Prokaryotic Translation

See also The Genetic Code, Structure of tRNAs, Initiation of Translation, Prokaryotic Translation Regulation, Lactose Operon Regulation (from Chapter 26), Promoter Organization... 

In eukaryotes the introns are removed from the primary transcript by processing, to produce the mRNA that is translated. Prokaryotes lack the machinery to perform this processing consequently, the translation product of the primary transcript would not be functional because it would encode amino acid sequences that are specified by the in-tron sequences. The problem can be circumvented by using cDNA prepared from the... 

FIGURE 11.24 The properties of mRNA molecules in prokaryotic versus eukaryotic cells during transcription and translation. 

The Shine-Salgano interaction is a base pairing interaction that occurs during translation initiation in prokaryotes between the Shine-Dalgarno sequence on messenger RNA (mRNA) and the anti-Shine-Dalgamo... 

This chapter presents methods and protocols suitable for the identification and characterization of inhibitors of the prokaryotic and/or eukaryotic translational apparatus as a whole or targeting specific, underexploited targets of the bacterial protein synthetic machinery such as translation initiation and amino-acylation. Some of the methods described have been used successfully for the high-throughput screening of libraries of natural or synthetic compounds and make use of model universal mRNAs that can be translated with similar efficiency by cellfree extracts of bacterial, yeast, and HeLa cells. Other methods presented here are suitable for secondary screening tests aimed at identifying a ... 

Brandi, L., Fabbretti, A., La Teana, A., Abbondi, M., Losi, D., Donadio, S., and Gualerzi, C. O. (2006b). Specific, efficient, and selective inhibition of prokaryotic translation initiation by a novel peptide antibiotic. Proc. Nat. Acad. Sci. USA 103, 39-44. 

Kolb, V. A., Makeyev, E. V., and Spirin, A. S. (2000). Co-translational folding of a eukaryotic multidomain protein in a prokaryotic translation system. J. Biol. Chem. 275, 16597-16601. 

The protein synthesis machinery reads the RNA template starting from the 5 end (the end made first) and makes proteins beginning with the amino terminus. These directionalities are set up so that in prokaryotes, protein synthesis can begin even before the RNA synthesis is complete. Simultaneous transcription-translation can t happen in eukaryotic cells because the nuclear membrane separates the ribosome from the nucleus. 

The next process is similar in both eukaryotes and prokaryotes, and involves the translation of mRNA molecules into polypeptides. This procedure involves many enzymes and two further types of RNA transfer RNA (tRNA) and ribosomal RNA (rRNA). There is a specific tRNA for each of the amino acids. These molecules are involved in the transportation and coupling of amino acids into the resulting... 

Traditionally, prokaryotic expression, especially employment of E. coli-based vectors, has been the system of choice. However, bacteria are unable to provide many vital components required for post-translational modifications including various forms of glycosyla-tion or lipid attachment and protein processing, all of which can also be important for proper protein folding. For this reason, it is not surprising that much time and effort has been dedicated to the development of alternative systems, summarized in Tab. 1.2. 

Obviously, prokaryotic systems are easy to use, the costs for their large-scale applications are low, and no safety risks are involved. The drawbacks are their limited capacity for post-translational modifications and generally low yields of complex mammalian... 

Gribskov, M. (1992). Translational initiation factors IF-1 and eIF-2 alpha share an RNA-binding motif with prokaryotic ribosomal protein SI and polynucleotide phosphoryl-ase. Gene 119, 107-111. 

Most frequently, extracts of either prokaryotic or eukaryotic origin as such from Escherichia coli, wheat germ or rabbit reticulocytes are employed for cost reasons and availability. While those based on E. coli are unable of post-translational protein modification, eukaryotic extracts do allow synthesis of glycosylated or phosphorylated proteins to some extent when additional components, such as microsomes for glycosylation are added. Care needs to be taken with cell-free systems recombinated from the individual components when a native protein is to be produced that does not fold spontaneously... 

Technically, in vitro transcription is achieved from standard expression plasmids typically carrying SP6 or T7 promoters using marketed kits. Translation into the polypeptide may be either coupled directly to the transcription (in vitro TnT) or require isolation of the RNA. Again, a large number of suitable prokaryotic and eukaryotic cell extracts as well as complementation factors are freely available. 

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