Comparative 3-D Modeling of tmRNA

To facilitate experimental testing of the molecular intricacies of trans-translation, which often require appropriately modified tmRNA derivatives, we developed a procedure for building three-dimensional models of tmRNA. Using comparative sequence analysis, phylogenetically-supported 2-D structures were obtained to serve as input for the program ERNA-3D. Motifs containing loops and turns were extracted from the known structures of other RNAs and used to improve the tmRNA models. Biologically feasible 3-D models for the entire tmRNA molecule could be obtained. The models were characterized by a functionally significant close proximity between the tRNA-like domain and the resume codon. Potential conformational changes which might lead to a more open structure of tmRNA upon binding to the ribosome are discussed. The method, described in detail for the tmRNAs of Escherichia coli, Bacillus anthracis, and Caulobacter crescentus, is applicable to every tmRNA.Improved molecular models of biological significance were obtained. These models will guide in the design of experiments and provide a better understanding of trans-translation. The comparative procedure described here for tmRNA is easily adopted for the modeling the members of other RNA families.Transfer-messenger RNA (tmRNA), also known as 10Sa RNA or ssrA RNA, is a hybrid of a tRNA-like domain (TLD) and a mRNA-like domain (MLD) connected by a variable number of pseudoknots [1]. TmRNA is a stable and essential component of trans-translation, a quality-control process that rescues ribosomes stalled on mRNAs lacking stop codons. During trans-translation, ribosomes switch from a defective mRNA (lacking its translation-termination signal) to the MLD of tmRNA. Because a stop codon is provided by the tmRNA, the ribosomes can dissociate and recycle [2]. As an additional advantage, the tandem translation of the two templates generates a tagged polypeptide which is degraded by housekeeping proteases [3,4].For tagging, tmRNA h