Nonsense suppression

Most of the aforementioned efforts are based on the nonsense suppression methodology as the method for peptide altering [100]. In this approach, a nonsense codon is introduced into the enzyme-coding mRNA in the site that has to be altered. Simultaneously, the tRNA-noncoded amino acid hybrids are prepared with nonsense anticodons. Finally, the translation of modified mRNA is performed in vivo [101] or in vitro [102]. 

PSI] is the prion of Sup35p, a translation termination factor (Ter-Avanesyan et al., 1994 Wickner, 1994). Conversion of wild-type yeast cells to the infected state results in reduction of the termination activity and, consequendy, to a nonsense suppression phenotype. This property can be used to detect [PSI] by genetic selection (Fig. 2). Sup35p is an essential gene whose knockout leads to cell death. Therefore, it appears that the [PSI] condition corresponds to only partial inactivation of Sup35p and enough of the normal protein is left to avert cell death. 

Figure 10 Alteration of the genetic code for incorporation of non-natural amino acids, (a) In nonsense suppression, the stop codon UAG is decoded by a non-natural tRNA with the anticodon CUA. In vivo decoding of the UAG codon by this tRNA is in competition with termination of protein synthesis by release factor 1 (RFl). Purified in vitro translation systems allow omission of RF1 from the reaction mixture, (b) A new codon-anticodon pair can be created using four-base codons such as GGGU. Crystal structures of these codon-anticodon complexes in the ribosomal decoding center revealed that the C in the third anticodon position interacts with both the third and fourth codon position (purple line) while the extra A in the anticodon loop does not contact the codon.(c) Non-natural base pairs also allow creation of new codon-anticodon pairs. Shown here is the interaction of the base Y with either base X or (hydrogen bonds are indicated by red dashes).

While readthrough is usually a detrimental process, in some cases it can help to suppress problems, e.g. arising from premature stop codons present on the DNA level. This type of readthrough, also termed nonsense suppression, leads to the generation of a fraction of the full length protein in addition to the shortened version. Omnipotent suppressors cause nonsense suppression of all three termination codons. In this process, a near cognate tRNA successfully competes with the termination factors such that amino acid incorporation rather than premature termination of translation occurs. Omnipotent suppression can be caused by mutations in various factors involved in the process of translation termination. Nonsense suppression can also result from an aa-tRNA that decodes a termination codon (suppressor tRNA) in this case only one of the termination codons is efficiently suppressed (Hawthorne and Leupold 1974 Stansfield and Tuite 1994). 

The effect of mutations in eRFl and eRF3 on the efficiency of translation termination in yeast has been extensively studied. A variety of mutations in both translation termination factor subunits results in a nonsense suppression phenotype (Eustice et al. 1986 Song and Liebman 1989 All-Robyn et al. 1990 Wakem and Sherman 1990 Stansfield et al. 1995a, 1997 Bertram et al. 2000 Velichutina et al. 2001 Cosson et al. 2002 Bradley et al. 2003 Chabelskaya et al. 2004 Salas-Marco and Bedwell 2004). 

Weng Y, Czaplinski K, Peltz SW (1996) Identification and characterization of mutations in the UPFl gene that affect nonsense suppression and the formation of the Upf protein complex but not mRNA turnover. Mol Cell Biol 16 5491-5506... 

Suppressor tRNAs constitute an experimentally induced variation in the genetic code to allow the reading of what are usually termination codons, much like the naturally occurring code variations described in Box 27-2. Nonsense suppression does not completely disrupt normal information transfer in a cell, because the cell usually has several copies of each tRNA gene some of these duplicate genes are weakly expressed and account for only a minor part of the cellular pool of a particular tRNA. Suppressor mutations usually involve a minor tRNA, leaving the major tRNA to read its codon normally. 

Suppression should lead to many abnormally long proteins, but this does not always occur. We understand only a few details of the molecular events in translation termination and nonsense suppression. 

The amber suppressor tRNA (tRNASuP) is introduced to overcome the limitation associated with uniform incorporation of the noncoded amino acid into the target protein. The amino acid-loaded tRNASup incorporates the nonnatural amino acid at a specific site in the protein, corresponding to the nonsense suppression site in the genel144 Phe(4-Tmd)-tRNASup is used to study the interactions of translocating preprolactin with lipids and proteins at the endoplasmic reticulum membranei134 145 ... 

Case Study 4 Nonsense Suppression Techniques for Unnatural Amino Acids in Genetic Encoding... 

Salnikova AB, Kryndushkin DS, Smirnov VN, Kushnirov W, Ter-Avanesyan MD (2005) Nonsense suppression in yeast cells overproducing Sup35 (eRF3) is caused by its non-heritable amyloids. J Biol Chem 280 8808-8812... 

Fig. 2. Sup35p and Sup45p constitute the translation release (termination) factor (Stansfield et al, 1995 Zhouravleva et al, 1995). Thus, mutation of SUP35 or conversion of Sup35p to a prion increases the efficiency of weak nonsense-suppressing tRNAs.

A particular form of CNB is (2-nitrophenyl)glycine (Npg). This artificial amino acid (Npg, Fig. 3.2-ID) was successfully incorporated into ion channels like the nicotinic acetylcholine receptor [22] by nonsense suppression, a technique developed by Peter Schultz and coworkers [23-26]. Irradiation (4 h, >360 nm) of proteins containing Npg led to peptide backbone cleavage in Xenopus oocytes [22],... 

All these studies were performed in vitro. Some in vivo experiments with caged proteins engineered by nonsense suppression were successful, especially on the acetylcholine receptor. 

J. Schack, U.L. RajBhandary, A possible approach to site-specific insertion of two different unnatural amino acids into proteins in mammalian cells via nonsense suppression, Chem. Biol. 2003, 10, 1095-1102. 

Early work on the use of phenylalanine phosphonates in synthetic peptides as SH2 domain ligands and phosphotyrosine phosphatase inhibitors proved the efficicacy of these agents in medicinal chemistry [20, 22], Incorporation of phosphonomethylene alanine (Pma) and phosphonomethylene phenylalanine (Pmp) using nonsense-mediated suppression has also been shown to be feasible using in vitro translation [5], but this has not been used for practical applications, perhaps because of scale-up challenges. Pma and Pmp have not yet been used in vivo in nonsense suppression, presumably because of the limited cell permeability of the amino acids. 

One extreme view of chemical introduction of an extrinsic fluorescent probe is found in the case ofthe alanine derivative of the fluorophore 6-dimethylamino-2-acylnaphthalene (DAN) (Figure 4.23). This derivative fluorophore, given the trivial name Aladan, is incorporated into a polypeptide by solid-phase synthetic chemistry (although a molecular biology technique known as nonsense suppression is now available for the introduction of unnatural amino-acid residues into recombinant proteins). The fluorescent emission maximum (Tnax) of Aladan shifts dramatically on different solvent exposures, from 409 nm in heptane to 542 nm in water, yet at the same time remains only mildly changed by variations in pH or salt concentration. This compares to a maximum environment-mediated shift of around 40 nm for intrinsic tryptophan fluorescence. In addition, there is little spectral overlap between extrinsic Aladan fluorescence and intrinsic fluorescence from tryptophan or tyrosine. 

In this view, several different strategies have been pursued to incorporate noncoded amino acids, including peptide synthesis [7] native chemical ligation [8, 9], enzyme-catalyzed semisynthesis [10, 11], biosynthetic incorporation via auxotrophic bacterial strain expression [12,13], and nonsense suppression methodologies in cell-free [4] or whole-cell [14] expression systems. Both native chemical ligation and enzymatic semi-synthesis require careful tailoring of the experimental procedures, whereas biosynthetic incorporation methods are not site-specific and are... 

---