Selection of the simplest RNA that binds isoleucine

Abstract

We have identified the simplest RNA binding site for isoleucine using selection-amplification (SELEX), by shrinking the size of the randomized region until affinity selection is extinguished. Such a protocol can be useful because selection does not necessarily make the simplest active motif most prominent, as is often assumed. We find an isoleucine binding site that behaves exactly as predicted for the site that requires fewest nucleotides. This UAUU motif (16 highly conserved positions; 27 total), is also the most abundant site in successful selections on short random tracts. The UAUU site, now isolated independently at least 63 times, is a small asymmetric internal loop. Conserved loop sequences include isoleucine codon and anticodon triplets, whose nucleotides are required for amino acid binding. This reproducible association between isoleucine and its coding sequences supports the idea that the genetic code is, at least in part, a stereochemical residue of the most easily isolated RNA-amino acid binding structures.

FIGURE 1.

(A) Summary structure of the UAUU motif based on results from the initial selection (Majerfeld and Yarus 1998). Initially, random nucleotides are capitalized and fixed nucleotides are in lowercase. Absolutely conserved nucleotides are in bold. Nonbold capitals designate highly conserved nucleotides (>70% identity in sequence alignment) and nonconserved nucleotides are indicated (N). (B) Refined summary structures of the UAUU motif based on 62 of the 72 sequenced UAUU isolates. Capitalization and boldface are the same as in A, except that lowercase letters were only sometimes derived from the constant regions and could also be selected from randomized nucleotides. Structures B1 and B2 summarize the two permutations of the motif. B1 is the preferred permutation (82%) in which the AC module occurs 5′ of the UAUUGG module.

FIGURE 2.

Representative elution profiles for representatives of the VG1, VG8, and UAUU sequence families. VG1 represented ~70% of the final 16-mer pool, and variant VG8 has the strongest K_d for isoleucine-Sepharose among the 16-mer sequence families (Table 3 ▶). Both show affinity for the column (delayed elution), but no peak with isoleucine.