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Strategies for Generating Modified Nucleosomes: Applications within Structural Biology Studies.

ABSTRACT: Post-translational modifications on histone proteins play critical roles in the regulation of chromatin structure and all DNA-templated processes. Accumulating evidence suggests that these covalent modifications can directly alter chromatin structure, or they can modulate activities of chromatin-modifying and -remodeling factors. Studying these modifications in the context of the nucleosome, the basic subunit of chromatin, is thus of great interest; however, the generation of specifically modified nucleosomes remains challenging. This is especially problematic for most structural biology approaches in which a large amount of material is often needed. Here we discuss the strategies currently available for generation of these substrates. We in particular focus on novel ideas and discuss challenges in the application to structural biology studies.

SUBMITTER: Musselman CA

PROVIDER: S-EPMC6476189 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Strategies for Generating Modified Nucleosomes: Applications within Structural Biology Studies.

Musselman Catherine A CA Kutateladze Tatiana G TG

ACS chemical biology 20190312 4

Post-translational modifications on histone proteins play critical roles in the regulation of chromatin structure and all DNA-templated processes. Accumulating evidence suggests that these covalent modifications can directly alter chromatin structure, or they can modulate activities of chromatin-modifying and -remodeling factors. Studying these modifications in the context of the nucleosome, the basic subunit of chromatin, is thus of great interest; however, the generation of specifically modifi ...[more]

PMID: 30817115

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Project description:Protein biosynthesis is a central process in all living cells that is catalyzed by a complex molecular machine─the ribosome. This process is termed translation because the language of nucleotides in mRNAs is translated into the language of amino acids in proteins. Transfer RNA (tRNA) molecules charged with amino acids serve as adaptors and recognize codons of mRNA in the decoding center while simultaneously the individual amino acids are assembled into a peptide chain in the peptidyl transferase center (PTC). As the nascent peptide emerges from the ribosome, it is threaded through a long tunnel referred to as a nascent peptide exit tunnel (NPET). The PTC and NPET are the sites targeted by many antibiotics and are thus of tremendous importance from a biomedical perspective and for drug development in the pharmaceutical industry.Researchers have achieved much progress in characterizing ribosomal translation at the molecular level; an impressive number of high-resolution structures of different functional and inhibited states of the ribosome are now available. These structures have significantly contributed to our understanding of how the ribosome interacts with its key substrates, namely, mRNA, tRNAs, and translation factors. In contrast, much less is known about the mechanisms of how small molecules, especially antibiotics, affect ribosomal protein synthesis. This mainly concerns the structural basis of small molecule-NPET interference with cotranslational protein folding and the regulation of protein synthesis. Growing biochemical evidence suggests that NPET plays an active role in the regulation of protein synthesis.Much-needed progress in this field is hampered by the fact that during the preparation of ribosome complexes for structural studies (i.e., X-ray crystallography, cryoelectron microscopy, and NMR spectroscopy) the aminoacyl- or peptidyl-tRNAs are unstable and become hydrolyzed. A solution to this problem is the application of hydrolysis-resistant mimics of aminoacyl- or peptidyl-tRNAs.In this Account, we present an overview of synthetic methods for the generation of peptidyl-tRNA analogs. Modular approaches have been developed that combine (i) RNA and peptide solid-phase synthesis on 3'-aminoacylamino-adenosine resins, (ii) native chemical ligations and Staudinger ligations, (iii) tailoring of tRNAs by the selective cleavage of natural native tRNAs with DNAzymes followed by reassembly with enzymatic ligation to synthetic peptidyl-RNA fragments, and (iv) enzymatic tailing and cysteine charging of the tRNA to obtain modified CCA termini of a tRNA that are chemically ligated to the peptide moiety of interest. With this arsenal of tools, in principle, any desired sequence of a stably linked peptidyl-tRNA mimic is accessible. To underline the significance of the synthetic conjugates, we briefly point to the most critical applications that have shed new light on the molecular mechanisms underlying the context-specific activity of ribosome-targeting antibiotics, ribosome-dependent incorporation of multiple consecutive proline residues, the incorporation of d-amino acids, and tRNA mischarging.Furthermore, we discuss new types of stably charged tRNA analogs, relying on triazole- and squarate (instead of amide)-linked conjugates. Those have pushed forward our mechanistic understanding of nonribosomal peptide synthesis, where aminoacyl-tRNA-dependent enzymes are critically involved in various cellular processes in primary and secondary metabolism and in bacterial cell wall synthesis.

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Strategies for Generating Modified Nucleosomes: Applications within Structural Biology Studies.

Publications

Strategies for Generating Modified Nucleosomes: Applications within Structural Biology Studies.

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