Coupled transcription

Figure 2 Comparison of cloning and expression methods. In the conventional strategy (left), dehydrogenase genes obtained by PCR amplification of the original source DNAs are cloned into overexpression plasmids and verified by sequencing. Those with the desired structure are individually transformed into suitable host strains and the proteins are obtained, either as crude extracts or as purified samples. In the proposed streamlined approach (right), full-length dehydrogenase genes obtained by chemical synthesis are used directly in coupled transcription/translation reactions to obtain the proteins of interest.

The RTS system includes two different technology platforms for cell-free protein expression as well as a number of tools for finding optimal conditions (Scheme 1.1). All expression systems use the T7-polymerase for transcription and an E. coli lyzate with reduced nuclease and protease activity for translation. The conditions are optimized for a coupled transcription/translation reaction so that the DNA can be directly used as the template. 

Operons that produce the enzymes of amino acid synthesis have a regulatory circuit called attenuation, which uses a transcription termination site (the attenuator) in the mRNA. Formation of the attenuator is modulated by a mechanism that couples transcription and translation while responding to small changes in amino acid concentration. 

Figure 7.15. Functions of cold-shock proteins (Csp s) as RNA chaperones. The model shows how Csp s assist in coupling transcription to translation. Cold-shock proteins bind relatively weakly to nascent mRNA extending from the RNA polymerase complex (RNAP) and maintain the mRNA in a linear form that can be bound to ribosomes and translated into protein. Under nonstressful conditions, the weakly binding Csp s are present at adequate concentrations to perform this chaperoning function. However, during cold stress, the propensity for RNA to form secondary structures that block translation becomes greater. This necessitates that a higher level of Csp s be present in the cell, to ensure that chaperoning of mRNA is effective. (Figure modified after Graumann and Marahiel, 1998.)...

Coupled transcription-translation in prokaryotes refers to the commencement of translation of an RNA molecule before its transcription from the DNA template is complete. Could such a situation arise in eukaryotes ... 

The mRNA required for in vitro translation can itself be produced by in vitro synthesis. Commercially available kits allow DNA cloned downstream of T7-, T3 or SP6-promoters to be transcribed effectively in vitro by the relevant RNA polymerases. In coupled transcription-translation, it should be remembered that translation of eukaryotic mRNA requires a 5 cap upstream of the initiation codon, and similarly, for prokaryotic translation there should be an appropriately positioned ribosome binding site. Commercial kits are also available for combined in vitro transcription and translation. 

Two genes (psbE and psbF) for polypeptides of Cyt b-559 have been,located and characterized from spinach [56], wheat [57], tobacco [58], Oenothera hookeri [58] and pea (D.L. Willey, unpublished). The gene for the 9 kDa polypeptide known to be associated with Cyt b-559 preparations was first of all located in spinach [59] and wheat [57] by hybrid-release translation or by coupled transcription-translation of fragments of chloroplast DNA. Nucleotide sequencing [56,57] revealed an open reading frame of 83 codons, encoding a protein with the known N-... 

Before embarking on a particular expression strategy it is useful to have some information regarding the hands-on time required before the first results can be obtained. The fastest turnaround times are achieved with cell lysates, such as the reticulocyte and wheat germ lysates, which only need to be primed with in vitro synthesized RNA (or DNA in the case of a coupled transcription/translation system). These systems potentially provide answers within a few hours but are rarely used because of the small quantities that they yield. Plasmid-based systems used for expression in E. coli, yeast, and in combination with the vaccinia-T7 vector system are relatively fast as well. These strategies require subcloning of the gene(s) of interest into a plasmid vector, which is then transfected into the... 

Figure 29.34 The CTD Coupling transcription to pre-mRNA processing. The transcription factor TFIIH phosphorylates the car boxy I-terminal domain (CTD) of RNA polymerase II, signaling the transition from transcription initiation to elongation. The phosphorylated CfD binds factors required for pre-mRNA capping, splicing, and polyadenyiation. These proteins are brought in close proximity lo their sites of action on the nascent pre-mRNA as it is transcribed during elongation.

Endo and co-workers at Ehime University, Matsuyama, Japan, have led the development of the most promising eukaryotic cell-free system to date, based on wheat embryos. A significant advance made by this group was the development of pEU expression vectors that have overcome many of the difficulties associated with mRNA synthesis for translation in a eukaryotic system [8]. In addition to extensive optimization of reaction conditions that have seen improvements in protein synthesis rates, Endo and colleagues have improved wheat extract embryo preparation protocols to enhance the stability of these systems to a remarkable extent [9]. When coupled with the dialysis mode of reaction, Endo et al. were able to maintain translational activity in a coupled transcription/ translation wheat embryo reaction for 150 hours, producing 5 mg of enzymatically active protein per mb reaction mixture [10]. This again represents a serious alternative to in vivo methods of large-scale protein production. 

Table 15.3 Conditions for coupled transcription/translation systems from . coli under reducing and oxidizing conditions...

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-... 

In addition to mRNA and tRNA, the third major class of RNA molecule required for protein synthesis is rRNA. Together with as many as 70 ribosomal proteins, rRNA folds into a two-subunit macromolecule complex called a ribosome (Chapter 5). In bacteria, the ribosomes attach to mRNA as it is being synthesized, thereby coupling transcription and translation. In eukaryotes, protein synthesis occurs in the cytoplasm, either by free ribosomes in the cytosol or by membrane-bound ribosomes associated with the endoplasmic reticulum. The differences between prokaryotic and eukaryotic protein synthesis are illustrated in Figure 26.3. 

Iborra F.J., Jackson D.A., Cook P.R. (2001). Coupled transcription and translation within nuclei of mammalian cells. Science 293 1139-1142. 

It has recently been found that there is some coupled transcription and translation in the nucleus of eukaryotic cells. 

Iborra, F. J., D. A. Jackson, and P. R. Cook. Coupled Transcription and Translation within Nuclei of Mammalian Cells. Science293, 1139-1142 (2001). [The primary research showing translation in the nucleus.]... 

The authors next turn to the more complex process of transcription in eukaryotes. The impossibility of coupling transcription and translation in eukaryotes as it occurs in prokaryotes (because of eukaryotic subcellular separation in the nucleus and cytoplasm) is pointed out. The three eukaryotic RNA polymerases that carry out transcription are described and related to the kinds of RNA they synthesize. The role of the eukaryotic TATA box and the TATA-box-binding protein in basal transcription are explained, as are other eukaryotic promoters and enhancers and some of the proteins... 

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