Improvement of RNA secondary structure prediction using RNase H cleavage and randomized oligonucleotides

RNA secondary structure prediction using free energy minimization is one method to gain an approximation of structure. Constraints generated by enzymatic mapping or chemical modification can improve the accuracy of secondary structure prediction. We report a facile method that identifies single-stranded regions in RNA using short, randomized DNA oligonucleotides and RNase H cleavage. These regions are then used as constraints in secondary structure prediction. This method was used to improve the secondary structure prediction of Escherichia coli 5S rRNA. The lowest free energy structure without constraints has only 27% of the base pairs present in the phylogenetic structure. The addition of constraints from RNase H cleavage improves the prediction to 100% of base pairs. The same method was used to generate secondary structure constraints for yeast tRNAPhe, which is accurately predicted in the absence of constraints (95%). Although RNase H mapping does not improve secondary structure prediction, it does eliminate all other suboptimal structures predicted within 10% of the lowest free energy structure. The method is advantageous over other single-stranded nucleases since RNase H is functional in physiological conditions. Moreover, it can be used for any RNA to identify accessible binding sites for oligonucleotides or small molecules

The Arabidopsis thaliana ATP-binding cassette proteins: an emerging superfamily

Solute transport systems are one of the major ways in which organisms interact with their environment, Typically, transport is catalysed by integral membrane proteins, of which one of the largest groups is the ATP-binding cassette (ABC) proteins. On the basis of sequence similarities, a large family of ABC proteins has been identified in Arabidopsis, A total of 60 open reading frames (ORFs) encoding ABC proteins were identified by BLAST homology searching of the nuclear genome. These 60 putative proteins include 89 ABC domains. Based on the assignment of transmembrane domains (TMDs), at least 49 of the 60 proteins identified are ABC transporters, Of these 49 proteins, 28 are full-length ABC transporters (eight of which have been described previously), and 21 are uncharacterized half-transporters. Three of the remaining proteins identified appear to be soluble, lacking identifiable TMDs, and most likely have non-transport functions. The eight other ORFs have homology to the nucleotide-binding and transmembrane components of multi-subunit permeases. The majority of ABC proteins found in Arabidopsis can, on the basis of sequence homology, be assigned to subfamilies equivalent to those found in the yeast genome, This assignment of the Arabidopsis ABC proteins into easily recognizable subfamilies (with distinguishable subclusters) is an important first step in the elucidation of their functional role in higher plants