Structural basis for the second step of group II intron splicing

The group II intron and the spliceosome share a common active site architecture and are thought to be evolutionarily related. Here we report the 3.7 Å crystal structure of a eukaryotic group II intron in the lariat-3′ exon form, immediately preceding the second step of splicing, analogous to the spliceosomal P complex. This structure reveals the location of the intact 3′ splice site within the catalytic core of the group II intron. The 3′-OH of the 5′ exon is positioned in close proximity to the 3′ splice site for nucleophilic attack and exon ligation. The active site undergoes conformational rearrangements with the catalytic triplex having dif- ferent configurations before and after the second step of splicing. We describe a complete model for the second step of group II intron splicing that incorporates a dynamic catalytic triplex being responsible for creating the binding pocket for 3′ splice site capture

Structural Biology of RNA by X-ray Crystallography, Chemical Probing, and Cryo-EM

There are a wide variety of non-coding RNAs that fold into well-defined 3D shapes and play important roles in the cell. Despite the importance of these non-coding RNAs in biology, the field of RNA structural biology is not as well-developed as protein structural biology. The main goal of this dissertation is to elucidate the structures of RNA molecules using several approaches. Other work on nucleic acid-related proteins is also presented.The first part of the dissertation focuses on the group II intron RNA. A crystal structure of the group II intron in the intermediate lariat-3′ exon state was determined to elucidate the mechanism of the second step of splicing and led to a model of the second step in which several junction nucleotides undergo dynamic rearrangements. These dynamic rearrangements are supported by splicing assays of mutants and SHAPE chemical probing. The SHAPE data also revealed that κ-κ′, a tertiary interaction in a different part of the intron, has dynamics that are necessary for splicing.Chapter 4 looks at the mechanism of selective fidelity in diversity-generating retroelements, a class of genetic elements that can generate a large amount of sequence variability in a protein. This work shows that selective fidelity was due to the low catalytic efficiency of the reverse transcriptase and depended on certain substituents in the nucleobase template.The next part of the dissertation explores the use of bacterial nanocompartments as a chaperone for cryo-electron microscopy (cryo-EM) structure determination of RNA . First, a high-resolution cryo-EM structure of a thermostable bacterial nanocompartment is presented, illustrating several of its interesting features. Second, a method to assemble RNA inside a nanocompartment is demonstrated and a cryo-EM dataset of this complex was collected, resulting in a 5 Å reconstruction of the encapsulated RNA.Chapter 7 explores the structure and mechanism of a DNA phosphorothioation complex. This complex is active in vivo and the recombinantly purified proteins bind to DNA in vitro. A cryo-EM dataset of this complex was collected and resulted in a 5 Å density map

Structural Biology of RNA by X-ray Crystallography, Chemical Probing, and Cryo-EM

There are a wide variety of non-coding RNAs that fold into well-defined 3D shapes and play important roles in the cell. Despite the importance of these non-coding RNAs in biology, the field of RNA structural biology is not as well-developed as protein structural biology. The main goal of this dissertation is to elucidate the structures of RNA molecules using several approaches. Other work on nucleic acid-related proteins is also presented.The first part of the dissertation focuses on the group II intron RNA. A crystal structure of the group II intron in the intermediate lariat-3′ exon state was determined to elucidate the mechanism of the second step of splicing and led to a model of the second step in which several junction nucleotides undergo dynamic rearrangements. These dynamic rearrangements are supported by splicing assays of mutants and SHAPE chemical probing. The SHAPE data also revealed that κ-κ′, a tertiary interaction in a different part of the intron, has dynamics that are necessary for splicing.Chapter 4 looks at the mechanism of selective fidelity in diversity-generating retroelements, a class of genetic elements that can generate a large amount of sequence variability in a protein. This work shows that selective fidelity was due to the low catalytic efficiency of the reverse transcriptase and depended on certain substituents in the nucleobase template.The next part of the dissertation explores the use of bacterial nanocompartments as a chaperone for cryo-electron microscopy (cryo-EM) structure determination of RNA . First, a high-resolution cryo-EM structure of a thermostable bacterial nanocompartment is presented, illustrating several of its interesting features. Second, a method to assemble RNA inside a nanocompartment is demonstrated and a cryo-EM dataset of this complex was collected, resulting in a 5 Å reconstruction of the encapsulated RNA.Chapter 7 explores the structure and mechanism of a DNA phosphorothioation complex. This complex is active in vivo and the recombinantly purified proteins bind to DNA in vitro. A cryo-EM dataset of this complex was collected and resulted in a 5 Å density map

Structure determination of group II introns

Group II introns are self-splicing catalytic RNAs that are able to excise themselves from pre-mRNAs using a mechanism identical to that utilized by the spliceosome. Both structural and phylogenetic data support the hypothesis that group II introns and the spliceosome share a common ancestor. Structures of group II introns have given insight into the active site required for the catalysis of RNA splicing. This review outlines crucial aspects of the structure determination of group II introns such as sample preparation and data processing. Given that group II introns are large RNAs that must be synthesized through in vitro transcription, there are special considerations that must be taken into account in terms of purification and crystallization, as compared to the isolation of large intact ribonucleoprotein complexes such as the ribosome. We specifically focus on the methodology used to determine the structure of the eukaryotic group II intron lariat from the brown algae Pylaiella littoralis. The techniques described in this review can also be applied for the structure determination of other large RNAs

Determinants of Adenine-mutagenesis in Diversity-Generating Retroelements

ABSTRACTDiversity-generating retroelements (DGRs) vary protein sequences to the greatest extent known in the natural world. These elements are encoded by constituents of the human microbiome and the microbial ‘dark matter’. Variation occurs through adenine-mutagenesis, in which genetic information in RNA is reverse transcribed faithfully to cDNA for all template bases but adenine. We investigated the determinants of adenine-mutagenesis in the prototypical Bordetella bacteriophage DGR through an in vitro system composed of the reverse transcriptase bRT, Avd protein, and a specific RNA. We found that the catalytic efficiency for correct incorporation during reverse transcription by the bRT-Avd complex was strikingly low for all template bases, with the lowest occurring for adenine. Misincorporation across a template adenine was only somewhat lower in efficiency than correct incorporation. We found that the C6, but not the N1 or C2, purine substituent was a key determinant of adenine-mutagenesis. bRT-Avd was insensitive to the C6 amine of adenine but recognized the C6 carbonyl of guanine. We also identified two bRT amino acids predicted to nonspecifically contact incoming dNTPs, R74 and I181, as promoters of adenine-mutagenesis. Our results suggest that the overall low catalytic efficiency of bRT-Avd is intimately tied to its ability to carry out adenine-mutagenesis

Determinants of adenine-mutagenesis in diversity-generating retroelements.

Diversity-generating retroelements (DGRs) vary protein sequences to the greatest extent known in the natural world. These elements are encoded by constituents of the human microbiome and the microbial 'dark matter'. Variation occurs through adenine-mutagenesis, in which genetic information in RNA is reverse transcribed faithfully to cDNA for all template bases but adenine. We investigated the determinants of adenine-mutagenesis in the prototypical Bordetella bacteriophage DGR through an in vitro system composed of the reverse transcriptase bRT, Avd protein, and a specific RNA. We found that the catalytic efficiency for correct incorporation during reverse transcription by the bRT-Avd complex was strikingly low for all template bases, with the lowest occurring for adenine. Misincorporation across a template adenine was only somewhat lower in efficiency than correct incorporation. We found that the C6, but not the N1 or C2, purine substituent was a key determinant of adenine-mutagenesis. bRT-Avd was insensitive to the C6 amine of adenine but recognized the C6 carbonyl of guanine. We also identified two bRT amino acids predicted to nonspecifically contact incoming dNTPs, R74 and I181, as promoters of adenine-mutagenesis. Our results suggest that the overall low catalytic efficiency of bRT-Avd is intimately tied to its ability to carry out adenine-mutagenesis

Determinants of adenine-mutagenesis in diversity-generating retroelements.

Diversity-generating retroelements (DGRs) vary protein sequences to the greatest extent known in the natural world. These elements are encoded by constituents of the human microbiome and the microbial 'dark matter'. Variation occurs through adenine-mutagenesis, in which genetic information in RNA is reverse transcribed faithfully to cDNA for all template bases but adenine. We investigated the determinants of adenine-mutagenesis in the prototypical Bordetella bacteriophage DGR through an in vitro system composed of the reverse transcriptase bRT, Avd protein, and a specific RNA. We found that the catalytic efficiency for correct incorporation during reverse transcription by the bRT-Avd complex was strikingly low for all template bases, with the lowest occurring for adenine. Misincorporation across a template adenine was only somewhat lower in efficiency than correct incorporation. We found that the C6, but not the N1 or C2, purine substituent was a key determinant of adenine-mutagenesis. bRT-Avd was insensitive to the C6 amine of adenine but recognized the C6 carbonyl of guanine. We also identified two bRT amino acids predicted to nonspecifically contact incoming dNTPs, R74 and I181, as promoters of adenine-mutagenesis. Our results suggest that the overall low catalytic efficiency of bRT-Avd is intimately tied to its ability to carry out adenine-mutagenesis

Structural basis for the second step of group II intron splicing.

The group II intron and the spliceosome share a common active site architecture and are thought to be evolutionarily related. Here we report the 3.7 Å crystal structure of a eukaryotic group II intron in the lariat-3' exon form, immediately preceding the second step of splicing, analogous to the spliceosomal P complex. This structure reveals the location of the intact 3' splice site within the catalytic core of the group II intron. The 3'-OH of the 5' exon is positioned in close proximity to the 3' splice site for nucleophilic attack and exon ligation. The active site undergoes conformational rearrangements with the catalytic triplex having different configurations before and after the second step of splicing. We describe a complete model for the second step of group II intron splicing that incorporates a dynamic catalytic triplex being responsible for creating the binding pocket for 3' splice site capture