Extensive alteration of codon usage alone, however, can introduce deleterious pauses into an open reading frame. In addition,
changing the host organism effectively randomizes the pauses, making expression unpredictable. Managing codon pair interactions
and simultaneously optimizing the entire set of parameters requires advanced computationally intensive design tools. Successful
applications to explore and exploit the incredible diversity of potential gene sequences have been developed using a branch–bound
algorithm.15 The design process called "translation engineering," for example, simultaneously satisfies the five parameters listed above.12
Continuing use of these techniques for ever-increasing numbers of genes in diverse organisms will further test their utility.
To date, genes have been tailored for expression in E. coli, gram positive bacteria, yeasts, human cells, and plants, with frequently dramatic increases in protein yield.
The Future of Translation Engineering and the Role of Pausing in Complex Proteins
A rich series of biological events takes place during the translation of a protein. Deconvoluting the various aspects of co-translational
modification critically depends on modulating the speed of the ribosome. One example is the independent domain folding of
multi-domain proteins. Experimental evidence shows that proper folding of a yeast TY3 GAG gene (which has two independent
domains) expressed in E. coli can be altered by the placement of pause sites between the two domains.16 By modifying the translation kinetics of complex multi-domain proteins, it may be possible to alter the time each domain
needs to organize. Although refolding studies indicate that the time required for a protein to settle into its final configuration
may take as long or longer that the translation of the protein,17 pausing may allow each domain to partially organize, committing to a particular, independent fold. Other co-translational
events, such as association with membranes, secretion, proteolysis, or association with other proteins, may all depend on
the kinetics of the emerging nascent peptide.
Evolution has favored the over-representation of codon pairs that encode pause sites. This overabundance presumably is renewed
as the set of pause sites change over evolutionary time. It appears that tremendous genome-wide selection pressure exists
for cells to avoid creating misfolded proteins.18 Perhaps these codon pair-encoded pauses are selected as a consequence of this pressure. Would-be designers of genes should
consider taking advantage of this rich and biologically-used method for controlling the translation of genes and the activity
of their encoded proteins.
The authors wish to thank GW Hatfield, RH Lathrop, S-P Hung, and A Fraudorf.
Joseph D. Kittle, Jr, Ph.D., Senior Vice President, Market Development, CODA Genomics, Inc., 4521 Campus Drive, Irvine, CA 92612, Office 949 348 1188
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