Characterization of Transition Metal-Sensing Riboswitches Otto Mossberg, Dr. Adam Saunders, Dr. Victoria DeRose University of Oregon, New Mexico State University Riboswitches Research Goals Analysis of riboswitch conformation by in-line probing Summary and Conclusions Examine the range of metal ion specificity within the NiCo class of riboswitches. Search for novel classes of riboswitch with specificity for other transition metals. Riboswitches have recently emerged as ubiquitous and essential components of genetic control in bacteria and archaea. Usually encoded in the 5'-untranslated region (5'-UTR) of certain genes, riboswitches are segments of RNA that can rapidly regulate the expression of their associated genes through binding of small molecule effectors. This binding influences secondary and tertiary structure of the RNA, controlling both transcription elongation and translation initiation. To test our structures for specificity to different metals, we used in-line probing assays. This assay takes advantage of the inherent instability of RNA, which results from nucleophilic attack from the 2’-hydroxyl on the phosphate. Because this occurs only when the strand is in the in-line conformation, secondary structure inhibits cleavage at certain sites. The assay itself involves incubating the RNA construct with varying concentration of transition metals and observing cleavage products on a denaturing polyacrylamide gel. Using the size of in-line cleavage products, we can locate sites where protection occurs on our predicted structure. Two of these occur in the inter-helix junction, supporting the idea that Mg2+ stabilizes folding in that region of NiCo riboswitches. Experimental Approach Cluster the 700+ putative NiCo riboswitches from bacterial genomes and metagenomes by secondary structure and assign to groups that could represent variant metal ion specificity. Search for novel classes of riboswitch in the 5’UTRs of genes encoding metal ion efflux pumps with known metal specificity. Future directions Clustering and screening of candidate riboswitches by secondary structure A new class of riboswitch that senses Ni2+ and Co2+ ions Furukawa, 2015 P11 P21 P22 P31 P32 P41 P42 P12 Terminator ORF Compare env-155 in-line probing pattern with original Clostridium NiCo riboswitch (in preparation). Test for binding of env-155 to Ni2+, Co2+, as well as to Mn2+, Co2+, Fe2+, Cu2+, Zn2+,... by in-line probing. Expand this in-line probing assay to additional riboswitch variants Continue to refine search for alternative transition metal binding riboswitches within the NiCo class. Search for novel transition metal sensing riboswitches in the 5’UTRs of efflux proteins with known specificity for Cr2+, Mn2+, Fe2+, Cu2+, Zn2+, Mo2+, Ag2+, Cd2+ and other transition metals. A new class of riboswitches has been identified that specifically bind Nickel (Ni2+) and Cobalt (Co2+) ions, and promote expression of a family of efflux pumps known as Cation Diffusion Facilitator (CDF) proteins. On two separate gels, we were able to (1) map the secondary structure of env-155 and (2) observe conformational stabilization in the presence of Mg2+. Map of secondary structure Conformational Stabilization Observed Nickel or Cobalt Ions We used the mFold software service to predict the structures of candidate NiCo riboswitches. Searching for those with variations in stem-loop size, structure, and position, we selected two sequences originally retrieved from environmental metagenomes and two from the whole genomes of isolated and cultivated strains. Cbo-3-1: NiCo riboswitch from Clostridia sp. characterized by Furukawa et. al. (2015) env-76 env-155 Lgr-1-1 Fva-1-1 canonical non-canonical Architecture: environmental/ microbiome samples isolated, sequenced strains Source: These Nickel/Cobalt (NiCo) riboswitches are distinct from previously characterized riboswitches in that they bind their target metal ions with high specificity, and also show cooperative binding at multiple sites2. References: Mandal, M. & Breaker, R. R. Gene regulation by riboswitches. Nat Rev Mol Cell Biol 5, 451– 463 (2004). Furukawa, K. et al. Bacterial riboswitches cooperatively bind ni(2+) or co(2+) ions and control expression of heavy metal transporters. Mol Cell 57, 1088–1098 (2015). Soukup, G. A. & Breaker, R. R. Relationship between internucleotide linkage geometry and the stability of RNA. RNA 5, 1308–1325 (1999). Synthesis of riboswitch RNAs DNA template produced using overlap PCR In-vitro transcription Radiolabeling with 32P Denaturing PAGE gel Ladder env-76 env-155 Biological functions of Nickel and Cobalt ions Nickel and Cobalt are both essential nutrients. Nickel is present in the active sites of many enzymes, and Cobalt is an essential component in cobalamin, commonly known as vitamin B12. Nonetheless, both are metals toxic at high concentrations, necessitating well-regulated import/export mechanisms.