Albany 2015:Book of Abstracts

Albany 2015
Conversation 19
June 9-13 2015
©Adenine Press (2012)

Modifications Modulate Anticodon Loop Dynamics and Codon Recognition in E. coli tRNAArg1,2

In Escherichia coli, three of six arginine codons (CGU, CGC and CGA) are decoded by only two tRNAArg isoacceptors. The anticodon-stem loop (ASL) domains of tRNAArg1 and tRNAArg2 differ only at position 32: tRNAArg1 is post-transcriptionally modified to contain a 2-thiocytidine (s2C32), while the same cytidine in tRNAArg2 is unmodified. Both isoacceptors also contain naturally-occurring modifications at positions 34 (inosine, I34) and 37 (2-methyladenosine, m2A37). To investigate the functional roles of these modifications, six ASL constructs, differing in their array of modifications, were analyzed using binding studies and structural and computational methods. Ribosome filter binding assays showed that while I34, as expected, facilitates wobble codon binding, both s2C32 and m2A37). modulated that effect by negating binding to the rare CGA codon. When a non-naturally occurring s2C32 was introduced for C32 in an unrelated S. cerevisiae tRNA ASLLeu construct also containing C32 and I34 and capable of wobble pairing, the same functional restriction of CGA binding was observed. The structures of the ASLs, when probed by either by NMR spectroscopy or x-ray crystallography, showed only a minor anticodon loop perturbation upon inclusion of s2C32. The mechanism by which the modification affects codon binding was then investigated by molecular dynamics simulations, which suggested that both s2C32 and m2A37). afford the ASL greater flexibility and conformational accessibility and provide an explanation for their ability to adopt one structure free in solution and two others when bound to the cognate arginyl-tRNA synthetase or to codons on the ribosome.

This research has been supported by National Science Foundation award MCB1101859.

Kathryn L. Sarachan1
William A. Cantara1
Jessica L. Spears1
Hasan Dimirci2
Frank V. Murphy IV3
Srivathsan Ranganathan1
Alan Chen1
Paul F. Agris1

1The RNA Institute
University at Albany – SUNY
Albany, NY 12222, USA
2PULSE Institute
SLAC National Accelerator Laboratory
2575 Sand Hill Road MS 59
Menlo Park, CA 94025
3NE-CAT/Cornell University
9700 South Cass Ave
Bldg. 436 E004
Argonne, IL 60439, USA

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