title: Nucleotides that are essential but not conserved; a sufficient L-tryptophan site in RNA.
abstract: 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.
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder, Boulder, Colorado 80309, USA.
authors.forename: Irene
authors.initials: I
authors.lastname: Majerfeld
authors.forename: Jana
authors.initials: J
authors.lastname: Chocholousova
authors.forename: Vikas
authors.initials: V
authors.lastname: Malaiya
authors.forename: Jeremy
authors.initials: J
authors.lastname: Widmann
authors.forename: Daniel
authors.initials: D
authors.lastname: McDonald
authors.forename: Jens
authors.initials: J
authors.lastname: Reeder
authors.forename: Matthew
authors.initials: M
authors.lastname: Iyer
authors.forename: Mali
authors.initials: M
authors.lastname: Illangasekare
authors.forename: Michael
authors.initials: M
authors.lastname: Yarus
authors.forename: Rob
authors.initials: R
authors.lastname: Knight
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D019295:Computational Biology; D017124:Conserved Sequence; D019143:Evolution, Molecular; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D052156:SELEX Aptamer Technique; D012689:Sequence Homology, Nucleic Acid; D014364:Tryptophan / Q000378:metabolism*
chemical_list: D052157:Aptamers, Nucleotide; D012313:RNA; D014364:Tryptophan