Identification and analysis of frameshift sites.

There are several ways that genes may encode alternative products. The most widely recognized mechanism is alternative splicing. However, genes may also employ noncanonical translational events to produce such products. Some of these mechanisms operate at the level of translational initiation. In prokaryotes, genes may include alternative ribosome-binding sites directing the synthesis of products that differ at the N terminus. In eukaryotes, in which ribosome-binding sites do not exist, leaky scanning allows the same kind of variation. Noncanonical elongation events can also generate products that differ at their C terminus (–3). Such events include programmed readthrough of translational termination codons (4,5) translational frameshifts (–9), and translational hops (10,11). In each case, the ribosome fails to follow normal rules of decoding, leading to the synthesis of a protein that is not encoded, in the normal sense, in the DNA. In this chapter, we will describe the methods employed in the identification and analysis of programmed translational frameshift sites, including their discovery, measurement of the efficiency of the events, and determination of the mechanism of the frameshift.