Polymerase specific polymerases

The inhibitors of RNA polymerase, which generates RNA from DNA, inhibit a crucial step in gene expression. Inhibition of the eukaryotic form of RNA polymerase is used in cancer chemotherapy and is also an important experimental tool. For example, actinomy-cin D binds to the guanine residues in DNA and blocks the movement of the eukaryotic RNA polymerase. Specific inhibitors of bacterial RNA polymerase can be used as antibacterial agents. Most of these inhibitors like rifamycin bind to the prokaryotic enzyme. 

Toniutto P, Fabris C, Bitetto D, Fornasiere E, Rapetti R, Pirisi M (2007) Valopicitabine dihydrochloride a specific polymerase inhibitor of hepatitis C virus. Curr Opin Investig Drags 8 150-158... 

Samson-Himmelstjerna, G. von, Woidtke, S., Epe, C. and Schnieder, T. (1997) Species-specific polymerase chain reaction for the differentiation of larvae from Diclyocaulus viviparus and Dictyocaulus eckerti. Veterinary Parasitology 68, 119-126. 

Studies of the alphavirus life cycle have revealed how heavily the virus relies on cellular processes for replication. The paucity of functions that seem unique to the virus is striking. The binding of the virus to the cell surface and the fusion of its membrane intracellularly depend on the viral spike glycoproteins. RNA-dependent RNA polymerases specific for the virus catalyze the replication of the viral RNA. Exit from the cell requires the interaction of the viral spike proteins with the viral capsid... 

Polymerase Chain Reaction and Methylation-Specific Polymerase Chain Reaction... 

BDNF, brain-derived neurotrophic factor DAT, dopamine transporter DRD, dopamine receptor MAOA, monoamine oxidase A MB-catechol-O-methyltransferase QM-MSP, quantitative multiplex methylation-specific polymerase chain reaction RELN, reelin TH. These primers are suitable for QM-MSP. 

Fig. 9.2 Representative examples of the methylation-specific polymerase chain reaction (MSP) analyses for gene promoter regions. Lanes Lf and M indicate the presence of unmethylated and methylated template, respectively. Placental DNA (PDNA) and in vitro methylated DNA (IMD) served as negative and positive controls, respectively. Water (H) was used to detect contamination. Samples 1, 3, 4, and 7 indicate the presence of a methylated promoter DNA with various degrees of methylation, and samples 2, 5, and 6 represent an unmethylated promoter...

Fig. 9.4 Establishment of the quantitative methylation-specific polymerase chain reaction (MSP) analytical procedure. The ABI PRISM 7900 HT Sequence Detector system was used to perform real-time polymerase chain reaction (PCR) using MSP primers and bisulfite-modified template DNA. Upper panels Setting up the conditions to obtain the standard curves with 50% (A) or 25% (B) sequential dilution of the template. Lower panels The amplification curves on the left represent P-actin, unmethylated, and methylated MSP products, respectively, for reelin (RELN) (C). Amplification curves were compared at the set threshold before 40 cycles. Amplification curves from various samples are shown in the lower panel right (D)...

Galm, O., and Herman, J. G. (2005) Methylation-specific polymerase chain reaction. Methods Mol. Med. 113, 279-291. 

Roberts, R. L., Barclay, M. L., Gearry, R. B., and Kennedy, M. A. (2004) A multiplexed aUele-specific polymerase chain reaction assay for the detection of common thiopurine S-methyltransferase (TPMT) muta tions. Clin. Chim. Acta. 341, 49-53. 

Unlike DNA polymerase, RNA polymerase does not require a primer to initiate synthesis. Initiation occurs when RNA polymerase binds at specific DNA sequences called promoters (described below). The 5 -triphos-phate group of the first residue in a nascent (newly formed) RNA molecule is not cleaved to release PPj, but instead remains intact throughout the transcription process. During the elongation phase of transcription, the growing end of the new RNA strand base-pairs temporarily with the DNA template to form a short hybrid... 

At least three types of proteins regulate transcription initiation by RNA polymerase specificity factors alter the specificity of RNA polymerase for a given promoter or set of promoters repressors impede access of RNA polymerase to the promoter and activators enhance the RNA polymerase-promoter interaction. 

A satisfactory mathematical model for initiation of transcription supposes that the polymerase and DNA bind reversibly to form a complex with formation constant Kf. This initial specific polymerase-promoter complex is referred to as a closed complex because it is thought that the bases in the DNA chain are all still paired. It is postulated that in a rate-determining step the closed complex is converted into an open complex, which is ready to initiate mRNA synthesis (Eq. 28-1).26 67 In the open complex the hydrogen bonds... 

Baumann, R. E., and Henschen, A. H. (1993). Human fibrinogen polymorphic site analysis by restriction endonuclease digestion and allele-specific polymerase chain reaction amplification Identification of polymorphisms at positions A alpha 312 and B beta 448. Blood 82, 2117-2124. 

Polynucleotide polymerases, or nucleotidyl transferases, are enzymes that catalyze the template-instructed polymerization of deoxyribo- or ribonu-cleoside triphosphates into polymeric nucleic acid - DNA or RNA. Depending on their substrate specificity, polymerases are classed as RNA- or DNA-dependent polymerases which copy their templates into RNA or DNA (all combinations of substrates are possible). Polymerization, or nucleotidyl transfer, involves formation of a phosphodiester bond that results from nucleophilic attack of the 3 -OH of primer-template on the a-phosphate group of the incoming nucleoside triphosphate. Although substantial diversity of sequence and function is observed for natural polymerases, there is evidence that many employ the same mechanism for DNA or RNA synthesis. On the basis of the crystal structures of polymerase replication complexes, a two-metal-ion mechanism of nucleotide addition was proposed [1] during this two divalent metal ions stabilize the structure and charge of the expected pentacovalent transition state (Figure B.16.1). 

No. Eukaryotic RNA polymerases have been isolated from many tissues, and in all cases, three distinct enzymes have been found in the nucleus. All contain a number of polypeptide subunits and are complex in structure, RNA polymerase I is known to be involved specifically in the transcription of rRNA genes. RNA polymerase II gives rise to transcripts that are subsequently processed to yield mRNA. RNA polymerase 111 is responsible for the transcription of the tRNA genes and a small ribosomal RNA gene that yields a species called 55 RNA. The three polymerases are distinguishable from one another by their differential sensitivity to the drug a-amanitin (the toxic principle of the mushroom Amanita phalloides), which does not affect bacterial RNA polymerase. RNA polymerase... 

EXPERIMENT 22 In Vitro Transcription from a Plasmid Carrying a T7 RNA Polymerase-Specific Promoter... 

In this experiment, you will carry out an in vitro transcription reaction from a plasmid (pSP72, Fig. 22-4) containing a T7 RNA polymerase specific promoter. A 32P-labeled nucleoside triphosphate (labeled at the a position) will be included in the reaction to produce a radioactive RNA molecule. The size and sequence of the various transcripts that you will produce can be predicted by the restriction enzymes that the plasmid will be digested with prior to the beginning of the transcription reaction. Since the plasmid template for the transcription reaction will be digested, you will be preparing runoff transcripts from the T7 promoter. As a result,... 

The chemistry of RNA synthesis is identical for all forms of RNA, including messenger RNA, transfer RNA, and ribosomal RNA. The basic steps just outlined also apply to all forms. Their synthetic processes differ mainly in regulation, posttranscriptional processing, and the specific polymerase that participates. 

Holland, P.M. Abramson, R.D. Watson, R. Gelfand, D.H. Detection of specific polymerase chain reaction product by utilizing the 5 to 3 exonuclease activity of Thermus aquaticus. Proc. Natl. Acad. Sci. 1991, 88, 7276-7280. 

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