Dataset Information

Comparative structural analysis of transcriptionally competent and incompetent rotavirus-antibody complexes.

ABSTRACT: During genome transcription in rotavirus, as with many segmented double-stranded RNA viruses, mRNA is transcribed within the intact subviral particle and translocated through specific channels in the capsid. To understand how the conformation of the capsid affects the efficiency of transcriptional events in the viral core, we carried out a series of comparative structural and biochemical studies to characterize four different structural forms of the virus exhibiting differing transcriptional behavior. Two of these were virus-antibody complexes having contrasting transcriptional capabilities, and two were variant structural forms of the virus that exist during the life cycle and also exhibit contrasting transcriptional behavior. Three-dimensional structural studies using electron cryomicroscopy showed that the binding of one Fab (8H2/G5) does not affect the conformation of the capsid, and the efficiency of mRNA production is similar to that of the native subviral particle. The other Fab (2A11/E9) introduces conformational changes in the capsid similar to those seen in the transcriptionally incompetent mature particle. In both of the transcriptionally incompetent particle types, mRNA synthesis was arrested after limited elongation with the resulting oligonucleotide transcripts remaining trapped inside the particles. Our results indicate that the continuous translocation of nascent mRNA through the capsid is critical for efficient transcript elongation and that the blockage of translocation causes premature termination of transcription.

SUBMITTER: Lawton JA

PROVIDER: S-EPMC21876 | biostudies-literature | 1999 May

REPOSITORIES: biostudies-literature

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Comparative structural analysis of transcriptionally competent and incompetent rotavirus-antibody complexes.

Lawton J A JA Estes M K MK Prasad B V BV

Proceedings of the National Academy of Sciences of the United States of America 19990501 10

During genome transcription in rotavirus, as with many segmented double-stranded RNA viruses, mRNA is transcribed within the intact subviral particle and translocated through specific channels in the capsid. To understand how the conformation of the capsid affects the efficiency of transcriptional events in the viral core, we carried out a series of comparative structural and biochemical studies to characterize four different structural forms of the virus exhibiting differing transcriptional beh ...[more]

PMID: 10318900

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Project description:Ortholog protein complexes are responsible for equivalent functions in different organisms. However, during evolution, each organism adapts to meet its physiological needs and the environmental challenges imposed by its niche. This selection pressure leads to structural diversity in protein complexes, which are often difficult to specify, especially in the absence of high-resolution structures. Here, we describe a multilevel experimental approach based on native mass spectrometry (MS) tools for elucidating the structural preservation and variations among highly related protein complexes. The 20S proteasome, an essential protein degradation machinery, served as our model system, wherein we examined five complexes isolated from different organisms. We show that throughout evolution, from the Thermoplasma acidophilum archaeal prokaryotic complex to the eukaryotic 20S proteasomes in yeast (Saccharomyces cerevisiae) and mammals (rat - Rattus norvegicus, rabbit - Oryctolagus cuniculus and human - HEK293 cells), the proteasome increased both in size and stability. Native MS structural signatures of the rat and rabbit 20S proteasomes, which heretofore lacked high-resolution, three-dimensional structures, highly resembled that of the human complex. Using cryoelectron microscopy single-particle analysis, we were able to obtain a high-resolution structure of the rat 20S proteasome, allowing us to validate the MS-based results. Our study also revealed that the yeast complex, and not those in mammals, was the largest in size and displayed the greatest degree of kinetic stability. Moreover, we also identified a new proteoform of the PSMA7 subunit that resides within the rat and rabbit complexes, which to our knowledge have not been previously described. Altogether, our strategy enables elucidation of the unique structural properties of protein complexes that are highly similar to one another, a framework that is valid not only to ortholog protein complexes, but also for other highly related protein assemblies.

Dataset Information

Comparative structural analysis of transcriptionally competent and incompetent rotavirus-antibody complexes.

Publications

Comparative structural analysis of transcriptionally competent and incompetent rotavirus-antibody complexes.

Similar Datasets

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