Figure 2.

The 67 glnA RNA binds l-glutamine. (A) Sequence and secondary structural model for the 67 glnA RNA from S. elongatus. Circled positions indicate areas of the RNA where internucleotide linkages undergo reduced (red) or constant (yellow) scission as ligand concentrations are increased when subjected to in-line probing (data from B). Nucleotides depicted in lowercase identify guanosine residues added to the construct to facilitate efficient in vitro transcription. Asterisks indicate the boundaries of the annotations for in-line probing results that could be clearly resolved by PAGE. (B) In-line probing analysis of 5′ ³²P-labeled 67 glnA RNA. Precursor RNAs (Pre) were loaded onto gel lanes after treatment as follows: NR, no reaction; T1, partial digest with RNase T1 (cleaves after G residues); ⁻OH, partial alkaline-mediated degradation. Additional lanes were loaded with precursor RNAs subjected to in-line probing conditions without ligand (-), or were subjected to inline probing conditions in the presence of various concentrations of l-glutamine ranging from 1 µM to 10 mM. Vertical lines designate areas where band intensities decrease as the RNA is exposed to higher concentrations of ligand. Band intensities of numbered regions were quantified and used to assess the extent of ligand binding. (C) Plot of the normalized fraction of band modulation (interpreted as fraction of RNAs bound to ligand) versus the logarithm of the concentration of ligand. Regions are as depicted in (B). The line represents the curve expected for a 1-to-1 RNA-ligand interaction with a K_D of 575 µM.