scholarly journals Architecture of the maize mitochondrial atp1 promoter as determined by linker-scanning and point mutagenesis.

1993 ◽  
Vol 13 (12) ◽  
pp. 7232-7238 ◽  
Author(s):  
W D Rapp ◽  
D S Lupold ◽  
S Mack ◽  
D B Stern
Keyword(s):  
Promoter Region ◽  
Consensus Sequence ◽  
Point Mutations ◽  
In Vitro Transcription ◽  
Wild Type ◽  
Transcription Start ◽  
Transcription System ◽  
Mitochondrial Promoters ◽  
Point Mutagenesis

Plant mitochondrial promoters are poorly conserved but generally share a loose consensus sequence spanning approximately 17 nucleotides. Using a homologous in vitro transcription system, we have previously shown that an 11-nucleotide sequence within this region comprises at least part of the maize mitochondrial atp1 promoter (W. Rapp and D. Stern, EMBO J. 11:1065-1073, 1992). We have extended this finding by using a series of linker-scanning and point mutations to define the atp1 promoter in detail. Our results show that mutations at positions -12 to +5, relative to the major transcription start site, can decrease initiation rates to between < 10 and 40% of wild-type levels. Some mutations, scattered throughout this region, have lesser effects or no effect. Taken together, our data suggest a model in which the atp1 promoter consists of a central domain extending from -7 to +5 and an upstream domain of 1 to 3 bp that is centered around -11 to -12. Because many mutations within this promoter region are tolerated in vitro, the maize atp1 promoter is distinct from the highly conserved yeast mitochondrial promoters.

Architecture of the maize mitochondrial atp1 promoter as determined by linker-scanning and point mutagenesis

1993 ◽  
Vol 13 (12) ◽  
pp. 7232-7238
Author(s):  
W D Rapp ◽  
D S Lupold ◽  
S Mack ◽  
D B Stern
Keyword(s):  
Promoter Region ◽  
Consensus Sequence ◽  
Point Mutations ◽  
In Vitro Transcription ◽  
Wild Type ◽  
Transcription Start ◽  
Transcription System ◽  
Mitochondrial Promoters ◽  
Point Mutagenesis

Plant mitochondrial promoters are poorly conserved but generally share a loose consensus sequence spanning approximately 17 nucleotides. Using a homologous in vitro transcription system, we have previously shown that an 11-nucleotide sequence within this region comprises at least part of the maize mitochondrial atp1 promoter (W. Rapp and D. Stern, EMBO J. 11:1065-1073, 1992). We have extended this finding by using a series of linker-scanning and point mutations to define the atp1 promoter in detail. Our results show that mutations at positions -12 to +5, relative to the major transcription start site, can decrease initiation rates to between < 10 and 40% of wild-type levels. Some mutations, scattered throughout this region, have lesser effects or no effect. Taken together, our data suggest a model in which the atp1 promoter consists of a central domain extending from -7 to +5 and an upstream domain of 1 to 3 bp that is centered around -11 to -12. Because many mutations within this promoter region are tolerated in vitro, the maize atp1 promoter is distinct from the highly conserved yeast mitochondrial promoters.

scholarly journals CcpA Causes Repression of the phoPR Promoter through a Novel Transcription Start Site, PA6

2006 ◽  
Vol 188 (4) ◽  
pp. 1266-1278 ◽  
Author(s):  
Ankita Puri-Taneja ◽  
Salbi Paul ◽  
Yinghua Chen ◽  
F. Marion Hulett
Keyword(s):  
Response Regulator ◽  
Consensus Sequence ◽  
Protein A ◽  
Carbon Sources ◽  
In Vitro Transcription ◽  
Defined Medium ◽  
Wild Type ◽  
Transcription Start

ABSTRACT The Bacillus subtilis PhoPR two-component system is directly responsible for activation or repression of Pho regulon genes in response to phosphate deprivation. The response regulator, PhoP, and the histidine kinase, PhoR, are encoded in a single operon with a complex promoter region that contains five known transcription start sites, which respond to at least two regulatory proteins. We report here the identification of another direct regulator of phoPR transcription, carbon catabolite protein A, CcpA. This regulator functions in the presence of glucose or other readily metabolized carbon sources. The maximum derepression of phoPR expression in a ccpA mutant compared to a wild-type stain was observed under excess phosphate conditions with glucose either throughout growth in a high-phosphate defined medium or in a low-phosphate defined medium during exponential growth, a growth condition when phoPR transcription is low in a wild-type strain due to the absence of autoinduction. Either HPr or Crh were sufficient to cause CcpA dependent repression of the phoPR promoter in vivo. A ptsH1 (Hpr) crh double mutant completely relieves phoPR repression during phosphate starvation but not during phosphate replete growth. In vivo and in vitro studies showed that CcpA repressed phoPR transcription by binding directly to the cre consensus sequence present in the promoter. Primer extension and in vitro transcription studies revealed that the CcpA regulation of phoPR transcription was due to repression of PA6, a previously unidentified promoter positioned immediately upstream of the cre box. EσA was sufficient for transcription of PA6, which was repressed by CcpA in vitro. These studies showed direct repression by CcpA of a newly discovered EσA-responsive phoPR promoter that required either Hpr or Crh in vivo for direct binding to the putative consensus cre sequence located between PA6 and the five downstream promoters characterized previously.

Dual functions of a cis-acting element within the rat prolactin gene promoter

1989 ◽  
Vol 9 (2) ◽  
pp. 817-819
Author(s):  
E A Barron ◽  
Z Cao ◽  
B G Schneider ◽  
E Kraig ◽  
A J Carrillo ◽  
...  
Keyword(s):  
Promoter Region ◽  
Gene Promoter ◽  
In Vitro Transcription ◽  
Cis Acting ◽  
Transcription System ◽  
Prolactin Gene ◽  
Imperfect Palindrome ◽  
Dual Functions ◽  
Symmetrical Sequence

Within the promoter region of the rat prolactin gene lies a TA-rich imperfect palindrome. The possible functions of the 18-base-pair symmetrical sequence were investigated by using an in vitro transcription system. Prolactin templates with and without the palindrome were transcriptionally assayed in both pituitary and nonpituitary extracts. Our results indicated that the palindromic sequence has at least two functions in the regulation of prolactin transcription.

scholarly journals Ex Vivo and In Vitro Identification of a Consensus Promoter for VSG Genes Expressed by Metacyclic-Stage Trypanosomes in the Tsetse Fly

2002 ◽  
Vol 1 (6) ◽  
pp. 1000-1009 ◽  
Author(s):  
Michael L. Ginger ◽  
Patricia A. Blundell ◽  
Alyson M. Lewis ◽  
Alison Browitt ◽  
Arthur Günzl ◽  
...  
Keyword(s):  
Salivary Glands ◽  
Ex Vivo ◽  
In Vitro Transcription ◽  
Tsetse Fly ◽  
Surface Glycoprotein ◽  
Start Site ◽  
Transcription Start ◽  
Transcription System ◽  
Rapid Amplification

ABSTRACT The trypanosome variant surface glycoprotein (VSG) is first expressed during differentiation to the infective, metacyclic population in tsetse fly salivary glands. Unlike the VSG genes expressed by bloodstream form trypanosomes, metacyclic VSGs (MVSGs) have their own promoters. The scarcity of metacyclic cells has meant that only indirect approaches have been used to study these promoters, and not even their identities have been agreed on. Here, we isolated trypanosomes by dissection from salivary glands and used an approach involving 5′ rapid amplification of cDNA ends to identify the transcription start site of three MVSGs. This shows that the authentic start site is that proposed for the MVAT series of MVSGs (K. S. Kim and J. E. Donelson, J. Biol. Chem. 272:24637-24645, 1997). In the more readily accessible procyclic trypanosome stage, where MVSGs are normally silent, we used reporter gene assays and linker scanning analysis to confirm that the 67 bp upstream of the start site is a promoter. This is confirmed further by accurate initiation in a homologous in vitro transcription system. We show also that MVSG promoters become derepressed when tested outwith their endogenous, subtelomeric loci. The MVSG promoters are only loosely conserved with bloodstream VSG promoters, and our detailed analysis of the 1.63 MVSG promoter reveals that its activity depends on the start site itself and sequences 26 to 49 bp and 56 to 60 bp upstream. These are longer than those necessary for the bloodstream promoter.

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.

scholarly journals Determination of the promoter region of mouse ribosomal RNA gene by an in vitro transcription system.

1984 ◽  
Vol 81 (2) ◽  
pp. 299-303 ◽  
Author(s):  
O. Yamamoto ◽  
N. Takakusa ◽  
Y. Mishima ◽  
R. Kominami ◽  
M. Muramatsu
Keyword(s):  
Ribosomal Rna ◽  
Promoter Region ◽  
In Vitro Transcription ◽  
Ribosomal Rna Gene ◽  
Transcription System

scholarly journals Dual functions of a cis-acting element within the rat prolactin gene promoter.

1989 ◽  
Vol 9 (2) ◽  
pp. 817-819 ◽  
Author(s):  
E A Barron ◽  
Z Cao ◽  
B G Schneider ◽  
E Kraig ◽  
A J Carrillo ◽  
...  
Keyword(s):  
Promoter Region ◽  
Gene Promoter ◽  
In Vitro Transcription ◽  
Cis Acting ◽  
Transcription System ◽  
Prolactin Gene ◽  
Imperfect Palindrome ◽  
Dual Functions ◽  
Symmetrical Sequence

Within the promoter region of the rat prolactin gene lies a TA-rich imperfect palindrome. The possible functions of the 18-base-pair symmetrical sequence were investigated by using an in vitro transcription system. Prolactin templates with and without the palindrome were transcriptionally assayed in both pituitary and nonpituitary extracts. Our results indicated that the palindromic sequence has at least two functions in the regulation of prolactin transcription.

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