RNA polymerases from bacteriophages

Mitochondrial DNA is transcribed by a nuclear-encoded RNA polymerase composed of two subunits. One subunit is homologous to the monomeric RNA polymerase from bacteriophage T7 the other resembles bacterial ct factors. 

The RNA polymerase from bacteriophage T3 is structurally and functionally very similar to T7 RNA Pol, yet the T3 RNA Pol exhibits a distinct, nearly exclusive promoter specificity (1—3). 

In contrast to the single-subunit RNA polymerase found in bacteriophages, the model prokaryotic RNA polymerase from E. coli is a multisubunit enzyme. This polymerase has a five-subunit core that forms a constricted, tunnel-shaped catalytic site [97]. Prokaryotic RNA polymerases require an additional subunit, a, for promoter-specific initiation of transcription [98, 99]. 

Chamberlin, M., McGrath, J., Waskell, L. New RNA polymerase from Escherichia coli infected with bacteriophage T7. Nature (Lond.) 228, 227-231 (1970). 

Schafer, R., Zillig, W. The effects of ionic strength on termination of transcription of DNAs from bacteriophages T4, T5 and T7 by DNA-dependent RNA polymerase from Escherichia coli and the nature of termination by factor q, Europ. J. Biochem. 33, 215-226 (1973b). 

Na salts of ribonucleotide triphosphates (Roche or Sigma) bovine serum albumin RNase-free, 20 mg/ml (Roche) RNasin ribonuclease inhibitor, 40 U/ml (Promega) both bacteriophage T7 RNA polymerase and RNA Cap structure analog m7G(5/)ppp(5/)G are from BioLabs DNase-RNase-free (Roche) complete EDTA-free proteinase inhibitors cocktail (Roche) pyruvate kinase (PK) (Roche). 

The exogenous RNA template (350 bp) used in the Amp-RT assay is prepared from a plasmid-cloned sequence of the encephalomyocarditis virus (EMCV) (positions 7114-7516, GenBank accession number M81861), and is generated by in vitro transcription of a T7-tagged PCR product by using T7 bacteriophage RNA polymerase. 

This method is based on the observation that some bacteriophage RNA polymerases are extremely specific for their own promoter sequences and for a specific strand (less than 1/500 from opposite strand) (Melton et al., 1984). Moreover, these polymerases do not initiate from nicks in the double-stranded DNA. 

Gell-free protein synthesis refers to a family of techniques in which ribosomes and translation factors are isolated from cells to synthesize polypeptides in vitro from a messenger RNA template. Proteins can also be expressed in vitro from a DNA template by exploiting the ability of bacteriophage RNA polymerases to synthesize mRNA transcripts from DNA. 

Bacteriophage RNA polymerases are available from a range of commercial suppliers, and transcription-only reactions should be performed according to the manufacturers instructions. The concentration of mRNA added to a subsequent translation reaction must be established empirically. Conditions for coupled tran-scription/translation are described in Section 15.6. 

The conditions for performing a wheat embryo cell-free translation reaction from an mRNA template are listed in Table 15.4. Points discussed for E. coli optimization in Section 15.6.1 are relevant to expression in wheat embryo extracts. However, it is worth noting that the wheat embryo system is better suited to the translation of added mRNA template, whereas coupled transcription/translation is better in the E. coli system. The principal reason for this difference is that transcription with bacteriophage RNA polymerases requires a relatively high Mg concentration (ca. 16 mM) E. coli translation-only reac-... 

As discussed in Chapter 10, mitochondria and chloroplasts probably evolved from bacteria that were endocytosed into ancestral cells containing a eukaryotic nucleus. In modern-day eukaryotes, both organelles contain distinct circular DNAs that encode some of the proteins essential to their specific functions. The RNA polymerases that transcribe mitochondrial (mt) DNA and chloroplast DNA are similar to polymerases from bacteria and bacteriophages. 

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