3364 Nucleic Acids Research, 2011, Vol. 39, No. 8 Figure 1. Conditional control by neomycin-binding aptamers. (A) Scheme of aptamer-mediated inhibition of translation initiation. The aptamer is inserted directly upstream of the start codon. Left: without ligand the aptamer does not interfere with ribosomal scanning. Right: the aptamer ligand complex inhibits the small ribosomal subunit and leads to decreased gene expression. (B) Secondary structure of the ribosomal A-site and the neomycin-binding aptamers N1 and R23. The lines dissect the aptamers into the terminal loop, an internal asymmetrical loop and the closing stem. Conserved nucleotides between the A-site and N1 are shaded in gray. (C) Hybrid aptamers. (D) gfp expression in the absence (black bars) and presence (white bars) of 100 mM neomycin. The ﬂuorescence emission of the vector pWHE601 (6) expressing gfp without an aptamer in its 50UTR was set as 100%. Background level of a vector with no gfp expression was subtracted from all data. Values represent the mean of three independently grown cultures. Measurements were repeated at least twice. aptamers (13). Interestingly, these aptamers were highly underrepresented in the original in vitro selected aptamer pool [they were not among 21 different aptamers sequenced after the selection process (14)]. We identiﬁed a neomycin-binding aptamer N1 which is with 27 nt the smallest riboswitch reported to date. In the present study we compared N1 with further neomycin- binding aptamers as well as the ribosomal decoding site (A-site), a natural neomycin-binding motif. We performed a comprehensive study to obtain mechanistic insights into the regulation. The combination of genetic, biochemical and structural data lead to the identiﬁcation of a struc- tural element which is not necessary for ligand binding but for conferring regulation to the aptamer N1 by destabilizing its ground state. MATERIALS AND METHODS Plasmid construction The yeast 2 m plasmid pWHE601 was used to constitutive- ly express the gfp gene from a pADH1 promoter (6). The vector contains a restriction site for AﬂII immediately upstream of the start codon with a 50UTR length of 38 or 44 nt, respectively, depending on the two described transcriptional start sites (15) and a singular site for NheI directly after the start codon. For aptamer insertion, the vector was digested with AﬂII and NheI. Aptamer pools were generated by PCR using a single-stranded DNA oligonucleotide as template. The PCR products were digested with AﬂII and NheI prior to ligation. The start codon which was cut out of the vector after AﬂII/NheI