Abstract
The along-groove packing motif is a quasi-reciprocal arrangement of two RNA double helices in which a backbone of each helix is closely packed within the minor groove of the other helix. At the center of the inter-helix contact, a GU base pair in one helix packs against a Watson-Crick base pair in the other helix. Here, based on in vivo selection from a combinatorial gene library of 16 S rRNA and on functional characterization of the selected clones, we demonstrate that the normal ribosome performance requires that helices 3 and 12 be closely packed. In some clones the Watson-Crick and GU base pairs exchange in their positions between the two helices, which affects neither the quality of the helix packing, nor the ribosome function. On the other hand, perturbations in the close packing usually lead to a substantial drop in the ribosome activity. The functionality of the clones containing such perturbations may depend on the presence of particular elements in the vicinity of the area of contact between helices 3 and 12. Such cases do not exist in natural 16 S rRNA, and their selection enriches our knowledge of the constraints imposed on the structure of ribosomal RNA in functional ribosomes.
Highlights
We explored a possibility of alternative nucleotide arrangements in along-groove packing motif (AGPM) SU296, which is formed between helices 3 and 12 of 16 S rRNA
We used in vivo selection of functional clones of 16 S rRNA for analysis of general constraints imposed on AGPM SU296 in working ribosomes
The selection was based on the ability of the mutated ribosomes to synthesize chloramphenicol acetyl-transferase (CAT) that would allow the cells to survive in the presence of chloramphenicol
Summary
Bacterial Strains and Media—For cloning and selection, we used the E. coli strain DH5␣. Plasmids—For cloning of combinatorial 16 S rRNA gene libraries and for selection of functional clones, plasmid pAMMG carrying the specialized ribosome system was constructed. This plasmid is analogous to the ones described elsewhere (9 –17). Luciferase Assay—Each of the seven miscoding variants of plasmid pLuc (supplemental Tables S3 and S4) was cotransformed together with pAMMG-wt or with a mutant derivative of it into DH5␣ cells and plated on the LB agar containing 100 g/ml ampicillin and 50 g/ml kanamycin.
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