Nucleotides that are essential but not conserved; a sufficient L-tryptophan site in RNA

RNA. 2010 Oct;16(10):1915-24. doi: 10.1261/rna.2220210. Epub 2010 Aug 10.


Conservation is often used to define essential sequences within RNA sites. However, conservation finds only invariant sequence elements that are necessary for function, rather than finding a set of sequence elements sufficient for function. Biochemical studies in several systems-including the hammerhead ribozyme and the purine riboswitch-find additional elements, such as loop-loop interactions, required for function yet not phylogenetically conserved. Here we define a critical test of sufficiency: We embed a minimal, apparently sufficient motif for binding the amino acid tryptophan in a random-sequence background and ask whether we obtain functional molecules. After a negative result, we use a combination of three-dimensional structural modeling, selection, designed mutations, high-throughput sequencing, and bioinformatics to explore functional insufficiency. This reveals an essential unpaired G in a diverse structural context, varied sequence, and flexible distance from the invariant internal loop binding site identified previously. Addition of the new element yields a sufficient binding site by the insertion criterion, binding tryptophan in 22 out of 23 tries. Random insertion testing for site sufficiency seems likely to be broadly revealing.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Aptamers, Nucleotide / chemistry
  • Aptamers, Nucleotide / genetics
  • Aptamers, Nucleotide / metabolism
  • Base Sequence
  • Binding Sites / genetics
  • Computational Biology
  • Conserved Sequence
  • Evolution, Molecular
  • Models, Molecular
  • Molecular Dynamics Simulation
  • Molecular Sequence Data
  • Nucleic Acid Conformation
  • RNA / chemistry*
  • RNA / genetics
  • RNA / metabolism*
  • SELEX Aptamer Technique
  • Sequence Homology, Nucleic Acid
  • Tryptophan / metabolism*


  • Aptamers, Nucleotide
  • RNA
  • Tryptophan