Heterogeneous Folding and Function of Small RNA Motifs: The Hairpin Ribozyme and a Translational Riboswitch.

Abstract

Heterogeneous folding and function are observed commonly for RNA, DNA, and protein, yet remain poorly understood. For RNA, several molecular origins have been proposed, including UV cross-linking, slow sugar repuckering, and alternate folding associated with denaturation during most purification methods. Additionally, natural alternative folds for many RNAs serve to control the biological function of the RNA. With each of these cases, there results variations in RNA fold that effect local and global behaviors of the RNA of interest. The hairpin ribozyme is a well-studied example of a highly structured functional RNA that exhibits pronounced heterogeneity. We have assessed the relative contributions of UV cross-linking, sugar repuckering and alternative folding to molecular heterogeneity of the hairpin ribozyme and find that it is multifactorial and can be systematically suppressed. Exposure to short-wavelength UV irradiation introduces distributive cross-links between adjacent pyrimidines and is avoided by using longer-wavelength irradiation or staining with crystal violet for visualization. Differential selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) does not reveal strong evidence for slow sugar repuckering. The remaining folding heterogeneity is removed using a non-denaturing affinity purification method that maintains the co-transcriptional fold of the RNA. These results suggest that heterogeneous folding and thus function of RNA generally may have multiple origins and are minimized by the avoidance of short-range UV exposure and the segmental, 5’-to-3’-directed folding resulting from transcription. Moving to studying natural heterogeneities necessary for biological function, we establish a ribosome expression system capable of site-specific fluorescence labeling and translation. We show that these ribosomes are capable of translating gene products in vitro, as well as forming initiation complexes. These assays form the basis for single molecule studies of (pre)initiation complex formation on the Thermoanaerobacter tengcongensis (Tte) 1564/1563 mRNA. The coding sequence of this mRNA is preceded by the Tte preQ1 translational riboswitch. Acting in response to binding the small metabolite preQ1, the Shine Dalgarno sequence of the mRNA is sequestered into the aptamer domain. We show that preQ1 suppresses binding of the 30S ribosome to this mRNA, thus offering direct evidence for the biological function of the translational preQ1 riboswitch.