Visualizing the global secondary structure of a viral RNA genome with cryo-electron microscopy.
The lifecycle, and therefore the virulence, of single-stranded (ss)-RNA viruses is regulated not only by their particular protein gene products, but also by the secondary and tertiary structure of their genomes. The secondary structure of the entire genomic RNA of satellite tobacco mosaic virus (STMV) was recently determined by selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE). The SHAPE analysis suggested a single highly extended secondary structure with much less branching than occurs in the ensemble of structures predicted by purely thermodynamic algorithms. Here we examine the solution-equilibrated STMV genome by direct visualization with cryo-electron microscopy (cryo-EM), using an RNA of similar length transcribed from the yeast genome as a control. The cryo-EM data reveal an ensemble of branching patterns that are collectively consistent with the SHAPE-derived secondary structure model. Thus, our results both elucidate the statistical nature of the secondary structure of large ss-RNAs and give visual support for modern RNA structure determination methods. Additionally, this work introduces cryo-EM as a means to distinguish between competing secondary structure models if the models differ significantly in terms of the number and/or length of branches. Furthermore, with the latest advances in cryo-EM technology, we suggest the possibility of developing methods that incorporate restraints from cryo-EM into the next generation of algorithms for the determination of RNA secondary and tertiary structures.
Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, USA.
Rees F
RF
Garmann
Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, USA.
Ajaykumar
A
Gopal
School of Biology, Georgia Institute of Technology, Atlanta, Georgia 30332, USA School of Biology, Georgia Institute of Technology, Atlanta, Georgia 30332, USA.
Shreyas S
SS
Athavale
Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, USA.
Charles M
CM
Knobler
Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, USA.
William M
WM
Gelbart
School of Biology, Georgia Institute of Technology, Atlanta, Georgia 30332, USA School of Biology, Georgia Institute of Technology, Atlanta, Georgia 30332, USA.
Stephen C
SC
Harvey
D000465:Algorithms; D019295:Computational Biology / Q000379:methods; D020285:Cryoelectron Microscopy; D016679:Genome, Viral; D008968:Molecular Conformation; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000737:chemistry*; D019400:Tobacco mosaic satellite virus / Q000235:genetics*
D012367:RNA, Viral
cryo-electron microscopy; secondary structure; viral RNA genome