Communication between the 5' cap structure and 3' poly(A) tail of eukaryotic mRNA results in the synergistic enhancement of translation. The cap and poly(A) tail binding proteins, eIF4E and Pab1p, mediate this effect in the yeast S. cerevisiae through their interactions with different parts of the translation factor eIF4G. Here, we demonstrate the reconstitution of an eIF4E/eIF4G/Pab1p complex with recombinant proteins, and show by atomic force microscopy that the complex can circularize capped, polyadenylated RNA. Our results suggest that formation of circular mRNA by translation factors could contribute to the control of mRNA expression in the eukaryotic cell.

Tapping mode atomic force microscopy in liquids gives a substantial improvement in imaging quality and stability over standard contact mode. In tapping mode the probe-sample separation is modulated as the probe scans over the sample. This modulation causes the probe to tap on the surface only at the extreme of each modulation cycle and therefore minimizes frictional forces that are present when the probe is constantly in contact with the surface. This imaging mode increases resolution and reduces sample damage on soft samples. For our initial experiments we used a tapping frequency of 17 kHz to image deoxyribonucleic acid plasmids on mica in water. When we imaged the same sample region with the same cantilever, the plasmids appeared 18 nm wide in contact mode and 5 nm in tapping mode.

The yeast [PSI+] factor propagates by a prion-like mechanism involving self-replicating Sup35p amyloids. We identified multiple Sup35p mutants that either are poorly recruited into, or cause curing of, wildtype amyloids in vivo. In vitro, these mutants showed markedly decreased rates of amyloid formation, strongly supporting the protein-only prion hypothesis. Kinetic analysis suggests that the prion state replicates by accelerating slow conformational changes rather than by providing stable nuclei. Strikingly, our mutations map exclusively within a short glutamine/asparagine-rich region of Sup35p, and all but one occur at polar residues. Even after replacement of this region with polyglutamine, Sup35p retains its ability to form amyloids. These and other considerations suggest similarities between the prion-like propagation of [PSI+] and polyglutamine-mediated pathogenesis of several neurodegenerative diseases.