Biochemistry and Molecular Biophysics Graduate Group

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BMB 632 - Probing Structure and Function of Complex RNA-Protein Machines

Fall, even-numbered years

Course Director: Kristen W. Lynch, Ph.D.

RNA-Protein complexes or RNPs can range from simple assemblies to megadalton enzymatic machines. The latter include two of the most abundant and essential enzymatic complexes for converting genes to functional protein – the ribosome and the spliceosome. Understanding the molecular interactions that hold these RNPs together and how these complexes function has required the development of new techniques and pushed the boundaries of quantitative biochemistry. In this course we will take an in-depth look at general concepts common to many RNA binding proteins, the methods used to study protein-RNA and RNA-RNA interactions, and how the complex nature of large RNPs uniquely allow them to achieve their precise functions. The course will be a combination of both lectures and student-lead discussion of recent literature. Students will be evaluated based on their presentations of primary literature and their participation in class discussion.

Preliminary Syllabus: (note: precise order and/or substitution of papers with more recent publications will be completed shortly before the beginning of class to accommodate progress in a rapidly expanding field)

Deckert et al. (2006) Protein composition and electron microscopy structure of affinity-purified human spliceosomal B complexes isolated under physiological conditions Mol Cell Biol. 26:5528-5543.

Nastaran Behzadnia et al. (2007) Composition and three-dimensional EM structure of double affinity-purified, human prespliceosomal A complexes. The EMBO Journal 26:1737-1748.

Berthold Kastner et al. (2008) GraFix: sample preparation for single particle electron cryomicroscopy. Nat Methods 5:53-55.

Elmar Wolf et al. (2009) Exon, intron and splice site locations in the spliceosomal B complex. The EMBO Journal 28: 2283-2292.

Luhrmann et al. (2009) Structural mapping of spliceosomes by electron microscopy. Current Opinion in Structural Biology 19:96-102.

Patrizia Fabrizio et al. (2009) The evolutionarily conserved core design of the catalytic activation step of the yeast spliceosome. Molecular Cell 36:593-608.

Watts et al. (2009) Architecture and secondary structure of an entire HIV-1 RNA genome. Nature 460:711-719.

Kevin A Wilkinson, Edward J Merino & Kevin M Weeks. (2006) Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE): quantitative RNA structure analysis at single nucleotide resolution. Nat Protocols 1:1610-1616.

Priya Ramaswamy & Sarah A Woodson. (2009) S16 throws a conformational switch during assembly of 30S 5’ domain. Nat Struct Mol Biol 16:438-445.

Abelson et al. (2010) Conformational dynamics of single pre-mRNA molecules during in vitro splicing. Nat Struct Mol Biol 17: 504-512.

Sashital et al. (2008) U2-U6 RNA folding reveals a group II intron-like domain and a four helix junction. Nat Struc Mol Biol 11:11237-1242.

Dustin B. Ritchie, Matthew J. Schellenberg, Andrew M. MacMillan. (2009) Spliceosome structure: Piece by piece. Biochimica et Biophysica Acta 1789:624-633.

Daniel J. Crawford et al. (2008) Visualizing the splicing of single pre-mRNA molecules in whole cell extract. RNA 14:170-179.

Marc Schneider et al. (2010) Human PRP4 kinase is required for stable tri-snRNP association during spliceosomal B complex formation. Nat Struc Mol Biol 17(2):216-221.

Hanson et al. (2004) Mass Spectrometry of Ribosomes from Saccharomyces cerevisiae. J Biol Chem. 279:42750-42757.