Microcin J25 (MccJ25) is a 21-amino acid peptide inhibitor active against the DNA-dependent RNA polymerase of Gram negative bacteria. Previously, the structure of MccJ25 was reported to be a head-to-tail circle, cyclo(-G(1)GAGHVPEYF(10)VGIGTPISFY(20)G-). On the basis of biochemical studies, mass spectrometry, and NMR, we show that this structure is incorrect, and that the peptide has an extraordinary structural fold. MccJ25 contains an internal lactam linkage between the alpha-amino group of Gly1 and the gamma-carboxyl of Glu8. The tail (Tyr9-Gly21) passes through the ring (Gly1-Glu8), with Phe19 and Tyr20 straddling each side of the ring, sterically trapping the tail in a noncovalent interaction we call a lassoed tail.

Nucleosomes are stable DNA-histone protein complexes that must be unwrapped and disassembled for genome expression, replication, and repair. Histone posttranslational modifications (PTMs) are major regulatory factors of these nucleosome structural changes, but the molecular mechanisms associated with PTM function remains poorly understood. Here we demonstrate that histone PTMs within distinct structured regions of the nucleosome directly regulate the inherent dynamic properties of the nucleosome. Precise PTMs were introduced into nucleosomes by chemical ligation. Single molecule magnetic tweezers measurements determined that only PTMs near the nucleosome dyad increase the rate of histone release in unwrapped nucleosomes. In contrast, FRET and restriction enzyme analysis reveal that only PTMs throughout the DNA entry-exit region increase unwrapping and enhance transcription factor binding to nucleosomal DNA. These results demonstrate that PTMs in separate structural regions of the nucleosome control distinct dynamic events, where the dyad regulates disassembly while the DNA entry-exit region regulates unwrapping. These studies are consistent with the conclusion that histone PTMs may independently influence nucleosome dynamics and associated chromatin functions.histone acetylation | chromatin dynamics | native chemical ligation

Post-translational modification (PTM) of histones plays a central role in genome regulation. Engineering histones with defined PTMs on one or multiple residues is crucial for understanding their function within nucleosomes and chromatin. We introduce a sequential native chemical ligation strategy suitable for the preparation of fully synthetic histone proteins, which allows for site-specific incorporation of varied PTMs throughout the sequence. We demonstrate this method with the generation of histone H3 acetylated at lysine 56 [H3(K56ac)]. H3(K56ac) is essential for transcription, replication, and repair. We examined the influence of H3(K56ac) on the targeting of a model DNA binding factor (LexA) to a site ∼30 bp within the nucleosome. We find that H3(K56ac) increases LexA binding to its DNA target site by 3-fold at physiological ionic strength. We then demonstrate that H3(K56ac) facilitates LexA binding by increasing DNA unwrapping and not by nucleosome repositioning. Furthermore, we find that H3(K56Q) quantitatively imitates H3(K56ac) function. Together these studies introduce powerful tools for the analysis of histone PTM functions.

Histone post-translational modifications are essential for regulating and facilitating biological processes such as RNA transcription and DNA repair. Fifteen modifications are located in the DNA-histone dyad interface and include the acetylation of H3-K115 (H3-K115Ac) and H3-K122 (H3-K122Ac), but the functional consequences of these modifications are unknown. We have prepared semisynthetic histone H3 acetylated at Lys-115 and/or Lys-122 by expressed protein ligation and incorporated them into single nucleosomes. Competitive reconstitution analysis demonstrated that the acetylation of H3-K115 and H3-K122 reduces the free energy of histone octamer binding. Restriction enzyme kinetic analysis suggests that these histone modifications do not alter DNA accessibility near the sites of modification. However, acetylation of H3-K122 increases the rate of thermal repositioning. Remarkably, Lys 3 Gln substitution mutations, which are used to mimic Lys acetylation, do not fully duplicate the effects of the H3-K115Ac or H3-K122Ac modifications. Our results are consistent with the conclusion that acetylation in the dyad interface reduces DNA-histone interaction(s), which may facilitate nucleosome repositioning and/or assembly/disassembly.All eukaryotic genomes are organized into strings of nucleosomes, where 147 bp of DNA are tightly wrapped around a histone protein octamer (1). Many biological processes are dependent on DNA-protein interactions. However, access to DNA-binding sites is often restricted by the nucleosome structure. Alterations in nucleosome structure, dynamics, and positioning have been hypothesized to play a gatekeeper role in regulating biological processes such as DNA replication, repair, and transcription (2).The post-translational modification (PTM) 3 of core histones (3) plays a central role in regulating the biological processing of eukaryotic genomes. Until recently, known histone PTMs were almost exclusively located on the unstructured histone tail regions, which extend from the structured core of the nucleosomes. PTMs in the histone tails can function to directly alter nucleosome (4 -6) and/or chromatin structure and stability (7,8) and function as protein-binding sites (9) in the "histone code" model (10).During the past 5 years over 30 additional histone PTMs were identified within structured regions of the nucleosome (11-13). Many of these modifications are buried within the nucleosome core and thus are not readily accessible for protein binding. Fifteen of these histone PTMs are located in the DNAhistone interface, where the histone octamer contacts the phosphate backbone of the wrapped DNA (14). Studies have suggested that only mild structural perturbations occur in coremodified nucleosomes, which implies that modifications buried beneath the DNA are unlikely to provide a protein-binding site (15). This has led to two additional models for the function of nucleosome core PTMs.Modifications such as Lys acetylation that reduce the positive charge of the histone octamer surface may reduce the bindi...