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RNA molecules play essential roles in nearly every stage of gene expression, usually as part of RNA-protein complexes or RNPs.  Indeed, one of the most exciting developments in recent years is the discovery of small RNAs that shut off the expression of other genes during development or in response to environmental signals.  In order for RNA molecules to function inside a cell, they must fold into specific three-dimensional shapes.  These 3D structures are dynamic, undergoing specific changes during the assembly of an RNA-protein complex, or during regulatory cycles.

The study of how RNAs fold will help us understand how they work as catalysts or as genetic switches.  Recently, we have begun to apply the principles obtained from ribozyme folding to other biologically important problems, including ribosome assembly and target recognition by small regulatory RNAs.  Ultimately, we hope to produce tools to diagnose and treat human diseases that arise from RNA malfunction.

RNA FOLDING AND DYNAMICS
We have characterized the folding pathways of group I self-splicing introns or ribozymes using a combination of biochemical and physical methods such as time-resolved hydroxyl radical footprinting, stopped-flow fluorescence, neutron spectroscopy and small angle scattering.  We have also developed simple reporter assays for evaluating RNA folding in bacteria and yeast.

Some principles that have emerged from these studies are that RNAs collapse to structured intermediates within milliseconds after the association of cations, but that this collapse can produce metastable conformations that persist for minutes or hours.  Refolding of metastable conformations depends strongly on RNA sequence and on ionic conditions, with more distributed charge-charge interactions producing systems that are more dynamic.

RNA FOLDING DURING RIBOSOME ASSEMBLY
An important problem in molecular biology is how interactions between proteins and newly transcribed RNAs direct the assembly of functional RNPs.  We are studying the folding of 16 S rRNA during assembly of the 30 S ribosome, using time-resolved hydroxyl radical footprinting.

Key findings are that most RNA tertiary interactions present in the 30 S ribosome can form within 5 seconds, even in the absence of ribosomal proteins.  However, this process is inefficient because a fraction of the 16 S rRNA population misfolds.  As expected, ribosomal proteins stabilize the correct tertiary interactions.  A key function of ribosomal proteins in the early stages of ribosome assembly may be to exert “quality control” to ensure that initial RNA interactions are correctly established before subsequent steps in the assembly pathway.

TIME-RESOLVED HYDROXYL RADICAL FOOTPRINTING
A large experimental challenge is to obtain high resolution information about RNA structure in a short timespan.  Time-resolved hydroxyl radical footprinting provides “snapshots” of RNA tertiary interactions and RNA-protein contacts, at 10-50 millisecond intervals. This method was developed with Michael Brenowitz and Mark Chance at the Center for Synchrotron Biosciences (http://www.aecom.yu.edu/home/csb/).

In brief, the solvent accessibility of the RNA backbone is determined from its susceptibility to cleavage in the presence of hydroxyl radical.  The hydroxyl radicals are generated by 10-30 ms exposures to a synchrotron X-ray beam at Brookhaven National Laboratory, or Fe(II)-EDTA and hydrogen peroxide for 1-2 ms in a rapid quench apparatus. The fragmentation pattern of the RNA is determined by sequencing.  This provides information about which parts of the RNA have folded in the elapsed interval since the start of the folding reaction.

SMALL REGULATORY RNAs AND Sm-LIKE PROTEINS
Small regulatory RNAs turn the expression of mRNAs on and off in response to environmental signals.  How do they recognize their targets?  This is an interesting problem because both the regulator and the mRNA have to refold in order to form the anti-sense complex.  Bacterial sRNAs bind an Sm-like protein Hfq, which promotes target recognition and recruits cellular enzymes such as the degradosome and ribosome to the RNA.  Sm proteins are found in all kingdoms of life, and are necessary for RNA processing and translation.

We are studying how Hfq protein stimulates antisense pairing between DsrA sRNA and the 5’ leader of rpoS mRNA, using stopped-flow FRET, RNA structure mapping and genetics.  The answers will help us understand the function of this important class of RNA-binding proteins, and how enteric bacteria respond to their host environment.