Manipulating the 3D organization of the largest synthetic yeast chromosome

Whether synthetic genomes can power life has attracted broad interest in the synthetic biology field. Here, we report de novo synthesis of the largest eukaryotic chromosome thus far, synIV, a 1,454,621-bp yeast chromosome resulting from extensive genome streamlining and modification. We developed megachunk assembly combined with a hierarchical integration strategy, which significantly increased the accuracy and flexibility of synthetic chromosome construction. Besides the drastic sequence changes, we further manipulated the 3D structure of synIV to explore spatial gene regulation. Surprisingly, we found few gene expression changes, suggesting that positioning inside the yeast nucleoplasm plays a minor role in gene regulation. Lastly, we tethered synIV to the inner nuclear membrane via its hundreds of loxPsym sites and observed transcriptional repression of the entire chromosome, demonstrating chromosome-wide transcription manipulation without changing the DNA sequences. Our manipulation of the spatial structure of synIV sheds light on higher-order architectural design of the synthetic genomes. </p

Characteristics of interactions at protein segments without non-local intramolecular contacts in the Protein Data Bank.

The principle of three-dimensional protein structure formation is a long-standing conundrum in structural biology. A globular domain of a soluble protein is formed by a network of atomic contacts among amino acid residues, but regions without intramolecular non-local contacts are often observed in the protein structure, especially in loop, linker, and peripheral segments with secondary structures. Although these regions can play key roles for protein function as interfaces for intermolecular interactions, their nature remains unclear. Here, we termed protein segments without non-local contacts as floating segments and sought them in tens of thousands of entries in the Protein Data Bank. As a result, we found that 0.72% of residues are in floating segments. Regarding secondary structural elements, coil structures are enriched in floating segments, especially for long segments. Interactions with polypeptides and polynucleotides, but not chemical compounds, are enriched in floating segments. The amino acid preferences of floating segments are similar to those of surface residues, with exceptions; the small side chain amino acids, Gly and Ala, are preferred, and some charged side chains, Arg and His, are disfavored for floating segments compared to surface residues. Our comprehensive characterization of floating segments may provide insights into understanding protein sequence-structure-function relationships