Branch site bulge conformations in domain 6 determine functional sugar puckers in group II intron splicing

Abstract

Although group II intron ribozymes are intensively studied the question how structural dynamics affects splicing catalysis has remained elusive. We report for the first time that the group II intron domain 6 exists in a secondary structure equilibrium between a single- and a two-nucleotide bulge conformation, which is directly linked to a switch between sugar puckers of the branch site adenosine. Our study determined a functional sugar pucker equilibrium between the transesterification active C2′-endo conformation of the branch site adenosine in the 1nt bulge and an inactive C3′-endo state in the 2nt bulge fold, allowing the group II intron to switch its activity from the branching to the exon ligation step. Our detailed NMR spectroscopic investigation identified magnesium (II) ions and the branching reaction as regulators of the equilibrium populations. The tuneable secondary structure/sugar pucker equilibrium supports a conformational selection mechanism to up- and downregulate catalytically active and inactive states of the branch site adenosine to orchestrate the multi-step splicing process. The conformational dynamics of group II intron domain 6 is also proposed to be a key aspect for the directionality selection in reversible splicing.

Aromatic 19F–13C TROSY—[19F, 13C]‐Pyrimidine Labeling for NMR Spectroscopy of RNA

“…20 kD) ( Figure 1d). We further incorporated a [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] Our initial efforts focused on the production of the [5-19 F, 5-13 C]-uridine phosphoramidite 7.B ased on our recently published synthetic access to a5-13 C-uridine building block [9] we included af luorination step using the commercially available fluorination reagent Selectfluor TM .T he synthetic route starting from [5-13 C]-uracil is shown (Scheme 1). The compound 1 was produced from [2-13 C]-bromoacetic acid, potassium cyanide and urea as previously described.…”

“…Schwalbe and coworkers used the latter method to probe the effect of all 2-F-Al abeling in the 73 nt guanine sensing riboswitch from bacillus subtilis. [3a] Only four resonances of sixteen could be assigned illustrating that in larger RNAs resonance assignment issues arise.Thus,anexpansion of the 19 Fstable isotope labeling scheme to yield a 19 F- 13 C-spin topology,such as a [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] F, 5-13 C]-labeling pattern in pyrimidines,would be desirable to evade resonance overlap issues in larger RNAs by adding asecond 13 Cdimension. Theapplication of 19 F-NMR to study high molecular weight systems is also hampered by the fast transverse relaxation by the chemical shift anisotropy (CSA) mechanism.…”

“…In ar ecent work this issue was addressed by introducing aromatic 19 F- 13 C-spin topologies in proteins and DNA, in which the cancellation of the dipole-dipole interaction and the chemical shift anisotropy relaxation mechanisms lead to avery favorable NMR spectroscopic behavior in aTROSY type experiment. [6] Here,wereport on the synthetic access to [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] F, 5-13 C]-uridine (U) and -cytidine (C) phosphoramidites for solid phase RNAsynthesis.W edemonstrate the favorable spectroscopic behavior of the [5-19 F, 5-13 C]pyrimidines in two model RNAs:the 61 nt human hepatitis B virus encapsidation signal epsilon (hHBV e)a nd the 59 nt pre-microRNA21. [7] Thefeasibility of the 13 C/ 19 Flabeling by solid phase RNAsynthesis is shown and used for aresonance assignment protocol based on [5-19 F, 5-13 C]/[5-19 F]-uridine labeling schemes.F or the 61 nt hHBV e RNAw eo bserved afavorable TROSY effect and compared the performance of [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] F, 13 C]-U labels with [6-1 H, 6-13 C]-U labels.T he novel stable isotope (SI) labeling scheme will facilitate NMR studies of large RNAs and high molecular weight RNA protein complexes as it was recently demonstrated for high molecular weight proteins systems.…”

Aromatic 19F–13C TROSY—[19F, 13C]‐Pyrimidine Labeling for NMR Spectroscopy of RNA

“…Forschungsartikel phosphoramidites 7 and 11 were available RNAs with sizes between 30 and 60 nts were synthesized. First, the 30 nt HIV TAR-2 with six uridines and the 61 nt hHBV e RNAw ith eighteen uridines were synthesized and six and five [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] F, 5-13 C]-U labels were incorporated in each RNA, respectively. We obtained high quality 19 F- 13 CT ROSY spectra within as hort time on aP rodigy TCI probe with the proton coil tuned to the 19 Fr esonance frequency for 0.5 mm RNA samples.T he additional 13 Cd imension allowed to resolve the resonance overlap,w hich was earlier observed for U23, U25 and U31 in the 1D-19 Fs pectrum.…”

Comparative Chloroplast Genomics of Four Pilea Species (Urticaceae): High Levels of Sequences Divergence Provides New Insight into Interspecific Diversity in Pilea

“…Figure 1 shows the schematic diagram of the cp genomes of Pilea. Introns play a signi cant role in selective gene splicing [21]. Among the 79 protein coding genes annotated, nine unique genes (rps16, rpoC1, atpF, petB, petD, rpl16, rpl2, ndhB, ndhA) contain one intron and two unique genes (ycf3, clpP) contain two introns.…”