Nathan A. Siegfried

Structured RNA elements within messenger RNA often direct or modulate the cellular production of active proteins. As reviewed here, RNA structures have been discovered that govern nearly every step in protein production: mRNA production and stability; translation initiation, elongation, and termination; protein folding; and cellular localization. Regulatory RNA elements are common within RNAs from every domain of life. This growing body of RNA-mediated mechanisms continues to reveal new ways in which mRNA structure regulates translation. We integrate examples from several different classes of RNA structure-mediated regulation to present a global perspective that suggests that the secondary and tertiary structure of RNA ultimately constitutes an additional level of the genetic code that both guides and regulates protein biosynthesis.

Secondary structure plays critical roles in nucleic acid function. Mismatches in DNA can lead to mutation and disease, and some mismatches involve a protonated base. Among protonated mismatches, A(+).C wobble pairs form near physiological pH and have relatively minor effects on helix geometry, making them especially important in biology. Herein, we investigate effects of helix position, temperature, and ionic strength on pK(a) shifting in A(+).C wobble pairs in DNA. We observe that pK(a) shifting is favored by internal A(+).C wobbles, which have low cooperativities of folding and make large contributions to stability, and disfavored by external A(+).C wobbles, which have high folding cooperativities but make small contributions to stability. An inverse relationship between pK(a) shifting and temperature is also found, which supports a model in which protonation is enthalpically favored overall and entropically correlated with cooperativity of folding. We also observe greater pK(a) shifts as the ionic strength decreases, consistent with anticooperativity between proton binding and counterion-condensed monovalent cation. Under the most favorable temperature and ionic strength conditions tested, a pK(a) of 8.0 is observed for the A(+).C wobble pair, which represents an especially large shift ( approximately 4.5 pK(a) units) from the unperturbed pK(a) value of adenosine. This study suggests that protonated A(+).C wobble pairs exist in DNA under biologically relevant conditions, where they can drive conformational changes and affect replication and transcription fidelity.

Secondary structural motifs play essential roles in the folding and function of RNA and DNA molecules. Previous work from our lab compared the folding of small DNA and RNA hairpin loops containing a sheared GA pair [Moody, E. M., Feerar, J. C., and Bevilacqua, P. C. (2004) Biochemistry 43, 7992-7998]. We found that the small DNA hairpins fold in a highly cooperative manner with indirect coupling, while their RNA counterparts fold in a much less cooperative fashion and display direct coupling. Herein, we extend this study to the double-stranded helix. We carried out double mutant cycles on base pairs having identical nearest-neighbor contexts but located in either external or internal helical registers. In the external register, both RNA and DNA exhibit extensive folding cooperativity between the penultimate and terminal base pair, which is independent of mismatch identity. In contrast, DNA exhibits virtually no folding cooperativity in the center of the helix, while RNA maintains substantial coupling, which is dependent on mismatch identity. Two models account for these non-nearest-neighbor effects: one involves the unfavorable entropy of helix initiation common to DNA and RNA, and the other involves steric and electrostatic strain peculiar to RNA. These data show that RNA can display cooperativity less than, greater than, or equal to that of DNA depending on context and position.