Termination-reinitiation in yeast cells: further evidence for mRNA:18S rRNA interactions in ribosome tethering It is known that nucleotide substitutions within motif 1 that are predicted to destabilise the motif 1:18S rRNA interaction are inhibitory to termination-reinitiation, but it is possible that these mutations may affect TURBS structure or interaction with other translational components, like eIF3 [17] . In an attempt to resolve this issue, we assayed termination-reinitiation in yeast cells, whose 18S rRNA helix 26 equivalent has a somewhat different primary sequence. This experiment was carried out in the context of the yeast dual reporter vector pAC99, into which we had cloned the relevant BM2 information appropriately framed between bgalactosidase and firefly luciferase reporter genes. In pAC99-BM2wt, b-galactosidase is synthesised by cap-dependent translation, whereas firefly luciferase should only be synthesised following reinitiation on the BM2fluc ORF ( Figure 4A ). To confirm that firefly luciferase was being synthesised as a result of a genuine termination-reinitiation event (as opposed to internal ribosome entry, for example), we generated a premature stop (ps) mutant (pAC99-BM2-ps) in which a stop codon was inserted into the M1 sequence 233 nt upstream of the start codon of BM2fluc. In pAC99-BM2-yeast, motif 1 was changed to give full complementarity to the yeast 18S rRNA partner ( Figure 4B ). The plasmids were transformed into yeast strain Y349, and b-galactosidase and luciferase assays were carried out. Termination-reinitiation frequency was determined in comparison to the reporter gene activities observed in a vector in which the reporter ORFs were fused in-frame (pAC99-BM2IFC). As expected, termination reinitiation was significantly (p,0.01) reduced in pAC99-yeastps, although there was still some background synthesis of fluc ( Figure 4C ). Importantly, mutation of the motif 1 homologue such that it was fully complementary to yeast 18S rRNA led to a significant (p,0.01) increase in BM2fluc synthesis relative to the wild-type BM2 reporter ( Figure 4C ), supporting the view that mRNA:rRNA base pairing is a key determinant in BM2 ORF expression.