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 are highly prevalent and cluster in regions of the ribosome that have functional importance, a high level of nucleotide conservation, and that 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 ribosomal RNA regions, transfer RNA, messenger RNA, proteins, or ligands by influencing local ribosomal RNA 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.In all kingdoms of life, ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), small nucleolar RNAs (snoRNAs), and other RNAs undergo specific post-transcriptional modification by a wide variety of enzymes (1). To date, >100 different modifications of the four standard RNA nucleosides, adenosine, cytidine, guanosine, and uridine, have been identified (2). These modifications can be organized into four main types (Figure 1) (1). The first involves isomerization of uridine to pseudouridine (5-ribosyluracil, Ψ), which contains a C-rather than the typical N-glycosidic linkage, as well as an additional imino group that is available for unique hydrogen-bonding interactions. The second includes alterations to the bases, such as methylation (typically on carbon, primary nitrogen, or tertiary nitrogen), deamination (e.g., inosine), reduction (e.g., dihydrouridine), thiolation, or alkylation (e.g., isopentenylation or threonylation). The third involves methylation of the ribose 2′ hydroxyl (Nm). The fourth type includes more complex modifications, such as multiple modifications (e.g., 5-methylaminomethyl-2-thiouridine; 3-(3-amino-3-carboxypropyl)uridine, acp 3 U; 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, m 1 acp 3 Ψ) or "hypermodifications" that can be incorporated by specific exchange mechanisms (e.g., queuosine). The possible electronic and steric effects of the nucleoside modifications on base pairing, base stacking, and sugar pucker in RNA have been discussed in detail by Davis (3) and Agris (4), among others (1). The effects of modifications such as Ψ on RNA hydration and dynamics have also been considered (4).Modified nucleotides in the ribosome are varied in their identity, but highly localized in their positions (5). If the sites of modification are mapped on the secondary structures of the small and large subunit (SSU and LSU) rRNAs, they might appear to be random; however, if the same modifications are located within the ribosome tertiary structures from high-resolution Xray crystal structures (6,7), they occur in the mo...

C-terminal peptide thioesters are an essential component of the native chemical ligation approach for the preparation of fully- or semi-synthetic proteins. However, efficient generation of C-terminal thioesters via Fmoc solid phase peptide synthesis remains a challenge. The recent N-acylurea approach to thioester synthesis relies on 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 by 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 may be purified and converted in situ to thioester under ligation conditions. This method is compatible with automated preparation of peptide-Nbz conjugates. We demonstrate that Dbz protection improves synthetic purity of Gly-rich peptide sequences derived from histone H4, as well as a 44-residue peptide from histone H3.

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.

Both natural and unnatural modifications in RNA are of interest to biologists and chemists. More than 100 different analogs 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 sitespecifically 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.RNAs undergo specific post-transcriptional modification by a wide variety of enzymes and ribonucleoprotein complexes (1,2). More than 100 different modifications of the four standard RNA nucleosides, adenosine, cytidine, guanosine, and uridine, have been identified (3). Examples from each of the four major categories of base isomerization, base modification, sugar modification, and hypermodification are shown in Figure 1, panel a. Similarly, a large number of unnatural modifications have been synthesized and used for structure and mechanistic studies of RNA (4-6). A few examples are shown in Figure 1, panel b (7-10). Natural modified nucleotides are often found in the functionally important regions of RNA, such as the peptidyl transferase center of ribosomal RNA (rRNA), the anticodon loop of transfer RNAs, or the branch site of spliceosomal . Several modifications, such as pseudouridine (14), have been known for almost 50 years. Their locations in natural RNAs can, in some cases, be highly conserved; yet, our understanding of their biological roles is still incomplete (15). Through a combination of biological, chemical, and biophysical approaches, much can be learned regarding the roles of modified nucleotides. Similarly, the incorporation of unnatural modifications may be useful in order to study the biological roles and functions of RNA (5).In a number of cases, the enzymes or small nucleolar RNAs (snoRNAs) that are responsible for site-specific RNA modification have been identified (2). Knock-out studies of the individual modifying enzymes or snoRNAs then allow the function of the modification to be deduced. In cases where the enzymes are not known, or the modification is not a natural one, alternative approaches must be considered. Over the past several decades, several chemical methods have been developed to study the effects of modifications in small RNA model systems. These studies have led to newer, more powerful approaches that allow site-specific modification of large RNAs, reconstitution into biological systems, and studies of RNA structure and function. Chemical SynthesisChemical synthesis of RNA allows for site-selective incorporation of modified nucleotides. Five decades ago, Michelson and coworkers synthesized a thymidylate dinucleotide by the *Corresponding author csc@chem.wayne.edu. Figure 2): i) coupling at the 5′ site with a protected phosphoramidite, ii) capping of the unreacted 5′-hydroxyl groups, ...

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.