Tyler D. Ames

Structural basis of ligand binding by a c-di-GMP riboswitch

The second messenger signaling molecule bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) regulates many processes in bacteria, including motility, pathogenesis and biofilm formation. c-di-GMP–binding riboswitches are important downstream targets in this signaling pathway. Here we report the crystal structure, at 2.7 Å resolution, of a c-di-GMP riboswitch aptamer from Vibrio cholerae bound to c-di-GMP, showing that the ligand binds within a three-helix junction that involves base-pairing and extensive base-stacking. The symmetric c-di-GMP is recognized asymmetrically with respect to both the bases and the backbone. A mutant aptamer was engineered that preferentially binds the candidate signaling molecule c-di-AMP over c-di-GMP. Kinetic and structural data suggest that genetic regulation by the c-di-GMP riboswitch is kinetically controlled and that gene expression is modulated through the stabilization of a previously unidentified P1 helix, illustrating a direct mechanism for c-di-GMP signaling.

A widespread self-cleaving ribozyme class is revealed by bioinformatics

Ribozymes are noncoding RNAs that promote chemical transformations with rate enhancements approaching those of protein enzymes. Although ribozymes are likely to have been abundant during the RNA world era, only ten classes are known to exist among contemporary organisms. We report the discovery and analysis of an additional self-cleaving ribozyme class, called twister, which is present in many species of bacteria and eukarya. Nearly 2700 twister ribozymes were identified that conform to a secondary structure consensus that is small yet complex, with three stems conjoined by internal and terminal loops. Two pseudoknots provide tertiary structure contacts that are critical for catalytic activity. The twister ribozyme motif provides another example of a natural RNA catalyst and calls attention to the potentially varied biological roles of this and other classes of widely distributed self-cleaving RNAs.

Bacterial aptamers that selectively bind glutamine.

The continued expansion of microbial sequence data has allowed for the detection of an increasing number of conserved RNA motifs by using comparative sequence analysis. Recently, we reported the discovery of two structured non-coding RNA motifs, called glnA and Downstream-peptide, that have similarity in sequence and secondary structure. In this report, we describe data demonstrating that representatives of both RNA motifs selectively bind the amino acid L-glutamine. These glutamine aptamers are found exclusively in cyanobacteria and marine metagenomic sequences, wherein several glnA RNA representatives reside upstream of genes involved in nitrogen metabolism. These motifs have genomic distributions that are consistent with a gene regulation function, suggesting they are components of glutamine-responsive riboswitches. Thus, our findings implicate glutamine as a regulator of cyanobacterial nitrogen metabolism pathways. Furthermore, our findings expand the collection of natural aptamer classes that bind amino acids to include glycine, lysine and glutamine.

Engineering high-speed allosteric hammerhead ribozymes.

Abstract Full-length hammerhead ribozymes were subjected to in vitro selection to identify variants that are allosterically regulated by theophylline in the presence of a physiologically relevant concentration of Mg2+. The population of allosteric ribozymes resulting from 15 rounds of in vitro selection yielded variants with observed rate constants (k obs) as high as 8 min-1 in the presence of theophylline and maximal k obs increases of up to 285-fold compared to rate constants measured in the absence of effector. The selected ribozymes have kinetic characteristics that are predicted to be sufficient for cellular gene control applications, but do not exhibit any activity in reporter gene assays. The inability of the engineered RNAs to control gene expression suggests that the in vitro and in vivo folding pathways of the RNAs are different. These results provide several key pieces of information that will aid in future efforts to engineer allosteric ribozymes for gene control applications.

Detection of 224 candidate structured RNAs by comparative analysis of specific subsets of intergenic regions

Abstract The discovery of structured non-coding RNAs (ncRNAs) in bacteria can reveal new facets of biology and biochemistry. Comparative genomics analyses executed by powerful computer algorithms have successfully been used to uncover many novel bacterial ncRNA classes in recent years. However, this general search strategy favors the discovery of more common ncRNA classes, whereas progressively rarer classes are correspondingly more difficult to identify. In the current study, we confront this problem by devising several methods to select subsets of intergenic regions that can concentrate these rare RNA classes, thereby increasing the probability that comparative sequence analysis approaches will reveal their existence. By implementing these methods, we discovered 224 novel ncRNA classes, which include ROOL RNA, an RNA class averaging 581 nt and present in multiple phyla, several highly conserved and widespread ncRNA classes with properties that suggest sophisticated biochemical functions and a multitude of putative cis-regulatory RNA classes involved in a variety of biological processes. We expect that further research on these newly found RNA classes will reveal additional aspects of novel biology, and allow for greater insights into the biochemistry performed by ncRNAs.

Small molecule fluoride toxicity agonists.

Fluoride is a ubiquitous anion that inhibits a wide variety of metabolic processes. Here, we report the identification of a series of compounds that enhance fluoride toxicity in Escherichia coli and Streptococcus mutans. These molecules were isolated by using a high-throughput screen (HTS) for compounds that increase intracellular fluoride levels as determined via a fluoride riboswitch reporter fusion construct. A series of derivatives were synthesized to examine structure-activity relationships, leading to the identification of compounds with improved activity. Thus, we demonstrate that small molecule fluoride toxicity agonists can be identified by HTS from existing chemical libraries by exploiting a natural fluoride riboswitch. In addition, our findings suggest that some molecules might be further optimized to function as binary antibacterial agents when combined with fluoride.