Programmed ribosomal frameshifting: much ado about knotting!

R of the genetic code can be temporarily suspended to make room for a variety of specific tricks that direct synthesis of additional proteins from mRNAs that would not be predicted by their sequences. This ‘‘recoding’’ includes specification of the 21st encoded amino acid, selenocysteine (1, 2). In another form, recoding itself provides a sensing mechanism to regulate expression (3). In other cases mRNA can direct synthesis of a set ratio of two proteins that share some amino acid sequences. Kim, Su, Maas, O’Neill, and Rich, in this issue of PNAS (4), describe experiments that show the mRNA structural features important for one such case of ratio setting. We know little about the global contribution that recoding makes to the complexity of proteomes. It could be that many mRNAs encode, in addition to their standard products, minor products that so far would have escaped detection. These products are likely to have distinct functions and contribute to the biological complexity of the proteome. Clearly the complexity of the human proteome is far beyond the more than 100,000 human genes. The total mRNA population might be 250,000 from alternate splicing, editing, and use of alternate promoters. How much this complexity will be expanded by protein modification, protein splicing, and recoding is far from clear but a total of 500,000 might not be surprising. Certainly the contribution from recoding is completely unknown; however, specific examples being studied give some guide to what is to come. Recoding by redefinition of codons and translational bypassing are being studied in detail but the majority of known examples are programmed shifts in reading frame (3, 5). Depending on the shift site ribosomes can be instructed to slip into 11 or the 21 frame. Examples are found in all well-studied organisms from viruses to bacteria to humans, but in the case of human gene expression only one family of genes, antizyme, is known to use frameshifting and this is 11 (6, 7). Many more cases of 21 frameshifting are known partly because of their use by members of large families of viruses and bacterial insertion sequences. Programmed 21 frameshifting occurs at ‘‘slippery’’ shift sites. At these sites, initial codonanticodon pairing in the zero frame is followed by dissociation and re-pairing of the tRNA anticodon to mRNA at an overlapping codon. The great majority of cases involve realignment of the codon anticodon pairing of not just one tRNA but of two adjacent tRNAs in the ribosome (peptidyl tRNA and aminoacyl tRNA). Even though the pairing rules for re-pairing are more relaxed than those for initial pairing, tandem slippage gives signature shift sites of the general form X XXY YYZ where X, Y, and Z can even be the same nucleotides. Tandem slippage was first recognized for the programmed frameshifting that occurs close to the 39 end of the gag gene of many retroviruses (8). This frameshifting is required for synthesis of the GagPol fusion polyprotein as there is no independent ribosome entry to the pol gene. [In retroviruses that have a separate protease gene between gag and pol two frameshift events are required to synthesize the GagProPol fusion polyprotein (9, 10).] The potential for codon:anticodon alignment is not the only important feature for programmed 21 frameshifting. Frameshifting is stimulated by discrete mRNA structures that in nearly all cases are formed from contiguous sequence starting 5–9 nt 39 of the shift site. Although in HIV (11) and Escherichia coli dnaX (12, 13) this stimulating structure is a simple stem loop, and in IS911 it is a three-way junction (14), in most cases it is a pseudoknot as first recognized in studies on viruses (15–17). There are various types of pseudoknots, but the classical H-(hairpin) pseudoknot (18) requires pairing of the loop of a hairpin loop with downstream sequences, resulting in two stems that are connected by two loops. RNA pseudoknots are widely occurring structural motifs that are involved in various RNA functions (19). Pseudoknots were first recognized experimentally from studying the folding of the 39 end of the turnip yellow mosaic virus RNA (for review see ref. 20). They have since been found in many classes of RNA including: ribosomal RNA (21), catalytic and self-splicing RNA (22, 23), tmRNA (24–26), internal ribosome entry sites of some picornaviruses (27), and translational repression sites on mRNAs for some ribosomal proteins (28) and for certain T-even bacteriophage proteins (29, 30). However, the pseudoknots involved in recoding are unique in that, as they play their role as a structure, they are immediately unfolded and their now linear sequence serves as a template for decoding. Pseudoknots cause ribosomal pausing, raising the possibility that this delay at the crucial site allows more time for anticodon:mRNA realignment to occur. This hypothesis has been tested in two cases with the conclusion that pausing may be necessary for 21 frameshifting but it is not sufficient (31, 32)—something else must be going on. Much remains to be learned, including when the pseudoknot is melted out and with what the pseudoknot might interact (see ref. 33 for discussion). Kim et al. (4) describe experiments that functionally test the unique structural features of a frameshift stimulating pseudoknot from the plant virus beet western yellow virus (BWYV), a luteovirus. Replication of BYMV genomic RNA requires two virus-encoded functions, P1 and P2 (for review see ref. 34). The polymerase, P2, is expressed only as an extension of P1 when a few percent of the ribosomes, three-quarters of the way through the coding sequence for P1, shift reading frame into the overlapping ORF for P2 (16, 35, 36). This means that the P1 protein and the P1-P2 frameshifted protein share their first 461 aa. Then, each uses a different frame to decode the next 146 aa, and P1-P2 then continues for another 429 unique aa. The frameshift event is a 21 tandem shift at a G GGG AAC site (the same shift site used by simian retrovirus 1; ref. 37), stimulated by a pseudoknot 6 nt downstream (Fig. 1). Rich’s group (38) recently described a crystal structure of the BWYV pseudoknot.