![]() ![]() In a typical folding pathway ( Figure 1), local stem-loops form in ~10 μs while individual domains of tertiary structure or intermolecular interactions typically form in 1–100 ms, depending on the folding conditions ( 41– 46). The potential for misfolding in RNA is exacerbated by the varied time scales for forming secondary and tertiary interactions. As a result, stable local interactions outcompete long-range interactions, frustrating the search for the native conformation and allowing incorrect base pairing patterns to persist ( 40, 41). Nevertheless, interactions between nucleotides far apart in the RNA sequence are entropically less favorable than those between nearby nucleotides. Tertiary interactions between double helices make folding more specific by favoring folding intermediates in which the double helices are correctly aligned ( 38, 39). As an RNA grows longer, the odds that it can form more than one stable secondary structure increases substantially. For example, the free energy of forming a 10 bp stem-loop typically ranges from -10 to -20 kcal/mol at 37 C, yet a mismatched helix of the same length may be only 1–2 kcal/mol less stable than the fully matched helix. Hammond).Īlthough stable RNA base pairs create good switches, the small number of natural nucleobases limits the specificity of RNA regulatory interactions and increases the chance of RNA misfolding ( 16). It also presents synthetic biologists with an attractive platform for genetic engineering ( 36, 37). The potential simplicity of RNA-based regulation offers microbes an expedient means of evolving new regulatory circuits ( 35). Owing to the stability of the RNA double helix, RNA regulatory elements may be as small as a single stem-loop or anti-sense helix, or involve more elaborate tertiary structures ( 32– 34). These features of stability and interchangeability ideally suit RNA for creating metastable structures that can switch gene expression on or off. RNA double helices are stable and long-lived, yet able to interchange through the sequential migration of base pairs during strand transfer or branch migration reactions. RNA folding and the need for RNA chaperones coli Hfq, are beginning to provide a physical picture of how proteins remodel RNA structures during RNA regulation. coli CspA, HIV NCp7, ribosomal protein S1, and E. This chapter will discuss how well-studied examples, such as E. The diverse biological roles of the above examples highlight the broad importance of proteins that escort, facilitate or accelerate structural changes in non-coding RNA. Eukaryotic proteins with ATP-independent RNA chaperone properties include RNA recognition motif (RRM) proteins such as hnRNP A1 ( 27), viral proteins such as the well-studied retroviral nucleocapsid protein (NCp7) ( 28, 29), and La and Ro proteins ( 30, 31). H-NS and StpA, which interact with the bacterial nucleoid, also possess RNA chaperone activity ( 17). In bacteria, this type of passive RNA chaperone includes cold shock proteins (CSPs) ( 14), the Sm family protein Hfq ( 21), the FinO/ProQ family of RNA binding proteins ( 22), and ribosomal proteins S1 ( 23– 25) and S12 ( 26). This chapter, however, will focus on RNA binding proteins that “passively” remodel RNA structures without hydrolyzing ATP. Bacteria typically encode a handful (0–12) of DEAD-box proteins that mainly act in ribosome biogenesis and RNA turnover ( 12, 20). RNA helices can be actively unwound by DEAD-box proteins, which couple unfolding of the RNA structure to ATP hydrolysis ( 18, 19). RNA chaperones act by transiently binding and releasing RNA substrates, disrupting the RNA secondary and tertiary structure (unwinding and unfolding) or accelerating base pairing with a second RNA strand (annealing) ( 16, 17). coli ( 15), suggesting that such proteins broadly mitigate the effects of RNA misfolding. Moreover, over-expression of cold shock proteins and other RNA chaperones buffered deleterious mutations in E. For example, the up-regulation of cold shock domain RNA binding proteins during low temperature growth destabilizes RNA structures that would otherwise impair transcription elongation and translation initiation at low temperatures ( 14). These housekeeping and regulatory functions of RNA chaperones are particularly important at cold temperatures that hyperstabilize RNA structures. RNA chaperones also facilitate conformational rearrangements during ribosome biogenesis ( 12) and eukaryotic pre-mRNA splicing ( 13). These regulatory RNAs are chaperoned by diverse families of RNA binding proteins, and the loss of RNA chaperone proteins can lead to impaired growth, reduced tolerance to stress, and reduced virulence ( 6– 11). ![]() Non-coding RNA sequences fold into useful structures that regulate gene expression as ribozymes, metabolite binding sensors, or antisense RNAs ( 1– 5). ![]()
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