Force measurements show that uL4 and uL24 mechanically stabilize a fragment of 23S rRNA essential for ribosome assembly

Laurent Geffroy, Thierry Bizebard, Ryo Aoyama, Takuya Ueda and Ulrich Bockelmann

In vitro reconstitution studies have shown that ribosome assembly is highly cooperative and starts with the binding of a few ribosomal (r-) proteins to rRNA. It is unknown how these early binders act. Focusing on the initial stage of the assembly of the large subunit of the E. coli ribosome, we prepared a 79 nucleotide-long region of 23S rRNA encompassing the binding sites of the early binders uL4 and uL24. Force signals are measured in a DNA/RNA dumbbell configuration with a double optical tweezers setup. The rRNA fragment was stretched until unfolded, in the absence or in the presence of the r-proteins (either uL4, uL24 or both). We show that the r-proteins uL4 and uL24 individually stabilize the rRNA fragment, both acting as molecular clamps. Interestingly, this mechanical stabilization is enhanced when both proteins are bound simultaneously. Independently, we observe a cooperative binding of uL4 and uL24 to the rRNA fragment. These two aspects of r-proteins binding both contribute to the efficient stabilization of the 3D structure of the rRNA fragment under investigation. We finally consider implications of our results for large ribosomal subunit assembly.

DOI

Conformational dynamics of the frameshift stimulatory structure in HIV-1

Dustin B Ritchie, Tonia R Cappellano, Collin Tittle, Negar Rezajooei, Logan Rouleau, William KA Sikkema and Michael T Woodside

Programmed ribosomal frameshifting (PRF) in HIV-1 is thought to be stimulated by a hairpin in the mRNA, although a pseudoknot-like triplex has also been proposed. Because the conformational dynamics of the stimulatory structure under tension applied by the ribosomal helicase during translation may play an important role in PRF, we used optical tweezers to apply tension to the HIV stimulatory structure and monitor its unfolding and refolding dynamics. The folding and unfolding kinetics and energy landscape of the hairpin were measured by ramping the force on the hairpin up and down, providing a detailed biophysical characterisation. Unexpectedly, whereas unfolding reflected the simple two-state behavior typical of many hairpins, refolding was more complex, displaying significant heterogeneity. Evidence was found for multiple refolding pathways as well as previously unsuspected, partially folded intermediates. Measuring a variant mRNA containing only the sequence required to form the proposed triplex, it behaved largely in the same way. Nonetheless, very rarely, high-force unfolding events characteristic of pseudoknot-like structures were observed. The rare occurrence of the triplex suggests that the hairpin is the functional stimulatory structure. The unusual heterogeneity of the hairpin dynamics under tension suggests a possible functional role in PRF similar to the dynamics of other stimulatory structures.

DOI

Single-molecule measurements of viral ssRNA packaging

Kalle J. Hanhijärvi, Gabija Ziedaite, Dennis H. Bamford, Edward Hæggström and Minna M. Poranen

Genome packaging of double-stranded RNA (dsRNA) phages has been widely studied using biochemical and molecular biology methods. We adapted the existing in vitro packaging system of one such phage for single-molecule experimentation. To our knowledge, this is the first attempt to study the details of viral RNA packaging using optical tweezers. Pseudomonas phage phi6 is a dsRNA virus with a tripartite genome. Positive-sense (+) single-stranded RNA (ssRNA) genome precursors are packaged into a preformed procapsid (PC), where negative-strands are synthesized. We present single-molecule measurements of the viral ssRNA packaging by the phi6 PC. Our data show that packaging proceeds intermittently in slow and fast phases, which likely reflects differences in the unfolding of the RNA secondary structures of the ssRNA being packaged. Although the mean packaging velocity was relatively low (0.07–0.54 nm / s), packaging could reach 4.62 nm / s during the fast packaging phase.

DOI