LtrA protein and the LtrB intron RNA were well induced in active form. (A) LtrA expression. Total protein lysate corresponding to constructs 1–5 in Figure 1B was prepared from each strain and separated by 10% SDS–PAGE. Protein bands were visualized after Coomassie blue staining. All the LIC constructs showed a distinct protein band of ∼130 Kd, corresponding in size to LtrA-intein-CBD upon induction with nisin, whereas the constructs without the intein-CBD fusion showed a protein band of ∼70 Kd, corresponding to the size of LtrA. (B) Intron RNA expression. Total RNA was prepared from the same set of constructs as above, and 5 µg of total RNA from each, with (+) or without (−) nisin induction, was separated on 1.2% agarose gel containing 2.2 M formaldehyde. All the strains expressing LtrB intron RNA showed an RNA band of ∼1 kb with nisin induction (lanes 2, 3, 4 and 5, dots). The slight difference in migration of the spliced intron was due to the size difference between the intron precursor and the intron lariat (lanes 2 and 4, arrows). (C) Primer extension assay. A primer was designed to bind to the 5′-end of the LtrB intron. A 69-nt primer extension product was generated from LtrB intron precursor (lanes 2–5); and a 41 nt primer extension product was generated from LtrB intron lariat (lanes 2 and 4). Primer extension products were separated in 10% polyacrylamide/8 M urea gels, visualized by autoradiography.

Hydrodynamic comparison of precursor and product introns indicates a major conformational change. (A) SEC. The size-exclusion peaks for the +A and ΔA RNP particles are compared. The identity of the small 17-kDa peak with both samples is not known. (B and C) SV analysis. Shown in (B and C) are the experimental data for +A and ΔA RNP, respectively, rendered as black points on solid black lines that are the fits to the Lamm equation. Each boundary shown corresponds to a 30-s time interval, starting at time = 0 (data are shown only for the initial boundaries). Residuals, showing the agreement between the absorbance data collected and the theoretical fit to the Lamm equation, are shown below each panel as a function of the radius of the experimental cell. (D) RNP particles are compared on a concentration distribution of S values, c(S).

Purification scheme of intron RNP. (A) Purification strategy. Lysates from induced cells are passed over a CBD column, which traps the LtrA-intein-CBD (LIC) protein fusion and associated RNA. Cleavage of LtrA from the intein with DTT releases the RNP from the chitin column. (B) Constructs. The purification construct (3, marked with asterisk) (ΔA) and control constructs are illustrated.

Intron RNP precursor purification. (A) Purified intron RNP showed a single protein band, whose size corresponded to the intron-encoded protein, LtrA. Lane 1: purified LtrA; lanes 2–4: chitin elution fractions; lane 5: re-suspension of the pellet from sucrose cushion centrifugation; lane 6: purified intron RNP; lane 7: total cell lysate. (B) RNA gel of the purified intron RNP. A band of ∼1 kb corresponds to the size of LtrB intron precursor. The RNA content of each even-numbered sucrose gradient fraction was analyzed by 1.2% agarose gel containing 2.2 M formaldehyde. Fractions 13–16 were pooled and concentrated as the purified intron RNP. The RNA content of the pooled chitin elution and the re-suspension of the sucrose cushion pellet were also analyzed. (C) Negative-stain micrographs of ΔA and +A RNPs. Specimens were prepared in uranyl acetate.

(A) Cryo-EM images of the group II intron RNP precursor. (A) Top two rows are montages of individual, low-pass filtered particles corresponding to the dominant ‘face’ view. At the right is an average of 298 particles. Scale bar represents 100 Å. Bottom two rows are montages of individual, low-pass filtered particles corresponding to the ‘chain-link’ view. At the right is an average of 93 particles. (B) RCT reconstructions and merged reconstruction shown in front (first row), intermediate (second row) and side views (third row). (a₁, a₂, a₃) RCT reconstruction from 298 particles in face view, some of which are depicted in (A). (b₁, b₂, b₃) RCT reconstruction from 93 particles in side view, some of which are depicted in (A). (c_1, c_2, c₃) Reconstruction of 647 particle images using as a reference the face-view reconstruction shown in panels (a₁, a₂, a₃).