Evaluation and refinement of tmRNA structure using gene sequences from natural microbial communities

DNA harvested directly from complex natural microbial communities by PCR ha s been successfully used to predict RNase P RNA structure, and can potentia lly provide an abundant source of information for structural predictions of other RNAs. In this study, we utilized genetic variation in natural commun ities to test and refine the secondary and tertiary structural model for th e bacterial tmRNA. The variability of proposed tmRNA secondary structures i n different organisms and the lack of any predicted tertiary structure sugg ested that further refinement of the tmRNA could be useful. To increase the phylogenetic representation of tmRNA sequences, and thereby provide additi onal data for statistical comparative analysis, we amplified, sequenced, an d compared tmRNA sequences from natural microbial communities. Using primer s designed from gamma proteobacterial sequences, we determined 44 new tmRNA sequences from a variety of environmental DNA samples. Covariation analyse s of these sequences, along with sequences from cultured organisms, confirm ed most of the proposed tmRNA model but also provided evidence for a new te rtiary interaction. This approach of gathering sequence information from na tural microbial communities seems generally applicable in RNA structural an alysis.