SummaryLysine acetylation of histones defines the epigenetic status of human embryonic stem cells and orchestrates DNA replication, chromosome condensation, transcription, telomeric silencing, and DNA repair. A detailed mechanistic explanation of these phenomena is impeded by the limited availability of homogeneously acetylated histones. We report a general method for the production of homogeneously and site-specifically acetylated recombinant histones by genetically encoding acetyl-lysine. We reconstitute histone octamers, nucleosomes, and nucleosomal arrays bearing defined acetylated lysine residues. With these designer nucleosomes, we demonstrate that, in contrast to the prevailing dogma, acetylation of H3 K56 does not directly affect the compaction of chromatin and has modest effects on remodeling by SWI/SNF and RSC. Single-molecule FRET experiments reveal that H3 K56 acetylation increases DNA breathing 7-fold. Our results provide a molecular and mechanistic underpinning for cellular phenomena that have been linked with K56 acetylation.

Post-transcriptional control makes a major contribution to the overall regulation of gene expression pathway. Within the cytoplasm this is mediated by a combination of regulatory RNA motifs within the 5' and 3' untranslated regions of mRNAs and their interacting protein/RNA partners. One of the most common regulatory RNA elements in mammalian transcripts (present in approximately 40% of all mRNAs) are upstream open reading frames (uORFs). However, despite the prevalence of these RNA elements how they function is not well understood. In general, they act to repress translation of the physiological ORF under control conditions, and under certain pathophysiological stresses this repression can be alleviated. It is known that re-initiation following the translation of an uORF is utilised in some situations however there are numerous alternative mechanisms that control the synthesis of a protein whose mRNA contains uORFs. Moreover, the trans-acting factors that are also involved in this process are not well defined. In this review we summarise our current understanding of this area and highlight some common features of these RNA motifs that have been discovered to date.

Nucleosomes fulfill the apparently conflicting roles of compacting DNA within eukaryotic genomes while permitting access to regulatory factors. Central to this is their ability to stably associate with DNA while retaining the ability to undergo rearrangements that increase access to the underlying DNA. Here, we have studied different aspects of nucleosome dynamics including nucleosome sliding, histone dimer exchange, and DNA wrapping within nucleosomes. We find that alterations to histone proteins, especially the histone tails and vicinity of the histone H3 ␣N helix, can affect these processes differently, suggesting that they are mechanistically distinct. This raises the possibility that modifications to histone proteins may provide a means of fine-tuning specific aspects of the dynamic properties of nucleosomes to the context in which they are located.The organization of DNA into chromatin creates the functional template for any process requiring access to the genetic information such as transcription, DNA replication, recombination, and repair. The basic repeating unit of chromatin is the nucleosome core particle, which consists of 147 bp of DNA wrapped almost twice around an octamer of histone H2A, H2B, H3, and H4 proteins (12, 32). Chromatin packaging tends to restrict access to the underlying DNA, meaning that regulation of chromatin structure is a key feature of many genetic processes.Eukaryotes have adopted an assortment of different strategies by which they can manipulate chromatin structure. One means involves the posttranslational modification of histone proteins by, for example, acetylation, methylation, and phosphorylation. These are often correlated with particular chromatin states, and the levels of many of these modifications are observed to change during the course of gene regulation (38). To date, the majority of the best-characterized modifications occur in the N-terminal extensions of the histone proteins. These N-terminal tails extend beyond the globular core of the nucleosome and do not take up distinct conformations in highresolution crystal structures (12, 32). Modification of residues within the tail domains has been shown to affect chromatin structure by generating epitopes for the recruitment of external chromatin binding proteins. These include the chromodomains of HP1 and PRC1, which interact specifically with histones methylated at histone H3 lysine 9 and 27, respectively (4, 14, 27), and bromodomain-containing proteins which bind acetylated histone tails (26,42).An alternative means by which histone tails can influence chromatin structure is through direct alteration of the dynamic properties of nucleosomes. Chromatin is not a static entity but can undergo an assortment of dynamic alterations. Arrays of nucleosomes spontaneously condense to form chromatin fibers (5, 9, 57), the positions of nucleosomes on DNA fragments can change as a result of thermally driven nucleosome sliding (16,37), and the outer turns of DNA are prone to transient dissociation from the histone octamer via a ...