Deciphering the folding pathways and predicting the structures of complex three-dimensional biomolecules is central to elucidating biological function. RNA is single-stranded, which gives it the freedom to fold into complex secondary and tertiary structures. These structures endow RNA with the ability to perform complex chemistries and functions ranging from enzymatic activity to gene regulation. Given that RNA is involved in many essential cellular processes, it is critical to understand how it folds and functions in vivo. Within the last few years, methods have been developed to probe RNA structures in vivo and genome-wide. These studies reveal that RNA often adopts very different structures in vivo and in vitro, and provide profound insights into RNA biology. Nonetheless, both in vitro and in vivo approaches have limitations: studies in the complex and uncontrolled cellular environment make it difficult to obtain insight into RNA folding pathways and thermodynamics, and studies in vitro often lack direct cellular relevance, leaving a gap in our knowledge of RNA folding in vivo. This gap is being bridged by biophysical and mechanistic studies of RNA structure and function under conditions that mimic the cellular environment. To date, most artificial cytoplasms have used various polymers as molecular crowding agents and a series of small molecules as cosolutes. Studies under such in vivo-like conditions are yielding fresh insights, such as cooperative folding of functional RNAs and increased activity of ribozymes. These observations are accounted for in part by molecular crowding effects and interactions with other molecules. In this review, we report milestones in RNA folding in vitro and in vivo and discuss ongoing experimental and computational efforts to bridge the gap between these two conditions in order to understand how RNA folds in the cell.

RNA folding has been studied extensively in vitro, typically under dilute solution conditions and abiologically high salt concentrations of 1 M Na+ or 10 mM Mg2+. The cellular environment is very different, with 20–40% crowding and only 10–40 mM Na+, 140 mM K+ and 0.5–2.0 mM Mg2+. As such, RNA structures and functions can be radically altered under cellular conditions. We previously reported that tRNAphe secondary and tertiary structures unfold together in a cooperative two-state fashion under crowded in vivo-like ionic conditions, but in a non-cooperative multi-state fashion under dilute in vitro ionic conditions unless in non-physiologically high concentrations of Mg2+. The mechanistic basis behind these effects remains unclear, however. To address the mechanism that drives RNA folding cooperativity, we probe effects of cellular conditions on structures and stabilities of individual secondary structure fragments comprising the full-length RNA. We elucidate effects of a diverse set of crowders on tRNA secondary structural fragments and full-length tRNA at three levels: at the nucleotide level by temperature-dependent in-line probing (ILP), at the tertiary structure level by small angle X-ray scattering (SAXS), and at the global level by thermal denaturation. We conclude that cooperative RNA folding is induced by two overlapping mechanisms: increased stability and compaction of tertiary structure through effects of Mg2+, and decreased stability of certain secondary structure elements through effects of molecular crowders. These findings reveal that despite having very different chemical makeups RNA and protein can both have weak secondary structures in vivo leading to cooperative folding.

Gonzaga University is a small, private, liberal arts institution that values engaging, humanistic learning environments. During the coronavirus pandemic, the University moved its in-person learning to a distance-learning format, and an extra week of spring break was given to faculty to oversee this transition. This communication focuses on how our introductory biochemistry lab course was modified for students to complete the final 6 weeks of the semester in the distance-learning format. The instructors worked collaboratively on this transition and focused on reassessing the learning objectives, finding creative solutions for students to experience laboratory techniques, and supporting and engaging our students. A survey administered to students at the end of the semester highlighted the strengths and weaknesses of both our in-person and distance-learning lab formats. This sudden change to course delivery in spring 2020 highlighted that we need to be prepared to teach under many unexpected scenarios. The student responses, along with faculty reflections, are being used to plan how our lab can be improved upon and delivered in a variety of modalities, including in-person, distancing-learning, and hybrid models.

RNA folding is often studied by renaturing full-length RNA in vitro and tracking folding transitions. However, the intracellular transcript folds as it emerges from the RNA polymerase. Here, we investigate the folding pathways and stability of numerous late-transcriptional intermediates of yeast and Escherichia coli transfer RNAs (tRNAs). Transfer RNA is a highly regulated functional RNA that undergoes multiple steps of posttranscriptional processing and is found in very different lengths during its lifetime in the cell. The precursor transcript is extended on both the 5′ and 3′ ends of the cloverleaf core, and these extensions get trimmed before addition of the 3′-CCA and aminoacylation. We studied the thermodynamics and structures of the precursor tRNA and of late-transcriptional intermediates of the cloverleaf structure. We examined RNA folding at both the secondary and tertiary structural levels using multiple biochemical and biophysical approaches. Our findings suggest that perhaps nature has selected for a single-base addition to control folding to the functional 3D structure. In near-cellular conditions, yeast tRNAPhe and E. coli tRNAAla transcripts fold in a single, cooperative transition only when nearly all of the nucleotides in the cloverleaf are transcribed by indirectly enhancing folding cooperativity. Furthermore, native extensions on the 5′ and 3′ ends do not interfere with cooperative core folding. This highly controlled cooperative folding has implications for recognition of tRNA by processing and modification enzymes and quality control of tRNA in cells.

A diverse set of organisms has adapted to live under extreme conditions. The molecular origin of the stability is unclear, however. It is not known whether the adaptation of functional RNAs, which have intricate tertiary structures, arises from strengthening of tertiary or secondary structure. Herein we evaluate effects of sequence changes on the thermostability of tRNA using experimental and computational approaches. To separate out effects of secondary and tertiary structure on thermostability, we modify base pairing strength in the acceptor stem, which does not participate in tertiary structure. In dilute solution conditions, strengthening secondary structure leads to non-two-state thermal denaturation curves and has small effects on thermostability, or the temperature at which tertiary structure and function are lost. In contrast, under cellular conditions with crowding and Mg-chelated amino acids, where two-state cooperative unfolding is maintained, strengthening secondary structure enhances thermostability. Investigation of stabilities of each tRNA stem across 44 organisms with a range of optimal growing temperatures revealed that organisms that grow in warmer environments have more stable stems. We also used Shannon entropies to identify positions of higher and lower information content, or sequence conservation, in tRNA and found that secondary structures have modest information content allowing them to drive thermal adaptation, while tertiary structures have maximal information content hindering them from participating in thermal adaptation. Base-paired regions with no tertiary structure and modest information content thus offer a facile evolutionary route to enhancing the thermostability of functional RNA by the simple molecular rules of base pairing.