Factors Affecting Translation of Messenger RNA\u27s In Vitro: Use of a GTP Analogue to Investigate Rates of Polypeptide Chain Elongation

The order of addition of amino acids to a growing protein is determined by the codon sequence of a messenger RNA molecule. This translation process was studied in vitro with a cell-free protein synthesis system derived from Escherichia coli. The rate of protein synthesis was proportional to the amount of messenger RNA added to the system. However, it was observed that different messenger RNA\u27s were not equally effective in promoting protein synthesis. Experiments were conducted to determine why the rate of protein synthesis depends on the type of messenger RNA

Cell-Free Protein Synthesis

The Nobel Prize in Medicine 1968 for interpretation of the genetic code and its function in protein synthesis and in Chemistry 2009 for studies of the structure and function of the ribosome highlighted the ground-breaking experiment performed on May 15, 1961 by Nirenberg and Matthaei and their principal breakthrough on the creation of "cell-free protein synthesis (CFPS) system". Since then the continuous technical advances have revitalized CFPS system as a simple and powerful technology platform for industrial and high-throughput protein production. CFPS yields exceed grams protein per liter reaction volume and offer several advantages including the ability to easily manipulate the reaction components and conditions to favor protein synthesis, decreased sensitivity to product toxicity, batch reactions last for multiple hours, costs have been reduced orders of magnitude, and suitability for miniaturization and high-throughput applications. With these advantages, there is continuous increasing interest in CFPS system among biotechnologists, molecular biologists and medical or pharmacologists

Optimisation of a transcription-translation coupled in vitro system

Cell-free protein synthesis exploits the catalytic machinery of the cell to produce active proteins. An in vitro system is flexible and well controlled, and it offers several advantages over conventional in vivo technologies such as easy ways for purification, synthesis of regulatory and/or toxic proteins, incorporation of artificial or modified amino acids that might be doted with isotopes required for NMR. Here I describe experiments exploring optimisation possibilities concerning yield and quality of the synthesised protein. Some experimental strategies also include expression of eukaryotic genes in prokaryotic expression systems. The following results have been achieved: 1: Quality criteria developed that allow a critical evaluation of parameters important for the coupled transcription/translation system or improving the yield and quality of the synthesized protein exploiting the features of the green fluorescent protein GFP. 2: The standard transcriptase used in overexpression studies in vivo and in vitro is the T7 polymerase. The fundamental difficulty with this enzyme is the fact that it is about six times faster than the E. coli transcriptase and thus uncouples transcription from translation, a possible reason for the fact that in vitro systems usually produce proteins with an activity of 30 to 60% only. We tested some slow mutants of T7 polymerase that approached the rate of the E. coli transcriptase and observed indeed a significant improvement up to 100% of the active fraction, although at the cost of lower yields. 3: A similar improvement of the active fraction was observed at lower incubation temperatures down to 20°C, again at the cost of lower yields. 4: According to literature data some amino acids are metabolised during in vitro incubations and thus could cause a limitation of protein synthesis. Indeed, we demonstrate that a second addition of amino acids in the middle of the incubation triggers a burst of further protein synthesis. Using this trick at 20°C pushed the yield of protein to almost that seen at 30°C, but now with an active fraction of 100%. In contrast, our analysis revealed that NTPs are not limiting the gene expression in vitro in our system (modified Roche RTS). 5: It is known that the codon usage of highly and lowly expressed proteins in E. coli differs dramatically. When we examined this point with human genes, to our surprise a corresponding difference could not be observed. Due to this fact it was possible to identify 11 tRNAs the corresponding codon are quite often used in human genes but rarely in E. coli genes. Therefore, for a good expression of eukaryotic genes in E. coli systems these 11 tRNAs should be added (and not only the 7 tRNAs supplied in systems from Novagen). 6: I outlined some ways to improve further the expression system

Biological activity of avian myeloblastosis virus RNA in cell-free systems for protein synthesis

Avian myeloblastosis virus RNA was fractionated into a high molecular weight RNA and a low molecular weight RNA fraction by sucrose density centrifugation. The larger RNA component had a sedimentation constant of 65S and the low molecular weight RNA about 4S. The low molecular RNA has two biological properties of interest. First, it behaves like tRNA in that it will attach amino acids in the presence of aminoacyl synthetases and second, it is a potent inhibitor of in vitro protein synthesis. The properties of this RNA fraction with regard to in vitro peptide synthesis were investigated in the E. coli and chick polysome cell-free systems. Peptide synthesis using a S30 preparation from E. coli, is inhibited about 50% with 0.5 μg of virus RNA when either Qß phage RNA or E. coli RNA is used as a template. Peptide synthesis directed by the homopolymers poly U and poly A are not inhibited by the low molecular weight virus RNA. When poly C is used as a template, proline polymerization is inhibited. Excess ribosomes added to the in vitro system did not reverse the inhibition by virus RNA. Increasing either mRNA or the ribosome-free supernatant reversed the inhibition. Peptide synthesis, using chick polysome cell-free systems was inhibited to a lesser extent. The results of these studies are consistent with a mechanism by which inhibition occurs prior to the formation of the messenger-ribosome complex. The transfer activity of the low molecular weight virus RNA fraction was examined. Virus RNA charged with amino acid transferred amino acids to TCA-precipitable protein to a degree similar to that transferred by liver aminoacyl-tRNA. Low molecular weight virus RNA appears to contain an RNA fraction with properties identical to tRNA. The development of systems for protein synthesis from chick cells was described. A ribosome preparation, capable of using poly U and Qß RNA as templates for protein synthesis, contained low endogenous mRNA activity and incorporated phenylalanine linearly for 30 minutes in the presence of poly U. Results using a chick polysome cell-free system were described. The endogenous message-containing system required ATP and GTP for incorporation, was sensitive to the addition of puromycin and incorporated amino acids when added in the form of aminoacyl-tRNA. Biological activity of high molecular weight virus RNA was examined in cell-free systems for protein synthesis. No inhibition of protein synthesis was seen using intact, heat-treated and alkaline-treated virus RNA. Added virus RNA stimulated amino acid incorporation in cell-free systems from E. coli and chick cells. This is used as an evidence that high molecular weight virus RNA may serve as a template for protein synthesis in vivo

Single-Molecule Studies Of tRNA Dynamics During Ongoing Translation

The pre-translocation complex of the ribosome can undergo spontaneous fluctuations of mRNA and tRNAs between classical and hybrid states, and occupation of the hybrid tRNA positions has been proposed to precede translocation. The classic and hybrid state tRNA positions been extensively characterized when the ribosome is stalled along the messenger RNA by either the absence or the delayed addition of elongation factor G (EF-G), or by the presence of antibiotics or GTP analogs that block translocation. Surprisingly, during multiple ongoing elongation cycles when both EF-G and ternary complexes are present, we found that tRNA positions in PRE complex ribosome do not fluctuate. Instead, they adopt a stationary intermediate structure between the stalled classical and hybrid tRNA positions, as indicated by single molecule fluorescence resonance energy transfer (FRET) between adjacent tRNAs and between A-site tRNA and ribosomal protein L11. These results indicate that EF-G promotes the formation of an intermediate structure during ongoing translation. smFRET experiments using labeled EF-G at those concentrations requires zero-mode waveguides (ZMWs), arrays of nanoscale holes in a thin metal film that confine the observation volume to reduce prohibitive background fluorescence. However, the need for specialized nanofabrication equipment has precluded their widespread adoption by the biochemical community. In contrast, nanosphere lithography uses the self-assembly of polystyrene beads into a hexagonal array, forming a natural lithographic mask for the deposition of metallic posts in the interstices between beads, followed by a different metal cladding around the posts and dissolution of the posts to leave a well array. The cross-sectional size of those interstices (and thus subsequent posts and wells) can be finely tuned by fusing the beads at the polystyrene glass transition temperature. That bead lattice serves as a template for creating sub-wavelength ZMWs with little specialized equipment and at a low cost, enabling wide-scale adoption