Book of Abstracts: Albany 2011
June 14-18 2011
©Adenine Press (2010)
Anticodon loop modifications modulate structural flexibility in E. coli tRNAArg1,2 that lacks a U-turn conformation in solution
Three of the six codons that are decoded by tRNAArg in E. coli are read by the isoacceptors tRNAArg1,2. The anticodon stem and loop domain (ASLArgICG) of these isoacceptors differ only in the identity of the residue at position 32 in the loop as either 2-thiocytidine (s2C32) or cytidine for tRNAArg1 and tRNAArg2, respectively. These isoacceptors also contain important modifications at positions 34 (inosine, I34) and 37 (2-methyladenosine, m2A37). To investigate the roles of the modifications in proper folding of the ASLArgICG, six ASLArgICG constructs differing in their array of modifications were analyzed by biophysical spectroscopic methods as well as functional binding assays. Thermal denaturation and circular dichroism spectroscopy showed that the modifications contribute competing thermodynamic and base stacking properties. Spectroscopic methods indicated that ASLArgICG modifications contributed significant differences in structural properties. However, restrained molecular dynamics calculations of the ASLArgICG structures from NMR spectroscopy clearly showed that the equilibrium solution conformations of the ASLs are nearly identical, but do not possess the invariant U-turn structure needed for binding to the ribosomal A-site. Yet, all of the ASLArg constructs were able to bind to the ribosome in the presence of the cognate CGU codon. The m2A37 modification restricts binding to CGC, while both s2C32 and m2A37 restrict binding to CGA. Taken together, the results suggest that chemical modifications modulate the flexibility of the loop, allowing induced conformations on the ribosome that can restrict binding to specific codons.
This research is supported by NSF grant number 53855.
William A. Cantara
The RNA Institute
Department of Biological Sciences
University at Albany, SUNY
Albany, NY 12222