Predicting Cotranscriptional Folding Kinetics For Riboswitch

Abstract

On the basis of a helix-based transition rate model, we developed a new method for sampling cotranscriptional RNA conformational ensemble and the prediction of cotranscriptional folding kinetics. Applications to E. coli. SRP RNA and pbuE riboswitch indicate that the model may provide reliable predictions for the cotranscriptional folding pathways and population kinetics. For E. coli. SRP RNA, the predicted population kinetics and the folding pathway are consistent with the SHAPE profiles in the recent cotranscriptional SHAPE-seq experiments. For the pbuE riboswitch, the model predicts the transcriptional termination efficiency as a function of the force. The theoretical results show (a) a force-induced transition from the aptamer (antiterminator) to the terminator structure and (b) the different folding pathways for the riboswitch with and without the ligand (adenine). More specifically, without adenine, the aptamer structure emerges as a short-lived kinetic transient state instead of a thermodynamically stable intermediate state. Furthermore, from the predicted extension-time curves, the model identifies a series of conformational switches in the pulling process, where the predicted relative residence times for the different structures are in accordance with the experimental data. The model may provide a new tool for quantitative predictions of cotranscriptional folding kinetics, and results can offer useful insights into cotranscriptional folding-related RNA functions such as regulation of gene expression with riboswitches.

Figure 1:

The secondary structures of (A) aptamer and (B) terminator of pbuE riboswitch.^– The aptamer structure contains 3 helices (P1, P2 and P3) and 2 hairpin loops. The terminator and the aptamer structures share the same P2 helix. However, the terminator structure contains a termination hairpin that halts transcription.

Figure 2:

(A) Multiple pathways for the formation of a helix after the first nucleation stack is formed. For example, after stage 2, there are two folding pathways: 1→2→3 and 1→2→4. (B) The free energy landscape of the tunneling pathway between two overlapping helices A and B. k₁ denotes the transition rate of the unfolding of helix A to form the first stack of helix B. ${k^{\prime }}_1,k_2,{k^{\prime }}_2,\cdots ,k_n$ denote the transition rates between the neighboring intermediates along the tunneling pathways.

Figure 3:

Secondary structure of E. Coli. SRP RNA. The E. Coli. SRP RNA has 6 helices (H2, H3, H4, H5, H6 and H7), 4 internal loops, 2 bulge loops and a hairpin loop. Nucleotides whose SHAPE reactivity were measured in experiment are marked in blue.

Figure 4:

(A) The figure shows that major SHAPE reactivity change for each transition, where the horizontal axis shows the elongating chain length during transcription and the vertical axis shows the SHAPE reactivity. During the transition from HP1 to HP2, C31 is paired with G82, the SHAPE reactivity of C31 begins to decrease after the chain is transcribed to the 83th nucleotide. From HP2 to the native structure, U14 become paired with G103 and the SHAPE profile also indicates the transition. The 3D structures for the kinetically important structures are predicted using the Vfold3D software^– and the Coarse-Grained MD simulations. (B) The population kinetics of 3 kinetically important conformations during the cotranscriptional folding process. The major transitions during the folding process are extracted from the predicted population kinetics for the different conformations. HP1 is first formed in the early transcription stage, followed by the formation of HP2, and additional helices and loops are added to HP1. In the late stage of the folding process, the first hairpin (H1) in HP2 is disrupted and the native structure is formed. From the population kinetics, the major folding pathway of E.Coli. SRP RNA is HP1→HP2→Native.

Figure 5:

Free energy ∆G as a function of the force for the coil state, the terminator, and the aptamer structure. The free energy profiles give the equilibrium unfolding forces 11 pN and 8 pN for the terminator hairpin and the adenine-bound aptamer, respectively. In the free energy calculations, we use G_BIND = 5.3 kcal/mol for the ligand-induced additional stability of the aptamer.

Figure 6:

The theoretical and experimental results for the Termination Efficiency (TE). (A) The predicted TE under 20 nt/s transcription speed. The TE is shown as the fractional population of the terminator at the end of cotranscriptional folding as a function of force with and without adenine. (B) Experimentally determined termination efficiency as a function of force with and without adenine. It is important to note that for both cases (with and without the presence of adenine), the theoretical predictions in (A) and the experimental results in (B) for the termination efficiency show similar force-dependence.

Figure 7:

(A) The population kinetics for the cotranscriptional folding of the pbuE riboswitch under 5.8 pN pulling force and transcription speed 20 nt/s without adenine. There are 8 kinetically important states identified during the folding process, U (Black), C1 (Red), C2 (Blue), C3 (Dark Cyan), C4 (Magenta), C5 (Dark Yellow), C6 (Navy), and T (Wine). (B) The folding pathway inferred from the populational kinetics. The major folding pathway for the pbuE riboswitch under 5.8 pN pulling force and transcription speed 20 nt/s without adenine is U→C1→C3→C5 →C6→T with C2 and C4 as off-pathway kinetic intermediates (traps) connected to U and C3, respectively.

Figure 8:

(A) The population kinetics for the cotranscriptional folding of the pbuE riboswitch under 5.8 pN pulling force and transcription speed 20 nt/s with adenine. There are also 8 kinetically important states identified during the folding process, U (Black), C1 (Red), C2 (Blue), C3 (Dark Cyan), C4 (Magenta), C6 (Dark Yellow), and T (Navy). (B) The folding pathway inferred from the populational kinetics. The major folding pathway for the pbuE riboswitch under 5.8 pN pulling force and transcription speed 20 nt/s with adenine is U→C1→C3 →C4→T with C2 and C6 as off-pathway kinetic intermediates (traps) connected to U and C4, respectively.

Figure 9:

The predicted 3D structure for the kinetically important states during the cotranscription process of pbuE riboswitch. The 3D structures are predicted using the Vfold3D software^– and Coarse-Grained MD simulations.

Figure 10:

The extension as a function of time for the cotranscriptional folding under force (A) f = 5.8 pN and (B) f = 8.1 pN.