Ribosomal frameshifting and transcriptional slippage: From genetic steganography and cryptography to adventitious use

Atkins, John F.; Loughran, Gary; Bhatt, Pramod R.; Firth, Andrew E.; Baranov, Pavel V.

Single molecule FRET and small angle X-ray scattering experiments on the E. coli dnaX shift cassette (403, 518, 579) , optical tweezer experiments and detailed product characterization (392) and parallel ensemble FRET kinetic experiments on a variant of the infectious bronchitis virus frameshift cassette (450) , have greatly refined knowledge of −1 frameshifting, part of which will be discussed after some comments on the systems used (also see (580) ). One of the smFRET experiments used a strengthened variant of the dnaX stem loop structure with a G inserted opposite a bulged C, and the A of an A:G apposition changed to a C (518) , and the other used a stem with the same number of base pairs without any unpaired bases, though with a different sequence (403) . The single ribosome trajectory experiments studied by optical tweezers, and the associated mass spectrometric analayses of products were performed with mRNAs with potential to form a stem of either 25 or 55 base pairs (392) , instead of the 11 base pairs (including an A:U at the base of the stem) found to be functionally relevant in vivo (517) . The calculated strength of variant dnaX stem loop structures correlates directly with frameshift efficiency (517) and even without the 5 Shine Dalgarno stimulator just substituting one base in the otherwise WT stem to allow uninterrupted pairing, causes 160% of WT frameshifting (80% of ribosomes shift frame). While further strengthening the stem may facilitate molecular tweezer experiments, it may also approach the 'roadblocking' effect seen with extra strong pseudoknots, especially given the expected interchangeability of simple stem loops and pseudoknots. Accordingly, any extrapolations from the finding of −4 frameshifting in the studies of (392) to biological relevance needs to be treated with caution. The ensemble FRET experiments (450) used a shift site, U UUA AAG, that is different from that of IBV, U UUA AAC and of dnaX, A AAA AAG and the identity of the different P-site tRNAs influences the type of shift occurring (395) . [The whole pseudoknot or just its stem 1, can function in such heterologous expression systems (395, 581) though with differing effectiveness.] It is unknown to what extent these, or other differences in the systems used, account for the discrepancies reported. One set of the dnaX results is interpreted to imply uncoupling of EF-G catalyzed translocation from standard ribosomal reverse rotation, leaving the ribosomes in a non-canonical rotated state during which the incoming aminoacyl-tRNA has potential to pair in the 0 or −1 frame (403) (see also (108) ). In contrast, the other papers advocate slippage during tRNA-mRNA translocation at the second codon of the shift site (392, 450, 518) . Also relevant to these studies is the distance between the initiating Shine Dalgarno sequence and the internal frameshift stimulatory Shine Dalgarno sequence since pairing is maintained for translocation over several nts and adequate space has to be present to prevent interference (394, 491, 495) .