Conformational Transitions of an Unmodified tRNA:  Implications for RNA Folding

Abstract

Unmodified tRNAs are powerful systems to study the effects of posttranscriptional modifications and site-directed mutations on both the structure and function of these ribonucleic acids. To define the general limitations of synthetic constructs as models for native tRNAs, it is necessary to elucidate the conformational states of unmodified tRNAs as a function of solution conditions. Here we report the conformational properties of unmodified yeast tRNAPhe as a function of ionic strength, [Mg2+], and temperature using a combination of spectroscopic measurements along with chemical and enzymatic probes. We find that in low [Na+] buffer at low temperature, native yeast tRNAPhe adopts tertiary structure in the absence of Mg2+. By contrast, tertiary folding of unmodified yeast tRNAPhe has an absolute requirement for Mg2+. Below the melting temperature of the cloverleaf, unmodified yeast tRNAPhe exists in a Mg2+-dependent equilibrium between secondary and tertiary structure. Taken together, our findings suggest that although the tertiary structures of tRNAs are broadly comparable, the intrinsic stability of the tertiary fold, the conformational properties of intermediate states, and the stability of intermediate states can differ significantly between tRNA sequences. Thus, the use of unmodified tRNAs as models for native constructs can have significant limitations. Broad conclusions regarding "tRNA folding" as a whole must be viewed cautiously, particularly in cases where structural changes occur, such as during protein synthesis.

Mg2+ binding and archaeosine modification stabilize the G15–C48 Levitt base pair in tRNAs

“…Many studies reported how the lack of modification in tRNA in vitro transcripts can be compensated by an increased concentration of Mg 2+ ions to obtain functional structures (Sampson and Uhlenbeck 1988;Perret et al 1990;Maglott et al 1998;Serebrov et al 1998). We illustrate here the interchangeable stabilizing role of metal binding and post-transcriptional modification on a specific tRNA tertiary interaction, the G15-C48 Levitt base pair, provided with a physicochemical explanation of the phenomenon.…”

The La protein functions redundantly with tRNA modification enzymes to ensure tRNA structural stability

“…In vitro, modified tRNAs exhibit greater thermodynamic stability than the corresponding unmodified tRNAs (Hall et al 1989;Maglott et al 1998;Serebrov et al 1998;Vermeulen et al 2005). Consistent with a role for modified nucleotides in stabilizing tRNAs, pre-tRNA Met i is unstable in cells containing a mutation in the m 1 A methyltransferase (Kadaba et al 2004).…”

Selective quenching of fluorescence from unbound oligonucleotides by gold nanoparticles as a probe of RNA structure

“…premature aggregation of the colloid. Unfolding of tertiary structure occurs with half-lives ranging from 0.01 s as demonstrated for tRNA (Maglott et al 1998) and RNase P (Fang et al 2000(Fang et al , 2002 to at least between 3 and 35 s, as demonstrated for the Tetrahymena thermophila self-splicing intron (Bevilacqua et al 1992;Zhuang et al 2000). Thus, it is possible for the RNA to be partially unfolded during the course of the assay.…”