Nucleic Acids Research, 2011, Vol. 39, No. 8 3367 Figure 2. Saturating mutagenesis of the internal loop. (A) Secondary structure of N1. The GUC motif is shaded in gray. The lower part of the internal loop (boxed) was analyzed and randomized. Nucleotides are indicated with N (any nucleotide) or Y (pyrimidines). (B–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. (E) Schematic representation of the connection between loop size and regulation. All aptamers with a 30 nucleotide insertion between the GUC motif and the closing stem (this means all candidates from pool N1-3/1 and N1-2/1) are inactive. In contrast, all aptamers from the N1-3/0 and N1-2/0 pools were active (e.g. Figure 2B). However, the efﬁciency of regulation is dependent on the sequence (Figure 2B and C, Table 1 and Supplementary Table S2) with a clear preference for pyr- imidines over purines. Furthermore, adenines are preferred over guanines which is exemplarily shown for the CUN series from the N1-3/0 pool (Figure 2C). The wild-type sequence CUU is the most active sequence with 3.9-, followed by CUC with 3.5-, CUA with 2.6- and CUG with 1.6-fold regulation. Next we analyzed the dependence on the length of the inserted sequence at the 50 site of the GUC motif. In addition to N1-3/0 and N1-2/0 we questioned if four, one or no nucleotide at this position also confer regula- tion. We cloned the pools N1-4/0, N1-1/0, N1-0/0 but re- stricted the pools to pyrimidines only. The data are included in Figure 2D, E, Table 1 and Supplementary Table S2. Four nucleotides (e.g. CCUU) result in a similar activity as three (wild-type: CUU) or two nucleo- tides (e.g. CU). However, one nucleotide at this position (C or U) leads to a signiﬁcant reduction in regulation (Figure 2D, E and Table 1). The absence of a nucleotide between the GUC motif and the adjacent closing stem results in a complete loss of activity. These data clearly demonstrate that one to four nucleo- tides 50 of the GUC motif are necessary in conferring riboswitch activity to N1. Notably the dynamic range of the regulation is modulated by both the sequence and the length of the insertion. High ligand-binding afﬁnity and thermodynamic stabilization are important for regulation Aminoglycosides are known to exert a stabilizing effect upon binding to RNAs (21–23). Therefore, we questioned if an increase in the thermal stability of the aptamer– neomycin complex may be responsible for ligand- dependent translational inhibition.