Translational Efficiencies

Lindquist, S. (1980). Translational efficiency of heat induced messages in Drosophila melanogasler cells. Mol. Biol. 137, 151-158. 

Cell-free translation system, used for the identification of cloned genes and gene expression, has been investigated extensively as a preparative production system of commercially interesting proteins after the development of continuous-flow cell-free translation system. Many efforts have been devoted to improve the productivity of cell-free system [1], but the relatively low productivity of cell-free translation system still limits its potential as an alternative to the protein production using recombinant cells. One approach to enhance the translational efficiency is to use a condensed cell-free translation extract. However, simple addition of a condensed extract to a continuous-flow cell-free system equipped with an ultrafiltration membrane can cause fouling. Therefore, it needs to be developed a selective condensation of cell-free extract for the improvement of translational efficiency without fouling problem. 

Gallie, D. R. (1991). The cap and poly(A) tail function synergistically to regulate mRNA translational efficiency. Genes Dev. 5, 2108—2116. 

In the last few years, DNA microarrays have been utilized to perform simultaneous analysis of the translational status of thousands of mRNAs, thereby enabling a comprehensive view of translation efficiency and regulation. Analyses were performed on mRNAs isolated from various... 

No. Cap analog Kas x 1(T6 (M ly Ki (nM)b % Capping Relative translational efficiency in vitrod Relative translational efficiency in vivoe 5degradationg... 

Translational efficiency of luciferase mRNAs lacking a poly(A) tract and capped with anti reverse cap analogs in a RRL system. The relative translational efficiency was calculated as described in the text. Translational efficiency of luciferase mRNAs containing 60-nt poly (A) tract and capped with anti reverse cap analogs in MM3MG cells. Luciferase activity was normalized by the amount of luciferase RNA in the cells. Relative translational efficiency was calculated as noted previously. 

The orientation of the cap in synthetic mRNA is the major contributor to overall translational efficiency. Cap analogs blocked at the 3 -O position of the first nucleoside moiety [m73/dGp3G (4) and m27,3 °Gp3G (2)] as well as modified in the 2 -0 position (which does not participate in phosphodiester formation) [m72 dGp3G (3) and m27 2 °Gp3G (1)], are incorporated into RNA exclusively in the correct orientation (Stepinski et al., 2001), which... 

ARCAs are incorporated into RNA exclusively in the correct orientation to an extent that is similar to the standard cap (see previously), which makes them potentially useful compounds in terms of increasing translational efficiency when incorporated into RNA. Similarly, they should be effective for inhibiting protein synthesis as free analogs. To test the influence of the ARCAs on protein synthesis in vitro, we use the microccocal nuclease treated rabbit reticulocyte lysate system (RRL system) optimized for cap-dependent translation (Cai et al., 1999). Highly cap-dependent translation is achieved at 100 mM potassium acetate and 1.4 mM magnesium chloride. 

Grudzien, E., Stepinski, J., Jankowska-Anyszka, M., Stolarski, R., Darzynkiewicz, E., and Rhoads, R. E. (2004). Novel cap analogs for in vitro synthesis of mRNAs with high translational efficiency. RNA 10, 1479-1487. 

That this translational system can indeed detect a different (increased) translational activity when the extracts are programmed with capped mRNA can be seen from the results presented in Fig. 12.3C. On the other hand, a general reduction of the translational efficiency caused by... 

Clearly, the results emerging suggested that at least two nucleotides were modified, the absolute sequence position within the tRNA had yet to be established. Ching etalP showed that a Se U residue was present in the wobble position of the tRNA " from C. sticklandii. This study confirmed a notion that the modification probably affects the translation efficiency of certain transcripts, based on the level of modification by selenium. The authors speculated that the modification to seleno-tRNA (GAG) allowed for more efficient use of this tRNA species as compared to the tRNA (GAA). Even today, no definitive data exist to show that this modification alters the translation efficiency in these bacterial model systems. Nonetheless, these studies had established the chemical forms of Se U and mnm Se U, and established that they were derived from modifications to nucleotides that first required sulfur (S U and mnm S U), the mechanism by which selenium was inserted into the tRNA would not be definitively answered until many years later. 

Building on earlier work of Osawa and co-workers [55], Oliver and Kowal [52] tested the feasibility of introducing a noncoded amino acid at an unassigned codon in M. luteus. DNA templates were prepared which coded for 19-mer polypeptides containing either the unassigned codon AGA(Arg) or the termination codon TAG at position 13 under the control of a T7 RNA polymerase promoter. The corresponding tRNAs, produced as described in Sect. 2, were based on tRNA and acylated with phenylalanine. The tRNA was modified to prevent recognition by the alanine aminoacyl-tRNA synthetase and to increase translational efficiency. 

The addition of RNase inhibitor (50 units) to the translation reaction mixture (50 pL) may improve translational efficiency (Recommended products Promega Code No. N2611). 

The rate of vivo accumulation of both inhibitor proteins steadily Increases, reaching a steady state after nine hours. However, a second wound at nine hours results in a tripling of the steady state rate of inhibitor accumulation over the next several hours. The data Indicates that the second wound causes no change in the apparent translational efficiencies of the mRNA for Inhibitors I and II but causes Increased rates of inhibitor accumulation by providing more translatable inhibitor messages when the plant s translation system is operating at high efficiency. 

Figure 4. Time course analysis of the accumulation of Inhibitors I and II protein, translatable mRNAs and apparent translational efficiencies in leaves of singly and doubly wounded tomato plants. Key — —, Inhibitor I, single wound —O—, Inhibitor II, single wound — 9 —, Inhibitor I, double wound and — O —, Inhibitor II, double wound.

B In vivo accumulation of Inhibitors I and II proteins in wounded tomato leaves. C Apparent translational efficiencies. 

Alternatively, poor efficiencies of inhibitor mRNAs may be due to their Incorporation into rlbonucleoproteln particles (RNPs) such as found in sea urchin embryos (21). Newly made mRNA in the embryos is found in RNPs and they apparently have "weak" template activities while in these particles. The presence of newly synthesized tomato mRNA in similar particles might explain the apparently low translational efficiencies noted herein. The use of chaotropic buffers in the preparation of tomato leaf mRNA (11) would not differentiate between free or polysome-bound mRNAs and those complexed in RNPs. If an RNP or similar particle is involved, then its role must be a temporal one since a second wound does not repeat the phenomenon (Fig. 4). 

There should be no internal translation initiation signal (ATG) between a promoter sequence and a structural gene since an internal ATG sequence significantly reduces translation efficiency. The distance between the control regions (promoter and terminator) and the structural gene should not be too long. Otherwise a produced m-RNA would be less stable. 

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