitochondrial RNA 3'-end formation in vitro

1988 ◽  
Vol 8 (10) ◽  
pp. 4502-4509
Author(s):  
T W Christianson ◽  
D A Clayton
Keyword(s):  
Dna Sequence ◽  
Transcription Termination ◽  
In Vitro Transcription ◽  
Mitochondrial Rna ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Transcription System ◽  
Flanking Regions

Vertebrate mitochondrial genomes contain a putative transcription termination site at the boundary between the genes for 16S rRNA and leucyl-tRNA. We have described previously an in vitro transcription system from human cells with the capacity to generate RNA 3' ends with the same map positions as those synthesized in vivo. By assaying the ability of variously truncated templates to support 3'-end formation, we demonstrated that the tridecamer sequence 5'-TGGCAGAGCCCCGG-3', contained entirely within the gene for leucyl-tRNA, is necessary to direct accurate termination. When two tridecamer sequences and their immediate flanking regions were placed in tandem, termination occurred at both promoter-proximal and promoter-distal sites. Furthermore, termination was competitively inhibited, in a concentration-dependent manner, by DNA containing the tridecamer sequence. These results suggest a modest sequence requirement for transcription termination that is contingent on a factor capable of recognizing the presence of the tridecamer DNA sequence.

scholarly journals Control of the Arabinose Regulon in Bacillus subtilisby AraR In Vivo: Crucial Roles of Operators, Cooperativity, and DNA Looping

2001 ◽  
Vol 183 (14) ◽  
pp. 4190-4201 ◽  
Author(s):  
Luı́s Jaime Mota ◽  
Leonor Morais Sarmento ◽  
Isabel de Sá-Nogueira
Keyword(s):  
Regulatory Gene ◽  
Regulatory Elements ◽  
In Vitro Transcription ◽  
Dna Looping ◽  
Major Protein ◽  
Flexible Control ◽  
Transcription System ◽  
Single Operator

ABSTRACT The proteins involved in the utilization of l-arabinose by Bacillus subtilis are encoded by thearaABDLMNPQ-abfA metabolic operon and by thearaE/araR divergent unit. Transcription from the ara operon, araE transport gene, andaraR regulatory gene is induced by l-arabinose and negatively controlled by AraR. The purified AraR protein binds cooperatively to two in-phase operators within thearaABDLMNPQ-abfA (ORA1 and ORA2) and araE (ORE1 and ORE2) promoters and noncooperatively to a single operator in the araR (ORR3) promoter region. Here, we have investigated how AraR controls transcription from theara regulon in vivo. A deletion analysis of theara promoters region showed that the five AraR binding sites are the key cis-acting regulatory elements of their corresponding genes. Furthermore, ORE1-ORE2 and ORR3 are auxiliary operators for the autoregulation ofaraR and the repression of araE, respectively. Analysis of mutations designed to prevent cooperative binding of AraR showed that in vivo repression of the ara operon requires communication between repressor molecules bound to two properly spaced operators. This communication implicates the formation of a small loop by the intervening DNA. In an in vitro transcription system, AraR alone sufficed to abolish transcription from thearaABDLMNPQ-abfA operon and araEpromoters, strongly suggesting that it is the major protein involved in the repression mechanism of l-arabinose-inducible expression in vivo. The ara regulon is an example of how the architecture of the promoters is adapted to respond to the particular characteristics of the system, resulting in a tight and flexible control.

scholarly journals Development of an in vitro transcription system for Neurospora crassa mitochondrial DNA and identification of transcription initiation sites.

1989 ◽  
Vol 9 (9) ◽  
pp. 3603-3613 ◽  
Author(s):  
J C Kennell ◽  
A M Lambowitz
Keyword(s):  
Mitochondrial Dna ◽  
Neurospora Crassa ◽  
Transcription Initiation ◽  
Consensus Sequence ◽  
In Vitro Transcription ◽  
Transcription System ◽  
Group I ◽  
Genes Encoding

We have developed an in vitro transcription system for Neurospora crassa mitochondrial DNA (mtDNA) and used it to identify transcription initiation sites at the 5' ends of the genes encoding the mitochondrial small and large rRNA and cytochrome b (cob). The in vitro transcription start sites correspond to previously mapped 5' ends of major in vivo transcripts of these genes. Sequences around the three transcription initiation sites define a 15-nucleotide consensus sequence, 5'-TTAGARA(T/G)G(T/G)ARTRR-3', all or part of which appears to be an element of an N. crassa mtDNA promoter. A somewhat looser 11-nucleotide consensus sequence, 5'-TTAGARR(T/G)R(T/G)A-3', was derived by including two additional promoters identified recently. Group I extranuclear mutants, such as [poky] and [SG-3], have a 4-base-pair (bp) deletion in the consensus sequence at the 5' end of the mitochondrial small rRNA and are grossly deficient in mitochondrial small rRNA (R. A. Akins and A. M. Lambowitz, Proc. Natl. Acad. Sci. USA 81:3791-3795, 1984). We show here that the 4-bp deletion in the consensus sequence decreases in vitro transcription from this site by more than 99%. N. crassa mtDNA is similar to Saccharomyces cerevisiae mtDNA in having multiple promoters, including separate promoters for the genes encoding the mitochondrial small and large rRNAs. Our results suggest that the primary effect of the 4-bp deletion in group I extranuclear mutants is to inhibit transcription of the mitochondrial small rRNA, leading to severe deficiency of mitochondrial small rRNA and small ribosomal subunits.

Development of an in vitro transcription system for Neurospora crassa mitochondrial DNA and identification of transcription initiation sites

1989 ◽  
Vol 9 (9) ◽  
pp. 3603-3613
Author(s):  
J C Kennell ◽  
A M Lambowitz
Keyword(s):  
Mitochondrial Dna ◽  
Neurospora Crassa ◽  
Transcription Initiation ◽  
Consensus Sequence ◽  
In Vitro Transcription ◽  
Transcription System ◽  
Group I ◽  
Genes Encoding

We have developed an in vitro transcription system for Neurospora crassa mitochondrial DNA (mtDNA) and used it to identify transcription initiation sites at the 5' ends of the genes encoding the mitochondrial small and large rRNA and cytochrome b (cob). The in vitro transcription start sites correspond to previously mapped 5' ends of major in vivo transcripts of these genes. Sequences around the three transcription initiation sites define a 15-nucleotide consensus sequence, 5'-TTAGARA(T/G)G(T/G)ARTRR-3', all or part of which appears to be an element of an N. crassa mtDNA promoter. A somewhat looser 11-nucleotide consensus sequence, 5'-TTAGARR(T/G)R(T/G)A-3', was derived by including two additional promoters identified recently. Group I extranuclear mutants, such as [poky] and [SG-3], have a 4-base-pair (bp) deletion in the consensus sequence at the 5' end of the mitochondrial small rRNA and are grossly deficient in mitochondrial small rRNA (R. A. Akins and A. M. Lambowitz, Proc. Natl. Acad. Sci. USA 81:3791-3795, 1984). We show here that the 4-bp deletion in the consensus sequence decreases in vitro transcription from this site by more than 99%. N. crassa mtDNA is similar to Saccharomyces cerevisiae mtDNA in having multiple promoters, including separate promoters for the genes encoding the mitochondrial small and large rRNAs. Our results suggest that the primary effect of the 4-bp deletion in group I extranuclear mutants is to inhibit transcription of the mitochondrial small rRNA, leading to severe deficiency of mitochondrial small rRNA and small ribosomal subunits.

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