Cellfree Translation Systems

One of the most remarkable discovery of the 1950s was the understanding that protein synthesis does not require the integrity of the cell and can be performed after cell disruption. This laid the basis for the creation of the so-called cell-free translation systems. The incorporation of amino acids into proteins in cell homogenates, in cell extracts, and in cell-free fractions containing microsomes was demonstrated long time ago; perhaps the first examples were the cell-free systems from animal tissues, specifically from rat liver, described by Siekevitz and Zamecnik in 1951 and by Zamecnik in 1953. It was shown soon thereafter that the incorporation of amino acids corresponding to protein synthesis in a cell-free system proceeds on ribonucleoprotein particles or ribosomes (Zamecnik's group, 1955). Cell-free protein-synthesizing systems with bacterial (E. coli) ribosomes were developed almost simultaneously in Zillig's, Zamecnik's, and Tissieres' groups during 1959 and 1960 (Schachtschabel & Zillig, 1959; Lamborg & Zamecnik, 1960; Tissieres, Schlessinger & Gros, 1960). All of these systems were programmed by endogenous mRNAs; in these systems ribosomes simply continued to synthesize polypeptides upon the mRNA molecules to which they were attached at the time of cell disruption. In 1961 Nirenberg and Matthaei improved the system, separated ribosomes from endogenous messages, and introduced the exogenous template for polypeptide synthesis (Matthaei & Nirenberg, 1961; Nirenberg & Matthaei, 1961). One of their main achievements was the use of synthetic polynucleotide templates prepared by polynucleotide phosphorylase, including the simple templates, such as poly(U) and poly(A). It is this innovation that made possible to break the genetic code.

Today, cell-free protein-synthesizing systems may be reconstituted from well-characterized, highly purified components, including ribosomes, template polynucleotides, and a set of aminoacyl-tRNAs or a system of tRNA aminoacylation, i.e. tRNA, amino acids, ATP, and aminoacyl-tRNA synthetases. In addition, the system should be supplied with a set of special proteins called elongation factors, as well as with GTP. The simplest cell-free ribosomal system of polypeptide synthesis, which can be used to study the fundamental mechanisms of translation, includes only six high-molecular-mass components plus GTP; for example, the poly(U)-directed system may be reconstituted from the following ingredients:

E. coli 70S ribosomes,

Phe-tRNA,

EF-Tu (protein with a molecular mass of 47,000 daltons), EF-Ts, (protein with a molecular mass of 34,000 daltons), EF-G (protein with a molecular mass of 83,000 daltons), GTP.

As a result of poly(U) translation, polyphenylalanine is synthesized.

For translating natural cellular mRNA and viral RNA, the prokaryotic cell-free system should be supplemented by a complete set of aminoacyl-tRNA, three proteins necessary for initiating translation (IF1, IF2, and IF3), and three proteins necessary for terminating translation (RF1, RF2, and RF3). When eukaryotic 80S ribosomes are used for cell-free translation, all corresponding protein factors should be of eukaryotic origin. These include the elongation factors, namely eEF1 which is equivalent to bacterial EF-Tu plus EF-Ts, and eEF2 equivalent to bacterial EF-G, numerous initiation factors (eIF1, eIF2, eIF3, eIF4A, eIF4B, eIF4C, eIF5, etc.), and one high-molecular-mass termination factor (eRF). In addition, initiation in eukaryotic systems requires ATP.

Usually, however, crude cell extracts comprising all these endogenous components and factors are used in a routine laboratory practice. Pre-incubation of the cell extract at physiological temperature is often sufficient to remove the endogenous mRNA from the ribosomes, due to the digestion of it by endogenous nucleases. The vacant ribosomes of the extract accept either exogenous natural mRNA or synthetic polynucleotides as templates. The treated extract including ribosomes, tRNAs, ARSases, and translation factors, in addition to an exogenous message for polypeptide synthesis, should be also supplemented with amino acids, ATP, GTP and ATP/GTP regenerating system (either phosphoenol pyruvate and pyruvate kinase, or creatine phosphate and creatine kinase, or acetyl phosphate and acetyl kinase).

An alternative strategy is the use of partially fractionated cell extract. Thus, ribosomes and all RNA are removed from the extract by ultracentrifugation with subsequent DEAE cellulose treatment, and the remaining extract fraction (the so-called S100 fraction which means "supernatant prepared at 100,000 g") is combined with purified ribosomes, total tRNA, and mRNA. In this case the S100 fraction contains all necessary protein translation factors and ARSases. Again, amino acids, ATP, GTP and ATP/GTP regenerating system should be added.

Sometimes it is expedient to produce mRNA immediately in the translation system, rather than to add an isolated mRNA (DeVries & Zubay, 1967; Gold & Schweiger, 1969). It is found to be easy in the case of prokaryotic systems, since prokaryotic cell extracts contain RNA polymerase. Then a corresponding DNA species, such as plasmid, isolated gene, synthetic DNA fragment, or viral DNA, is added to the DNA-free extract instead of mRNA, and the proper mRNA is synthesized by the endogenous RNA polymerase in situ. In this case ribosomes start to translate the nascent chains of mRNA, even prior to the completion of their synthesis. That is why such systems are called coupled transcription-translation systems. Of course, the coupled systems should be supplemented with all four nucleoside triphosphate for RNA synthesis, rather then with just ATP and GTP required for translation alone.

The eukaryotic extracts are prepared from the cytoplasmic fraction, so that they lack an endogenous RNA polymerase activity. This limitation can be overcome by addition of a prokaryotic RNA polymerase - usually bacteriophage T7 or SP6 RNA polymerase - to the eukaryotic extract, in order to produce mRNA in situ using DNA species with corresponding T7 or SP6 promoters. In this case, however, no real coupling between transcription and translation takes place, since the bacteriophage RNA polymerases work much faster than the translation system. Nevertheless, the eukaryotic transcription-translation systems of this type are found to be practical and productive.

The ionic strength and specifically the Mg2+ concentration are important factors for the cell-free systems. The usual range of Mg2+ concentrations, within which ribosomes are active in the cell-free system, extends from 3 to 20 mM; the optimum is somewhere between these values and depends on the ribosome origin and monovalent cation (K+ or NH4+) concentration, as well as on the concentration of di-and polyamines; it also depends on the incubation temperature. As a rule, SH-compounds, such as mercaptoethanol, dithiotreitol or glutathione, should be present in the translation mixture in order to maintain the reduced state of translation factors.

One principal shortcoming of all cell-free translation and transcription-translation systems should be mentioned: in contrast to the in vivo protein synthesis, they have short lifetimes and, as a consequence, give a low yield of the protein synthesized. This makes them useful only for analytical purposes and inappropriate for preparative syntheses of polypeptides and proteins. Indeed, the bacterial (E. coli) cellfree systems are usually active during 10 to 60 min at 37°C. The systems based on rabbit reticulocyte lysate or wheat germ extract are capable of working during one hour, although in some cases the lifetime may be prolonged up to 3 or 4 hours.

It has been found (Spirin et al., 1988) that the above shortcoming can be conquered, if the incubation is performed under conditions of continuous removal of the products (synthesized polypeptide, AMP, GDP, inorganic phosphates, etc.) and continuous supply with the consumable substrates (amino acids, ATP and GTP). This can be achieved with the use of a porous barrier limiting the reaction mixture. One way is to pass the flow of the substrate-containing solution through the reactor (continuous-flow cellfree system); the outflow will remove the products through the barrier including the protein synthesized, provided the proper membrane is selected. It is interesting that the components involved in translation (or transcription-translation) are retained in the reactor volume under these conditions, even when some of them (in an individual state) are smaller than the barrier (membrane) pores. From this it is likely that the components of the protein-synthesizing system in a functional state are present as large dynamic complexes with each other. Another way, but still based on the same principle, is to put the reaction mixture into a dialysis bag or other dialysis device against a large volume of the substrate-containing solution; during incubation there will be the removal of the low-molecular-mass products and the provision with new portions of the consumable substrates through the dialysis membrane (continuous-exchange cell-free system). In this case, however, the protein synthesized is retained in the reactor. The lifetimes of the systems described, especially of the flow system, increases up to 50 hours at least, both for prokaryotic and eukaryotic ones. The yields of proteins synthesized are typically around 100 to 200 mg, and may be up to 1 mg in some cases, from 1 ml reactor.

The most important information regarding translation and its molecular mechanisms has been obtained with the aid of cell-free systems of different types.

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  • tanja foerster
    What does cell free fraction contain?
    8 years ago

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