Monthly Featured Lab

Each month the RNA Society of North Carolina
highlights the research of a member laboratory.

The featured group for July, 2004 is the laboratory of

Dr. Rebecca Alexander

We are investigating the molecular interactions that contribute to efficient aminoacylation of tRNA by methionyl-tRNA synthetase. In addition to being key players in translation, the aminoacyl-tRNA synthetases (aaRSs) are good models for understanding signal transduction-like events. Many aaRSs bind to the anticodon portion of their matching (cognate) tRNA molecules, and the anticodon-binding site is often some distance removed from the enzyme active site, where amino acid attachment occurs. Efficient catalysis therefore depends on communication between protein domains.

A comparison of the crystal structure of MetRS (shown here, PDB# 1QQT) with glutaminyl-tRNA synthetase (GlnRS) identified a similar peptide motif at the interface of catalytic and anticodon-binding domains in each protein. In order to determine whether the structure of this motif contributes to communication between the domains, we are using cassette mutagenesis to construct chimeric proteins containing the linker peptide of GlnRS within the context of MetRS (and vice versa).

A microhelix mimic of the tRNAMet acceptor stem is sequence-specifically aminoacylated by MetRS, although the catalytic efficiency of the reaction is significantly decreased. Both the microhelixMet and an anticodon stem-loop mimic bind to MetRS, but the presence of the anticodon stem-loop does not increase the rate of microhelix aminoacylation. At a minimum, therefore, the two portions of tRNAMet must be covalently linked. In an attempt to re-establish communication between the anticodon and acceptor stem portions of tRNAMet, we are connecting the RNA microhelices with a flexible linker of varying length and sequence and using in vitro selection to identify improved RNA substrates. It is anticipated that such a selection will provide clues as to which regions of MetRS are involved in long-range signaling between anticodon binding and active site domains.

RNA helicase activity of E. coli CsdA, a cold shock protein:In collaboration with Pamela Jones at Winston-Salem State University, we are investigating the RNA helicase properties of CsdA, a protein that is highly overproduced during the bacterial cold shock response. We are using a molecular beacon assay to detect RNA unwinding activity of wild-type CsdA and mutants that should be diminished in helicase activity.