Dynamic unwrapping of nucleosomes by HsRAD51 that includes sliding and rotational motion of histone octamers.

Wrapping of genomic DNA into nucleosomes poses thermodynamic and kinetic barriers to biological processes such as replication, transcription, repair and recombination. Previous biochemical studies have demonstrated that in the presence of adenosine triphosphate (ATP) the human RAD51 (HsRAD51) recombinase can form a nucleoprotein filament (NPF) on double-stranded DNA (dsDNA) that is capable of unwrapping the nucleosomal DNA from the histone octamer (HO). Here, we have used single molecule Förster Resonance Energy Transfer (smFRET) to examine the real time nucleosome dynamics in the presence of the HsRAD51 NPF. We show that oligomerization of HsRAD51 leads to stepwise, but stochastic unwrapping of the DNA from the HO in the presence of ATP. The highly reversible dynamics observed in single-molecule trajectories suggests an antagonistic mechanism between HsRAD51 binding and rewrapping of the DNA around the HO. These stochastic dynamics were independent of the nucleosomal DNA sequence or the asymmetry created by the presence of a linker DNA. We also observed sliding and rotational oscillations of the HO with respect to the nucleosomal DNA. These studies underline the dynamic nature of even tightly associated protein-DNA complexes such as nucleosomes.

Hybrid phase ligation for efficient synthesis of histone proteins.

We introduce a hybrid solid-solution phase ligation approach that combines the efficiency of solid phase ligation with solution phase ligation in the total synthesis of modified histone proteins. A two linker strategy allows analysis throughout work on the solid phase and maximizes yields through cleavage at an external Rink, while an internal HMBA linker allows the native carboxyl terminus for any protein sequence. We demonstrate this approach for two histone proteins: triple-acetylated H4-K5ac, K12ac, K91ac and CENP-A-K124ac.

Probing the stabilizing effects of modified nucleotides in the bacterial decoding region of 16S ribosomal RNA.

The bacterial decoding region of 16S ribosomal RNA has multiple modified nucleotides. In order to study the role of N(4),2'-O-dimethylcytidine (m(4)Cm), the corresponding phosphoramidite was synthesized utilizing 5'-silyl-2'-ACE chemistry. Using solid-phase synthesis, m(4)Cm, 5-methylcytidine (m(5)C), 3-methyluridine (m(3)U), and 2'-O-methylcytidine (Cm) were site-specifically incorporated into small RNAs representing the decoding regions of different bacterial species. Biophysical studies were then used to provide insight into the stabilizing roles of the modified nucleotides. These studies reveal that methylation of cytidine and uridine has different effects. The same modifications at different positions or sequence contexts within similar RNA constructs also have contrasting roles, such as stabilizing or destabilizing the RNA helix.

A reversible protection strategy to improve Fmoc-SPPS of peptide thioesters by the N-Acylurea approach.

C-terminal peptide thioesters are an essential component of the native chemical ligation approach for the preparation of fully or semisynthetic proteins. However, the efficient generation of C-terminal thioesters by Fmoc solid-phase peptide synthesis remains a challenge. The recent N-acylurea approach to thioester synthesis relies on the deactivation of one amine of 3,4-diaminobenzoic acid (Dbz) during Fmoc SPPS. Here, we demonstrate that this approach results in the formation of side products through the over-acylation of Dbz, particularly when applied to Gly-rich sequences. We find that orthogonal allyloxycarbonyl (Alloc) protection of a single Dbz amine eliminates these side products. We introduce a protected Fmoc-Dbz(Alloc) base resin that may be directly used for synthesis with most C-terminal amino acids. Following synthesis, quantitative removal of the Alloc group allows conversion to the active N-acyl-benzimidazolinone (Nbz) species, which can be purified and converted in situ to thioester under ligation conditions. This method is compatible with the automated preparation of peptide-Nbz conjugates. We demonstrate that Dbz protection improves the synthetic purity of Gly-rich peptide sequences derived from histone H4, as well as a 44-residue peptide from histone H3.

Synthesis and solution conformation studies of the modified nucleoside N(4),2'-O-dimethylcytidine (m(4)Cm) and its analogues.

The dimethylated ribosomal nucleoside m(4)Cm and its monomethylated analogues Cm and m(4)C were synthesized. The conformations (syn vs anti) of the three modified nucleosides and cytidine were determined by CD and 1D NOE difference spectroscopy. The ribose sugar puckers were determined by the use of proton coupling constants. The position of modification (e.g., O vs N methylation) was found to have an effect on the sugar pucker of cytidine.

Combined Approaches to Site-Specific Modification of RNA.

Both natural and unnatural modifications in RNA are of interest to biologists and chemists. More than 100 different analogues of the four standard RNA nucleosides have been identified in nature. Unnatural modifications are useful for structure and mechanistic studies of RNA. This Review highlights chemical, enzymatic, and combined (semisynthesis) approaches to generate site specifically modified RNAs. The availability of these methods for site-specific modifications of RNAs of all sizes is important in order to study the relationships between RNA chemical composition, structure, and function.

Expanding the nucleotide repertoire of the ribosome with post-transcriptional modifications.

In all kingdoms of life, RNAs undergo specific post-transcriptional modifications. More than 100 different analogues of the four standard RNA nucleosides have been identified. Modifications in ribosomal RNAs (rRNAs) are highly prevalent and cluster in regions of the ribosome that have functional importance, have a high level of nucleotide conservation, and typically lack proteins. Modifications also play roles in determining antibiotic resistance or sensitivity. A wide spectrum of chemical diversity from the modifications provides the ribosome with a broader range of possible interactions between rRNA regions, transfer RNA, messenger RNA, proteins, or ligands by influencing local rRNA folds and fine-tuning the translation process. The collective importance of the modified nucleosides in ribosome function has been demonstrated for a number of organisms, and further studies may reveal how the individual players regulate these functions through synergistic or cooperative effects.