We went on to examine the possibility that multiple copies of motif 1 would stimulate termination-reinitiation, through the provision of an increased number of tethering sites. As a precedent for this, the murine Gtx mRNA (also known as Nkx 6-2) contains a short 9 nt IRES element that is known to act more efficiently when present in multiple copies. Similarly to motif 1, ribosomes are recruited through interactions between the Gtx IRES and helix 26 of the 18S rRNA [28] [29] [30] , although with the arm of the helix [31] rather than the apical loop ( Figure 3C , left panel). Two sets of constructs were prepared. In the first, one or two additional copies of the 6 nt (AUGGGA) core of motif 1 were introduced alongside the original ( Figure 3B ). Control constructs were also generated in which one or two copies of the hexanucleotide AUGCGA were looped in as a negative control, since this motif 1 mutation was shown previously to be unable to support termination-reinitiation, presumably due to the abolition of mRNA:rRNA base pairing [11] . In the second set of constructs, the bases downstream of the wild-type AUGGGA sequence were substituted to generate one or two extra copies of the AUGGGA motif ( Figure 3B ). In vitro translations revealed, however, that none of the mutations had a stimulatory effect on reinitiation on the BM2 ORF. Looping in a single extra copy of the AUGGGA (or AUGCGA) hexanucleotide had very little effect on the process, whilst insertion of two copies led to inhibition in both contexts ( Figure 3B ). In constructs where motif 1 was duplicated by virtue of nucleotide substitutions, termination-reinitiation was inhibited to a similar extent in constructs containing either one or two copies of the 18S complementary motif ( Figure 3B ). These data indicate that there is no additive effect on termination-reinitiation of adding extra copies of the 18S complementary region. In fact, the inhibitory effects seen most likely reflect the importance of the precise spacing of the ribosome with respect to motif 1 (in the loop-in mutants) or, alternatively, some effect on TURBS RNA secondary structure.