scholarly journals Precise assignment of the light-strand promoter of mouse mitochondrial DNA: a functional promoter consists of multiple upstream domains.

1986 ◽  
Vol 6 (9) ◽  
pp. 3253-3261 ◽  
Author(s):  
D D Chang ◽  
D A Clayton
Keyword(s):  
Mitochondrial Dna ◽  
Initiation Site ◽  
Upstream Region ◽  
Mitochondrial Rna ◽  
Base Pairs ◽  
Transcriptional Initiation ◽  
Mitochondrial Rna Polymerase ◽  
Template Dna ◽  
Light Strand ◽  
Transcriptional Initiation Site

Using deletion mutagenesis we localized the promoter for the light strand of mouse mitochondrial DNA to a 97-base-pair region, from -88 to +9 nucleotides of the transcriptional initiation site. Within this region the light-strand promoter could be dissected into at least three different functional domains. The specificity region, a maximum of 19 base pairs between -10 and +9 of the transcriptional initiation site, was essential and sufficient for accurate transcriptional initiation. A second region, extending to -29 nucleotides from the initiation site, facilitated the formation of a preinitiation complex between the template DNA and factor(s) present in the mitochondrial RNA polymerase fraction and was required for efficient transcription. A third, ill-defined upstream region, which extended up to -88 nucleotides from the initiation site, appeared to influence template transcriptional efficiencies in competition assays. Without the specificity domain, the upstream regions were incapable of supporting any transcription. The presence of multiple upstream domains was confirmed by disrupting nucleotide sequences in the upstream region by using linker insertion and linker replacement techniques.

Precise assignment of the light-strand promoter of mouse mitochondrial DNA: a functional promoter consists of multiple upstream domains

1986 ◽  
Vol 6 (9) ◽  
pp. 3253-3261
Author(s):  
D D Chang ◽  
D A Clayton
Keyword(s):  
Mitochondrial Dna ◽  
Initiation Site ◽  
Upstream Region ◽  
Mitochondrial Rna ◽  
Base Pairs ◽  
Transcriptional Initiation ◽  
Mitochondrial Rna Polymerase ◽  
Template Dna ◽  
Light Strand ◽  
Transcriptional Initiation Site

Using deletion mutagenesis we localized the promoter for the light strand of mouse mitochondrial DNA to a 97-base-pair region, from -88 to +9 nucleotides of the transcriptional initiation site. Within this region the light-strand promoter could be dissected into at least three different functional domains. The specificity region, a maximum of 19 base pairs between -10 and +9 of the transcriptional initiation site, was essential and sufficient for accurate transcriptional initiation. A second region, extending to -29 nucleotides from the initiation site, facilitated the formation of a preinitiation complex between the template DNA and factor(s) present in the mitochondrial RNA polymerase fraction and was required for efficient transcription. A third, ill-defined upstream region, which extended up to -88 nucleotides from the initiation site, appeared to influence template transcriptional efficiencies in competition assays. Without the specificity domain, the upstream regions were incapable of supporting any transcription. The presence of multiple upstream domains was confirmed by disrupting nucleotide sequences in the upstream region by using linker insertion and linker replacement techniques.

scholarly journals Sequences required for transcriptional initiation of the Saccharomyces cerevisiae CYC7 genes.

1987 ◽  
Vol 7 (10) ◽  
pp. 3785-3791 ◽  
Author(s):  
A M Healy ◽  
T L Helser ◽  
R S Zitomer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Spatial Relationship ◽  
Initiation Site ◽  
Tata Box ◽  
Upstream Region ◽  
Base Pairs ◽  
Transcriptional Initiation ◽  
Consensus Sequences ◽  
Tata Sequence ◽  
Yeast Genes

A series of BAL 31 deletions were constructed in the upstream region of the Saccharomyces cerevisiae CYC7 gene to determine sequences required for transcriptional initiation. These deletions identified the TATA box as an alternating A-T sequence at -160 and the initiation sequences as well as the spatial relationship between them. The TATA box was necessary for wild-type levels of expression of the CYC7 gene. Decreasing the distance between the TATA sequence and the initiation site did not alter gene expression, but the site of transcription was shifted 3'-ward. In most cases, transcription initiated at a number of sites, the 5'-most of which was the first suitable site greater than 45 base pairs 3' of the TATA sequence, suggesting a spatial relationship between these sequences. Consensus sequences previously proposed for initiation sites were evaluated with respect to the start sites identified in this study as well as the start sites of other yeast genes.

Sequences required for transcriptional initiation of the Saccharomyces cerevisiae CYC7 genes

1987 ◽  
Vol 7 (10) ◽  
pp. 3785-3791
Author(s):  
A M Healy ◽  
T L Helser ◽  
R S Zitomer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Spatial Relationship ◽  
Initiation Site ◽  
Tata Box ◽  
Upstream Region ◽  
Base Pairs ◽  
Transcriptional Initiation ◽  
Consensus Sequences ◽  
Tata Sequence ◽  
Yeast Genes

A series of BAL 31 deletions were constructed in the upstream region of the Saccharomyces cerevisiae CYC7 gene to determine sequences required for transcriptional initiation. These deletions identified the TATA box as an alternating A-T sequence at -160 and the initiation sequences as well as the spatial relationship between them. The TATA box was necessary for wild-type levels of expression of the CYC7 gene. Decreasing the distance between the TATA sequence and the initiation site did not alter gene expression, but the site of transcription was shifted 3'-ward. In most cases, transcription initiated at a number of sites, the 5'-most of which was the first suitable site greater than 45 base pairs 3' of the TATA sequence, suggesting a spatial relationship between these sequences. Consensus sequences previously proposed for initiation sites were evaluated with respect to the start sites identified in this study as well as the start sites of other yeast genes.

A downstream-element-binding factor facilitates assembly of a functional preinitiation complex at the simian virus 40 major late promoter

1990 ◽  
Vol 10 (7) ◽  
pp. 3635-3645
Author(s):  
D E Ayer ◽  
W S Dynan
Keyword(s):  
Rna Polymerase Ii ◽  
Simian Virus 40 ◽  
Initiation Site ◽  
Simian Virus ◽  
Recognition Sequence ◽  
Base Pairs ◽  
Transcriptional Initiation ◽  
Nuclear Extracts ◽  
Major Late Promoter ◽  
Time Required

Recent work has shown that many promoters recognized by eucaryotic RNA polymerase II contain essential sequences located downstream of the transcriptional initiation site. We show here that the activity of a promoter element centered 28 base pairs downstream of the simian virus 40 major late initiation site appears to be mediated by a DNA-binding protein, which was isolated by affinity chromatography from HeLa cell nuclear extracts. In the absence of the other components of the transcriptional machinery, the protein bound specifically but weakly to its recognition sequence, with a Kd of approximately 10(-8) M. Analysis of kinetic data showed that mutation of the downstream element decreased the number of functional preinitiation complexes assembled at the promoter without significantly altering the time required for half the complexes to assemble. This suggests that in the absence of the downstream activating protein, preinitiation complexes are at least partially assembled but are not transcriptionally competent.

scholarly journals Distinct roles for two purified factors in transcription of Xenopus mitochondrial DNA.

1995 ◽  
Vol 15 (12) ◽  
pp. 7032-7042 ◽  
Author(s):  
I Antoshechkin ◽  
D F Bogenhagen
Keyword(s):  
Mitochondrial Dna ◽  
Rna Polymerase ◽  
Sequence Similarity ◽  
Protein A ◽  
Mitochondrial Rna ◽  
Basal Transcription ◽  
Hmg Box ◽  
Mitochondrial Rna Polymerase ◽  
Dnase I Footprinting

Transcription of Xenopus laevis mitochondrial DNA (xl-mtDNA) by the mitochondrial RNA polymerase requires a dissociable factor. This factor was purified to near homogeneity and identified as a 40-kDa protein. A second protein implicated in the transcription of mtDNA, the Xenopus homolog of the HMG box protein mtTFA, was also purified to homogeneity and partially sequenced. The sequence of a cDNA clone encoding xl-mtTFA revealed a high degree of sequence similarity to human and Saccharomyces cerevisiae mtTFA. xl-mtTFA was not required for basal transcription from a minimal mtDNA promoter, and this HMG box factor could not substitute for the basal factor, which is therefore designated xl-mtTFB. An antibody directed against the N terminus of xl-mtTFA did not cross-react with xl-mtTFB. xl-mtTFA is an abundant protein that appears to have at least two functions in mitochondria. First, it plays a major role in packaging mtDNA within the organelle. Second, DNase I footprinting experiments identified preferred binding sites for xl-mtTFA within the control region of mtDNA next to major mitochondrial promoters. We show that binding of xl-mtTFA to a site separating the two clusters of bidirectional promoters selectively stimulates specific transcription in vitro by the basal transcription machinery, comprising mitochondrial RNA polymerase and xl-mtTFB.

scholarly journals Non-DNA-Templated Addition of Nucleotides to the 3′ End of RNAs by the Mitochondrial RNA Polymerase of Physarum polycephalum

2008 ◽  
Vol 28 (18) ◽  
pp. 5795-5802 ◽  
Author(s):  
Mara L. Miller ◽  
Dennis L. Miller
Keyword(s):  
Mitochondrial Dna ◽  
Rna Polymerase ◽  
Rna Editing ◽  
Physarum Polycephalum ◽  
Mitochondrial Gene ◽  
In Vitro Transcription ◽  
Open Reading Frames ◽  
Mitochondrial Rna ◽  
Mitochondrial Rna Polymerase

ABSTRACT Mitochondrial gene expression is necessary for proper mitochondrial biogenesis. Genes on the mitochondrial DNA are transcribed by a dedicated mitochondrial RNA polymerase (mtRNAP) that is encoded in the nucleus and imported into mitochondria. In the myxomycete Physarum polycephalum, nucleotides that are not specified by the mitochondrial DNA templates are inserted into some RNAs, a process called RNA editing. This is an essential step in the expression of these RNAs, as the insertion of the nontemplated nucleotides creates open reading frames for the production of proteins from mRNAs or produces required secondary structure in rRNAs and tRNAs. The nontemplated nucleotide is added to the 3′ end of the RNA as the RNA is being synthesized during mitochondrial transcription. Because RNA editing is cotranscriptional, the mtRNAP is implicated in RNA editing as well as transcription. We have cloned the cDNA for the mtRNAP of Physarum and have expressed the mtRNAP in Escherichia coli. We have used in vitro transcription assays based on the Physarum mtRNAP to identify a novel activity associated with the mtRNAP in which non-DNA-templated nucleotides are added to the 3′ end of RNAs. Any of the four ribonucleoside triphosphates (rNTPs) can act as precursors for this process, and this novel activity is observed when only one rNTP is supplied, a condition under which transcription does not occur. The implications of this activity for the mechanism of RNA editing are discussed.

A cloned human CCAAT-box-binding factor stimulates transcription from the human hsp70 promoter

1990 ◽  
Vol 10 (12) ◽  
pp. 6709-6717
Author(s):  
L S Lum ◽  
L A Sultzman ◽  
R J Kaufman ◽  
D I Linzer ◽  
B J Wu
Keyword(s):  
Initiation Site ◽  
Dependent Manner ◽  
Hsp70 Gene ◽  
Hsp70 Promoter ◽  
Transcriptional Initiation ◽  
Enhancer Binding Protein ◽  
Binding Factor ◽  
Ccaat Box ◽  
Transcriptional Initiation Site ◽  
Human Hsp70

The basal promoter of the human hsp70 gene is predominantly controlled by a CCAAT element at position -70 relative to the transcriptional initiation site. We report the isolation of a novel cDNA clone encoding a 114-kDa polypeptide that binds to the CCAAT element of the hsp70 promoter. Expression of this CCAAT-binding factor (CBF) cDNA activated transcription from cotransfected hsp70 promoter-reporter gene constructs in a CCAAT-dependent manner. CCAAT-binding factor shows no homology to the previously identified human CCAAT transcription factor or rat CCAAT/enhancer-binding protein.

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