Control of T even bacteriophage RNA synthesis in vitro

by Loren R. Snyder

Written in English
Published: Pages: 128 Downloads: 940
Share This
Classifications
LC ClassificationsMicrofilm 23772
The Physical Object
FormatMicroform
Paginationvii, 128 l.
Number of Pages128
ID Numbers
Open LibraryOL1368040M
LC Control Number92895336

  Using in vitro RNA replication by the transcription polymerase of T7 bacteriophage as an experimental model, we identify hundreds of new replicating RNAs, define three mechanistic hallmarks of replication (subterminal de novo initiation, RNA shape-shifting, and interrupted rolling-circle synthesis), and describe emergence from DNA seeds as a. This approach, together with RNA synthesis, for investigation of the sequence-dependent aminoacylation of RNA oligonucleotides in vitro has proved most useful for studying aminoacyl tRNA synthetases-tRNA recognitions in vivo. Totally synthetic genes lend themselves well to systematic mutagenesis by the principle of fragment (cassette) replacement. Template optimization for In Vitro Transcription. Bio-Synthesis provides enzymatic long RNA synthesis using transcription from DNA templates. In vitro transcribed RNA can be used as control template for Taqman SNP genotyping assays, drug metabolism genotyping assays or as positive control template for basic clinical research. A procedure for large-scale isolation of plasmid DNA without the use of RNase has been developed to obtain a DNA template for preparative in vitro RNA synthesis catalyzed by phage RNA polymerases.

  Characterization of RNA primer synthesis in the T4 bacteriophage in vitro DNA replication system. J Biol Chem , – Medline, Google Scholar; Liu CC, Burke RL, Hibner U, Barry J, Alberts BM (). Probing DNA replication mechanisms with the T4 bacteriophage in vitro system. Cold Spring Harb Symp Quant B – In Vitro Synthesis of RNA SP6, T7 and T3 phage RNA polymerases have high specificity for their respective 23 base promoters (Figure 1). The development of cloning vectors containing promoters for these polymerases has made the in vitro synthesis of single stranded RNA molecules a routine laboratory procedure. Antitermination is the prokaryotic cell's aid to fix premature termination of RNA synthesis during the transcription of occurs when the RNA polymerase ignores the termination signal and continues elongating its transcript until a second signal is reached. Antitermination provides a mechanism whereby one or more genes at the end of an operon can be switched either on or off, depending on. Small linear DNA molecules containing histone octamers (nucleosome cores) are transcribed by SP6 RNA polymerase with only slightly lower efficiency than naked DNA templates. The position of the histone octamer on the DNA remains unchanged even after multiple passages by the polymerase. The histone octamer is not released even transiently from the DNA by polymerase transit.

  Compared to bacteriophage transcription, the structure of bacterium promoters allows for a much larger modularity of transcription, even with a single transcription factor. The recent development of an E. coli cell-free system driven by the endogenous RNA polymerase as efficient as conventional bacteriophage cell-free systems opens previously. The RNA world is a hypothetical stage in the evolutionary history of life on Earth, in which self-replicating RNA molecules proliferated before the evolution of DNA and term also refers to the hypothesis that posits the existence of this stage. Alexander Rich first proposed the concept of the RNA world in , and Walter Gilbert coined the term in Numerous key discoveries in biology have emerged from studies of RNA (ribonucleic acid), including seminal work in the fields of biochemistry, genetics, microbiology, molecular biology, molecular evolution and structural of , 30 scientists have been awarded Nobel Prizes for experimental work that includes studies of RNA. Specific discoveries of high biological significance are.   A widely used molecular biology technique, in vitro transcription uses bacteriophage DNA-dependent RNA polymerases to synthesize template-directed RNA molecules. Enzymes like bacteriophage SP6, T3 and T7 RNA polymerases are used to produce synthetic RNA transcripts, which can be used as hybridization probes, as templates for in vitro translation applications, or in structural .

Control of T even bacteriophage RNA synthesis in vitro by Loren R. Snyder Download PDF EPUB FB2

Cystoviruses are the only bacteriophage that are more closely related to viruses of eukaryotes than to other phage. Bacteriophage Φ6 is a member of the Cystoviridae family.

It infects Pseudomonas bacteria (typically plant-pathogenic P. syringae). It has a three-part, segmented, double-stranded RNA genome, totalling ~ kb in length. Ikeda RA, Warshamana GS () In vivo and in vitro activities of point mutants of the bacteriophage T7 RNA polymerase promoter.

Biochemistry – PubMed CrossRef Control of T even bacteriophage RNA synthesis in vitro book Scholar Joho KE, Gross LB () Identification of a region of the bacteriophage T3 and T7 RNA polymerases that determines promoter : Elita Avota, Normunds Licis. Mol. Biol. () 19, Selective Synthesis of T-even BacteriophagEarly Messenger in vitro E.

GEIDUSCHEK, L. SNYI)ER, A. COLVILL AND M. SARNAT Department of Biophysics University of Chicago Ccago, Illinois, U.S.A. (Received 29 Marchand in revised forro 5 June ) Only rostricted portions of the native T2 and T4 DNA templates aro transcribed by Escherichia coli RNA Cited by: the bacteriophage @Opsu&r contains the gene for a single bac- terial suppressor tRNA (9).

Several laboratories have studied the process of tRNA transcription in an in vitro system consisting of $8Ops&r DNA and purified DNA-dependent RNA polymerase and p. Synthesis of specific RNA sequences in vitro is simplified because of the availability of bacteriophage RNA polymerases and specially designed DNA vectors.

RNA polymerases encoded by SP6, T7, or T3 bacteriophage genomes recognize particular phage promoter sequences of their respective viral genes with a high degree of specificity (1, 2, 3).Cited by: 3.

Synthesis of specific RNA sequences in vitro is simplified because of the availability of bacteriophage RNA polymerases and specially designed DNA vectors.

RNA polymerases encoded by SP6, T7, or T3 bacteriophage genomes recognize particular phage promoter sequences of therr respective viral genes with a high degree of specificity (1, 2, 3).

DNA synthesis in vitro using intact duplex T7 DNA as template is dependent on a novel group of three phage T7-induced proteins: DNA-priming protein (activity which complements a cell extract lacking the T7 gene 4-protein), T7 DNA polymerase (gene 5-protein plus host factor), and T7 DNA-binding protein.

The reaction requires, in addition to the four deoxyribonucleoside triphosphates, all four. Synthesis of RNA by In Vitro T can be used to inform vaccine design and FMD control strategies. In vitro transcription using bacteriophage RNA polymerases and linearised plasmid or.

First, further cost reduction is required for the in vitro synthesis of mRNAs. As compared to the protein production, in vitro synthesis offers much economical production of mRNAs in the laboratory scale.

However, for a large industrial production, there are unmet needs for the supply of rNTP, T7 RNA polymerase, and other enzymes. The event that occurs in bacteriophage multiplication that does not occur in animal virus replication is A.

adsorption to the host cells. injection of only the viral nucleic acid into the host cell. host cell synthesis of viral enzymes and capsid proteins. assembly of. nitiation of DNA replication in phage T7 chromosome. The primary origin of phage T7 (position –) contains two T7 RNA polymerase promoters, ϕA and ϕB, an AT-rich region, and a.

DNA synthesis was detected in the extract prepared from the sample collected 12min after infection. Optimal T7-DNA-directed DNA synthesis was ob- served in the 22 and min samples.

No incorpora- tion was seen when T7 DNA was omitted from the reaction mixture. The phage-induced RNA polymerase and DNA polymerase were also assayed. Enzymatic RNA Synthesis using Bacteriophage T7 RNA Polymerase.

Heike Gruegelsiepe. Search for more papers by this author. Book Editor(s): Description of Method‐T7 Transcription in vitro. Transcription Protocols.

Troubleshooting. Rapid Preparation of T7 RNA Polymerase. In bacterial transcription, diverse σ-family promoter recognition proteins compete for a common RNA polymerase core. Bacteriophage T4 infection ultimately reduces this competition to a duel between activated viral middle and enhanced late transcription, involving two σ proteins, two phage-encoded activator proteins and two phage-specific co-activators.

Geiduschek EP, Snyder L, Colvill AJ, Sarnat M. Selective synthesis of T-even bacteriophage early messenger in vitro. J Mol Biol. Aug; 19 (2)– HALL BD, NYGAARD AP, GREEN MH. CONTROL OF T2-SPECIFIC RNA SYNTHESIS. J Mol Biol. Jul; – NYGAARD AP, HALL BD. FORMATION AND PROPERTIES OF RNA-DNA COMPLEXES.

The RNA polymerase in E. coli cells infected with T4 phage is not identical with that in uninfected E. coli. A T4 phage protein is probably required to modify the host polymerase.

Materials and methods In vitro synthesis of labelled XmRNA (3H-AmRNA) was effected in the following conditions: 40 jug of iADNA (derived from strain C60OXCI avs) and 20 units of RNA polymerase; tris-HC M, pH ; MnClz M; MgC12 M; GTP M (30 pCi per mole); 3 other nucleoside triphosphates M; and 2.

VIROLOGY() In Vitro Synthesis of Biologically Active Beet Necrotic Yellow Vein Virus RNA l_. QUILLET,1 H. GUILLEY, G. JONARD, AND K. RICHARDS Institut de Biologie Moleculaire des Plantes, 12 rue du General Zimmer, Strasbourg, France Received Ma accepted Beet necrotic yellow vein virus (BNYVV) has a quadripartite plus-strand RNA.

It thus appears that the RNA phages f2 and Q, give rise to a less marked inhibition of ribosomal RNA synthesis than do phages R17 and R This observation must, therefore, be taken into account by any hypothesis attempting to explain the phage-induced inhibition of host RNA syn-thesis.

Demonstration that the rate of RNA synthesis determined by the fluorescence of molecular beacons in real time is proportional to the concentration of added bacteriophage T3 or bacteriophage T7 RNA polymerase. The DNA template used to initiate each group of transcription reactions contained a promoter that was specific for the added RNA polymerase.

The classic experiment presented in this test was designed to identify the template molecules of translation by analyzing the synthesis of phage proteins in Escherichia coli cells infected with bacteriophage T In a preliminary experiment, a small bacterial culture was grown in a medium of 15 N‐ and 13 C‐containing nutrients (15 N and 13 C are nonradioactive, heavy isotopes of nitrogen.

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. An in vitro T7 bacteriophage transcription system has been utilized in which the RNA was initiated to a specific length (defined by the absence of the appropriate nucleoside triphosphate).

Geiduschek EP, Snyder L, Colvill AJ, Sarnat M. Selective synthesis of T-even bacteriophage early messenger in vitro. J Mol Biol. Aug; 19 (2)– Milanesi G, Brody EN, Geiduschek EP.

Sequence of the in vitro transcription of T4 DNA. Nature. Mar 15; ()– Richardson JP. Enzymic synthesis of RNA from T7 DNA. One intriguing option is the reactivation of synthetic bacteriophage DNA, a process also known as “genome rebooting.” Some phage genomes can be rebooted either using cell-free systems (in vitro transcription-translation) (20, 21) or in E.

coli cells. Early m-RNA's code for early proteins which are needed for phage DNA synthesis and for shutting off host DNA, RNA and protein biosynthesis. In some cases the early proteins actually degrade the host chromosome.

After phage DNA is made late m-RNA's and late proteins are made. Studies on DNA Replication in the Bacteriophage T4 In Vitro System B.M. ALBERTS, J. BARRY, P. BEDINGER, T. FORMOSA, C.V. JONGENEEL, AND K.N. KREUZER Department of Biochemistry and Biophysics, University of California, San Francisco, California In vitro systems reconstituted from the purified com.

Tsugita A, Inouye M. Purification of bacteriophage T4 lysozyme. J Biol Chem. Jan 25; (2)– HALL BD, NYGAARD AP, GREEN MH.

CONTROL OF T2-SPECIFIC RNA SYNTHESIS. J Mol Biol. Jul; – Geiduschek EP, Snyder L, Colvill AJ, Sarnat M. Selective synthesis of T-even bacteriophage early messenger in vitro.

The in vitro synthesis of proteins in cell-free extracts is an important tool for molecular biologists and has a variety of applications, including the rapid identification of gene products (e.g., proteomics), localization of mutations through synthesis of truncated gene products, protein folding studies, and incorporation of modified or unnatural amino acids for functional studies.

Abstract. RNA was synthesized in vitro from a template of bacteriophage T4 DNA, in the presence of Mn 2+.A comparison was made of the RNA synthesized by purified RNA polymerase from two sources, Micrococcus lysodeikticus and Escherichia coli; these are referred to as Micrococcus cRNA and E.

coli cRNA respectively (where cRNA indicates RNA synthesized in vitro by using purified RNA. Bacteriophage Q protein engages σ-dependent paused RNA polymerase (RNAP) by binding to a DNA site embedded in late gene promoter and renders RNAP resistant to termination signals.A bacteriophage (/ b æ k ˈ t ɪər i oʊ f eɪ dʒ /), also known informally as a phage (/ f eɪ dʒ /), is a virus that infects and replicates within bacteria and term was derived from "bacteria" and the Greek φαγεῖν (phagein), meaning "to devour".Bacteriophages are composed of proteins that encapsulate a DNA or RNA genome, and may have structures that are either simple.

Geiduschek EP, Snyder L, Colvill AJ, Sarnat M. Selective synthesis of T-even bacteriophage early messenger in vitro.

J Mol Biol. Aug; 19 (2)– Rabussay D, Geiduschek EP. Phage T4-modified RNA polymerase transcribes T4 late genes in vitro. Proc Natl Acad Sci U S A. Dec; 74 (12)– [PMC free article].