Analysis of yeast mitochondrial RNA synthesis using a homologous in vitro transcription system by John Clark Edwards

Cover of: Analysis of yeast mitochondrial RNA synthesis using a homologous in vitro transcription system | John Clark Edwards

Published in 1983 .

Written in English

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Statementby John Clark Edwards.
Classifications
LC ClassificationsMicrofilm 84/2029 (Q)
The Physical Object
FormatMicroform
Paginationv, 127 leaves : ill.
Number of Pages127
ID Numbers
Open LibraryOL2911723M
LC Control Number84142771

Download Analysis of yeast mitochondrial RNA synthesis using a homologous in vitro transcription system

In Vitro Analysis of the Yeast Mitochondrial RNA Polymerase. Authors; Stohl, L. L., and Clayton, D. () Yeast mitochondrial RNA polymerase is homologous to those encoded by bacteriophages T3 and T7. () Nucleotides flanking the promoter sequence influence the transcription of the yeast mitochondrial gene coding for ATPase Author: Elizabeth A.

Amiott, Judith A. Jaehning. Edwards JC, Levens D, Rabinowitz M. Analysis of transcriptional initiation of yeast mitochondrial DNA in a homologous in vitro transcription system.

Cell. Dec; 31 (2 Pt 1)– Martin NC, Underbrink-Lyon K. A mitochondrial locus is necessary for the synthesis of mitochondrial tRNA in the yeast Saccharomyces by: Edwards JC, Levens D, Rabinowitz M. Analysis of transcriptional initiation of yeast mitochondrial DNA in a homologous in vitro transcription system.

Cell. Dec; 31 (2 Pt 1)– Edwards JC, Osinga KA, Christianson T, Hensgens LA, Janssens PM, Rabinowitz M, Tabak by: Thus, yeast and human mtDNA are transcribed by phage T7-like single-subunit RNA polymerases (RNAP) called Rpo41 and POLRMT, respectively.

They are structurally homologous to T7 RNAP, but both yeast and human RNAPs require transcription factors to initiate transcription which includes: Mtf1 (in yeast) and mitochondrial transcription factor A /TFAM and mitochondrial transcription Author: Urmimala Basu.

Edwards JC, Levens D, Rabinowitz M. Analysis of transcriptional initiation of yeast mitochondrial DNA in a homologous in vitro transcription system. Cell.

Dec; 31 (2 Pt 1)– Elble R. A simple and efficient procedure for transformation of yeasts. Biotechniques. Cited by:   Highlights Mitochondrial (mt) RNA polymerases (RNAP) serve as an intriguing link between single and multi-subunit RNAPs.

Although structurally similar to T7 RNAP, the mt RNAPs depend on accessory transcription factors. Both components bind the promoter and work cooperatively to catalyze promoter-specific initiation. Induced-fit mechanism involving DNA bending is employed to. Using various mutant strains and nutritional manipulations, we investigated a potential role for cyclic AMP (cAMP) in the regulation of mitochondrial (mt) gene expression in the yeast Saccharomyces cerevisiae.

In RAS mutants known to have either abnormally low or high cellular levels of this nucleotide, we show that both mt transcription rate and overall mt transcript levels vary directly with.

We have isolated the translation system from yeast mitochondria and have reconstructed it in vitro. This submitochondrial system, composed of mitochondrial ribosomes, tRNA, pH 5 fraction and mRNA, is maximally active at 10 mM Mg2+ and mM KCl or NH4Cl.

NH4+ is more stimulatory than K+. IN VITRO TRANSCRIPTION OF CLONED EUKARYOTIC tRNA GENES O. Schmidt, B. Hovemann, S. Silverman, H. Yamada, J. Mao and D.

Soil Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, ConnecticutU.S.A. ABSTRACT Extracts from Xenopus germinal vesicles have been used to transcribe faithfully cloned Drosophila or yeast tRNA.

Initiation of transcription in yeast mitochondria: analysis of origins of replication and of genes coding for a Messenger RNA and a transfer RNA March Nucleic Acids Research 12(4) The Mrpolymerase is immunologically related to an Mrprotein purified from yeast as a primase, although it is thought to be a functional unit of mitochondrial RNA polymerase.

For in organello transcription assays followed by chase of newly synthesized transcripts, RNA synthesis in isolated yeast mitochondria was performed in the presence of uridine 5 0 -triphosphate. Analysis of the nucleotide sequence of the genetic locus for yeast mitochondrial RNA polymerase (RPO41) reveals a continuous open reading frame with the coding potential for a polypeptide of amino acids, a size consistent with the electrophoretic mobility of this enzymatic activity.

The transcription product from this gene spans the singular reading frame. This unit describes methods for importing in vitro–translated or recombinant proteins into isolated yeast mitochondria and for exporting mitochondrial proteins translated in the yeast mitochondrial matrix into the inner mitochondrial membrane.

The methods use mitochondria isolated from yeast cells and mitochondrial protein precursors derived. The in vitro transcriptional initiation sites of four yeast mitochondrial tRNA genes have been investigated in a run-off transcription assay. Precise initiation originating within the 9-nucleotide.

The analysis of the enzymatic properties of the yeast mitochondrial degradosome reconstituted in vitro from Suv3 and Dss1 proteins expressed in E. coli, provided significant insights into its function, revealing a remarkable degree of functional interdependence of the two subunits. The complex purifies as a heterodimer with the apparent.

A diversity of promoter structures It is evident that tremendous diversity exists between the modes of mitochondrial transcription initiation in the different eukaryotic kingdoms, at least in terms of promoter structures.

Within vertebrates, a single promoter for each strand exists, which may be unidirectional or bidirectional. In fungi and plants, multiple promoters are found, and in each. 2. The mitochondrial translation system. The number of mitochondrially encoded proteins is very small compared to the total number of mitochondrial proteins: inthe manually validated list of mitochondrial proteins of the MitoP2 database showed entries for yeast, for human and for mic studies on the basis of mass spectrometry of mitochondria.

Edwards JC, Levens D, Rabinowitz M. Analysis of transcriptional initiation of yeast mitochondrial DNA in a homologous in vitro transcription system. Cell. Dec; 31 (2 Pt 1)– Levens D, Morimoto R, Rabinowitz M.

Mitochondrial transcription complex from Saccharomyces cerevisiae. J Biol Chem. Feb 10; (3)– In vitro synthesized prepro-alpha-factor was translocated across Aspergillus nidulans microsomal membranes in either the homologous A.

nidulans or a yeast cell-free system. Our understanding of protein factors that modulate mitochondrial transcription or couple RNA synthesis with RNA processing and degradation is limited, and is derived primarily from genetic and biochemical studies in the budding yeast Saccharomyces cerevisiae (Calvo et al., ; Shadel, ).

To assess whether mtRNAP can cap RNA with NAD + and NADH, we performed in vitro transcription experiments (Figure 1 and Figure 1—figure supplement 1).We analyzed S. cerevisiae mtRNAP and a DNA template carrying the S.

cerevisiae mitochondrial 21S promoter (Deshpande and Patel, ), and, in parallel, human mtRNAP and a DNA template containing a derivative of the human mitochondrial. Jean-Claude Farré, Alejandro Araya, in Methods in Enzymology, In vitro and in organello approaches to study mitochondrial RNA editing.

The mitochondrial RNA editing process is poorly understood because of the lack of appropriate experimental approaches. Most current studies on mitochondrial gene expression in plant mitochondria were either based on the analysis of.

RNAs move within cells and often reside at specific locations. The need to detect those movements and locales across the transcriptome is pressing. We report an approach in living cells that provides a record of localization across the entire collection of RNAs a cell contains.

It relies on covalently marking the RNA when it directly encounters a specific site. Controlling RNA levels (balance between RNA synthesis and degradation) is vital for both the regulation and functioning of the mitochondrial system (28–30).

RNA degradation is mediated principally by (arguably) small multiprotein complexes, like the exosome in the cytoplasm of eukaryotes (31) or the degradosome in bacteria (32). Directed Alteration of Saccharomyces cerevisiae Mitochondrial DNA by Biolistic Transformation and Homologous Recombination.

Pages In Vitro Analysis of the Yeast Mitochondrial RNA Polymerase. Pages Analysis of Mitochondrial Protein Synthesis in Yeast. Pages   Using an in vitro oligonucleotide transcriptional system, we have assayed transcription initiation activity by T7 RNAP.

These assays have not only defined the limits of conventional de novo initiation on truncated promoters, but have identified novel activities of initiation of RNA synthesis. Masters BS, Stohl LL, Clayton DA () Yeast mitochondrial RNA polymerase is homologous to those encoded by bacteriophages T3 and T7.

Cell –99 PubMed CrossRef Google Scholar McAllister WT () Structure and function of the bacteriophage T7 RNA. Second, in yeast and other fungi as well as in mammals, mitochondrial transcription is known to involve RNA polymerase core enzymes related to T7 and T3 RNA polymerases (reviewed in Tracy and Stern, ), suggesting that if mitochondrial RNA polymerase were organelle encoded in plants, the genes would be easily identified.

Abstract. A single-subunit RNA polymerase, POLRMT, transcribes the mitochondrial genome in human cells. Recently, a factor termed as the mitochondrial transcription elongation factor, TEFM, was shown to stimulate transcription elongation in vivo, but its effect in vitro was relatively modest.

In the current work, we have isolated active TEFM in recombinant form and used a reconstituted in. Abstract. DmTTF is a Drosophila mitochondrial DNA-binding protein, which recognizes two sequences placed at the boundary of clusters of genes transcribed in opposite directions.

To obtain in vivo evidences on the role of DmTTF, we characterized a DmTTF knock-down phenotype obtained by means of RNA interference in -2 cells. By a combination of RNase protection and real-time RT–PCR. Edwards JC, Levens D, Rabinowitz M. Analysis of transcriptional initiation of yeast mitochondrial DNA in a homologous in vitro transcription system.

Cell. Dec; 31 (2 Pt 1)– Levens D, Lustig A, Rabinowitz M. Purification of mitochondrial RNA polymerase from Saccharomyces cerevisiae. J Biol Chem. Feb 10; (3)– Yeast Mitochondrial RNA Polymerase Is Homologous Those Encoded by Bacteriophages T3 and T7 Brian S.

Masters, Lori L. Stahl, and David A. Clayton Department of Pathology Stanford University School of Medicine Stanford, California Summary Analysis. RNA from yeast is the total RNA obtained from Saccharomyces cerevisiae. Application RNA from yeast is used in studies which use natural RNA in an in vivo and in vitro protein-synthesizing system.

The product is also used as a carrier RNA for in situ hybridization. It has been used for RNA coimmunoprecipitation. Preparation Note. Figure 1. Schematic representations of the mitochondrial recombination process and of the experimental and analysis workflow.

(A) Mitochondrial recombination during mating (adapted from Chen and Butow ).When crossing two yeast cells of opposite mating type, mitochondria fuse to form a long reticulum, spreading within the zygote.

This chapter reviews the methods that have been successfully used to date for the identification of RNA modification and editing enzymes. The development of recombinant DNA and RNA techniques and techniques for chemical synthesis of DNA and RNA offers adequate alternatives for obtaining synthetic or semisynthetic unmodified or partially modified RNA substrates suitable for analyzing enzymatic.

For gene expression studies, ng or 1 μg of RNA was reverse-transcribed with the iScript cDNA synthesis kit (Bio-Rad) according to the manufacturer’s specifications. qRT-PCR results were normalized against three reference genes (AT1G, AT2G, and AT2G), except for the mitochondrial gene expression analysis for which five.

Abstract. The transcripts of a 6Kbp region of the mitochondrial genome of S. cerevisiae, localized in the 21S rRNA-OXI1 span and including 12 tRNA genes (from tRNA(thr) to tRNA(ala)) and several G+C clusters, have been studied by analysis of in vitro capped primary transcripts and by fine mapping of the 5' ends of transcripts.

Once transcription is initiated, RNA polymerase is released from the DNA. Cell-free protein expression is the in vitro synthesis of a protein using translation-compatible extracts of whole cells.

In principle, whole cell extracts contain all the macromolecules and components needed for transcription, translation and even post-translational. The T7 bacteriophage RNA polymerase (RNAP) performs all steps of transcription, including promoter recognition, initiation, and elongation as a single-polypeptide enzyme.

Arabidopsis thaliana possesses three nuclear-encoded T7 phage-type RNAPs that localize to mitochondria (RpoTm), plastids (RpoTp), or presumably both organelles (RpoTmp).

Their specific functions are as yet unresolved. Abstract. The RNA helicase SUV3 and the polynucleotide phosphorylase PNPase are involved in the degradation of mitochondrial mRNAs but their roles in vivo are not fully understood. Additionally, upstream processes, such as transcript maturation, have been linked to some of these factors, suggesting either dual roles or tightly interconnected mechanisms of mitochondrial RNA .Mitochondrial DNA (mtDNA) is highly compacted into DNA-protein structures termed mitochondrial nucleoids (mt-nucleoids).

The key mt-nucleoid components responsible for mtDNA condensation are HMG box-containing proteins such as mammalian mitochondrial transcription factor A (TFAM) and Abf2p of the yeast Saccharomyces cerevisiae. To gain insight into the function and organization of mt.

In general, yeast mRNAs have short and rather unstructured 5′ UTRs (Kertesz et al. ). A genome-wide analysis of transcription start sites in yeast enabled the characterization of the 5′ UTR for ∼80% of yeast genes (Nagalakshmi et al.

). These data revealed an average 5′ UTR length of 50 nucleotides with.

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