Snapshots of the RNA editing machine in trypanosomes captured at different assembly stages in vivo.

Mitochondrial pre-messenger RNAs in kinetoplastid protozoa are substrates of uridylate-specific RNA editing. RNA editing converts non-functional pre-mRNAs into translatable molecules and can generate protein diversity by alternative editing. Although several editing complexes have been described, their structure and relationship is unknown. Here, we report the isolation of functionally active RNA editing complexes by a multistep purification procedure. We show that the endogenous isolates contain two subpopulations of approximately 20S and approximately 35-40S and present the three-dimensional structures of both complexes by electron microscopy. The approximately 35-40S complexes consist of a platform density packed against a semispherical element. The approximately 20S complexes are composed of two subdomains connected by an interface. The two particles are structurally related, and we show that RNA binding is a main determinant for the interconversion of the two complexes. The approximately 20S editosomes contain an RNA-binding site, which binds gRNA, pre-mRNA and gRNA/pre-mRNA hybrid molecules with nanomolar affinity. Variability analysis indicates that subsets of complexes lack or possess additional domains, suggesting binding sites for components. Together, a picture of the RNA editing machinery is provided.

Coaxially stacked RNA helices in the catalytic center of the Tetrahymena ribozyme.

Coaxial stacking of helical elements is a determinant of three-dimensional structure in RNA. In the catalytic center of the Tetrahymena group I intron, helices P4 and P6 are part of a tertiary structural domain that folds independently of the remainder of the intron. When P4 and P6 were fused with a phosphodiester linkage, the resulting RNA retained the detailed tertiary interactions characteristic of the native P4-P6 domain and even required lower magnesium ion concentrations for folding. These results indicate that P4 and P6 are coaxial in the P4-P6 domain and, therefore, in the native ribozyme. Helix fusion could provide a general method for identifying pairs of coaxially stacked helices in biological RNA molecules.

Determination of the ribosome structure to a resolution of 2.5 Å by single-particle cryo-EM.

With the advance of new instruments and algorithms, and the accumulation of experience over decades, single-particle cryo-EM has become a pivotal part of structural biology. Recently, we determined the structure of a eukaryotic ribosome at 2.5 Å for the large subunit. The ribosome was derived from Trypanosoma cruzi, the protozoan pathogen of Chagas disease. The high-resolution density map allowed us to discern a large number of unprecedented details including rRNA modifications, water molecules, and ions such as Mg2+ and Zn2+ . In this paper, we focus on the procedures for data collection, image processing, and modeling, with particular emphasis on factors that contributed to the attainment of high resolution. The methods described here are readily applicable to other macromolecules for high-resolution reconstruction by single-particle cryo-EM.

Kinetics of an RNA conformational switch.

The spliced leader RNA from Leptomonas collosoma has two competing secondary structures of nearly equal free energy. Short, complementary oligonucleotides can drive the structure from one form of the other. We report stopped-flow rapid-mixing and temperature-jump measurements of the kinetics of the structural switch. At high concentrations of oligonucleotide, the rate of binding becomes limited by the rate of the structural switch, which occurs on a time scale of a fraction of a second. The low activation energy observed for the process implies a branch migration type of mechanism in which portions of the two competing helices transiently coexist.