Volume 19, Issue 1, Pages 27-37 (July 2005) Kinetics and Thermodynamics Make Different Contributions to RNA Folding In Vitro and in Yeast  Elisabeth M. Mahen, Jason W. Harger, Elise M. Calderon, Martha J. Fedor  Molecular Cell  Volume 19, Issue 1, Pages 27-37 (July 2005) DOI: 10.1016/j.molcel.2005.05.025 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Hairpin Ribozyme Sequences with the Potential to Form Functional Ribozymes and Nonfunctional Stem-Loop Structures (A) HP28 is a minimal self-cleaving hairpin ribozyme studied previously (Donahue et al., 2000; Nesbitt et al., 1999; Yadava et al., 2001). Base-paired helices H1 through H4 and loops A and B are indicated. The arrow indicates the reactive phosphodiester. (B) HP28-510 is a variant of HP28 that has a sequence added to the 5′ end (bold font) that can pair with ten bases at the 5′ end of the ribozyme to form an Alt5′H1 stem loop that is incompatible with assembly of a functional ribozyme. Alt5′H1 is expected to be more stable than H1 by 4 or 7 kcal/mol based on the difference in free energies calculated for Alt5′H1 and H1 helices in HP28-510 mRNA and in vitro transcripts, respectively (Mathews et al., 1999). (C) HP28-310 is a variant of HP28 that has a sequence added to the 3′ end (bold font) that can pair with ten bases at the 3′ end of the ribozyme to form an Alt3′H1 stem loop that is incompatible with assembly of a functional ribozyme. Free energy calculations (Mathews et al., 1999) suggest that Alt3′H1 is more stable than H1 by 6 kcal/mol in HP28-310 mRNA and by 7 kcal/mol in the HP28-310 sequence in in vitro transcripts. Molecular Cell 2005 19, 27-37DOI: (10.1016/j.molcel.2005.05.025) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Self-Cleavage Activity Reflects Partitioning between Assembly of Functional Ribozymes and Competing Stem-Loop Structures during Transcription In Vitro Self-cleavage kinetics are compared for the unmodified HP28 ribozyme and ribozyme variants with ten complementary nucleotides inserted upstream (HP28-510) or downstream (HP28-310) of the ribozyme during transcription in vitro with a low (A) or high (B) concentration of free Mg2+. Lines represent fits to a single-exponential (solid line) or double-exponential (HP28-310, dashed line) rate equation. Molecular Cell 2005 19, 27-37DOI: (10.1016/j.molcel.2005.05.025) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Chemical Protection Mapping of Ribozyme Structures Assembled In Vitro (A and B) (A) DMS modification sites in the 3′ strand of H1 (yellow) or (B) the 5′ strand of H1 (blue) are indicated with filled circles for RNA structures assembled during transcription or during cation-induced refolding of denatured transcripts. Asterisks indicate primer extension products that stop at positions preceding DMS modification sites. Open circles indicate DMS-independent blocks to reverse transcription. Molecular Cell 2005 19, 27-37DOI: (10.1016/j.molcel.2005.05.025) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Intracellular Self-Cleavage Activity Reflects Competition between Assembly of Functional Ribozymes and Competing Stem-Loop Structures in Yeast (A) Hairpin ribozyme sequences (green) were inserted into the 3′ untranslated region (yellow) of the yeast PGK1 gene. Chimeric HP mRNAs were expressed from the PGK1 promoter under the control of the GAL1-10 upstream activation sequence, UASGAL (aqua). HP mRNA decays both through self-cleavage (kcleavage) and through the normal mRNA degradation pathway (kdegradation), so self-cleavage accelerates HP mRNA decay by an amount that equals the intracellular cleavage rate. (B) The difference between decay rates measured for chimeric mRNAs with or without an inactivating mutation gives an HP28 self-cleavage rate of 0.034 m−1. (C and D) Chimeric HP28-510 (C) and HP28-310 (D) mRNAs with and without inactivating mutations decayed at the same rates in yeast, indicating that complementary insertions blocked assembly of functional ribozymes. Molecular Cell 2005 19, 27-37DOI: (10.1016/j.molcel.2005.05.025) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 Chemical Protection Mapping of Hairpin Ribozyme Structures Assembled in Yeast Ribozyme sequences in chimeric HP28m, HP28-39m, and HP28-310m mRNAs (A) and HP28-510m and HP28-59m mRNAs (B) that were modified by DMS are indicated with filled circles. Asterisks indicate primer extension products that stop at positions preceding DMS modification sites. Molecular Cell 2005 19, 27-37DOI: (10.1016/j.molcel.2005.05.025) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 Thermodynamic Stability Affects Partitioning between Functional Ribozymes and Stem-Loop Structures In Vitro and in Yeast (A) Hairpin ribozyme sequences with the potential to form nonfunctional stem-loop structures that vary in thermodynamic stability. ΔΔG values represent the difference in calculated free energy between AltH1 and H1 structures in kcal/mol (Mathews et al., 1999). When chimeric mRNA and in vitro transcript sequences vary, the in vitro transcript sequence and ΔΔG values are in parentheses. (B) Comparison of self-cleavage kinetics during transcription in vitro in reactions with low concentrations of free Mg2+ for ribozymes with eight (HP28-58) or nine (HP28-59) complementary nucleotides inserted upstream of the ribozyme sequence and for variants with seven (HP28-37) or nine (HP28-39) complementary nucleotides inserted downstream of the ribozyme sequence. Solid lines represent fits to a single-exponential rate equation. (C) Intracellular self-cleavage kinetics for chimeric mRNAs that contain a self-cleaving ribozyme with nine complementary nucleotides inserted upstream or downstream of the ribozyme sequence. Self-cleavage rates were calculated from the difference between decay rates measured for chimeric mRNAs with and without an inactivating mutation. Molecular Cell 2005 19, 27-37DOI: (10.1016/j.molcel.2005.05.025) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 7 Models for RNA Folding In Vivo (A) In a rapid conformational exchange model, secondary structures form during transcription and subsequently undergo exchange at a rate that is determined by the rate at which the initial secondary structure unfolds, which might be accelerated through the action of an RNA chaperone (purple). The folding outcome reflects relative rates of dissociation of alternative secondary structures, which correlates with thermodynamic stability. (B) In a delayed folding model, secondary structure assembly is not coincident with transcription, perhaps because single-stranded RNA binding proteins (blue) are deposited on nascent transcripts. Secondary structures form only after an entire folding domain has been transcribed. Once a domain is free to fold, partitioning among alternative structures reflects the relative kinetics of secondary structure formation. Stable secondary structures remain fixed after initial assembly, while unstable secondary structures rearrange at rates determined by their relative rates of dissociation as in the rapid conformational exchange model. Molecular Cell 2005 19, 27-37DOI: (10.1016/j.molcel.2005.05.025) Copyright © 2005 Elsevier Inc. Terms and Conditions