Calvin Yip

The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

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Conserved from yeast to humans and composed of six subunits (Elp1 – Elp6), Elongator catalyzes the modification of the anticodon loop of transfer RNAs (tRNAs) and in turn regulates messenger RNA (mRNA) decoding efficiency. By examining human Elongator and its subunits by single-particle electron microscopy (EM), co-purification and pulldown assays, and substrate binding assays, we found that the complex shares similar overall morphologies as the yeast counterpart, and the accessory proteins serve to stabilize ELP3 and improve the binding of substrate tRNAs. Collectively, our work generated insights into the assembly process of this complex and established a platform for characterizing human Elongator and its variants.The heterotrimeric TINTIN complex is an important regulator of transcriptional elongation. Composed of the Eaf3, Eaf5, and Eaf7 proteins, TINTIN exists as both as a module within the NuA4 histone acetyltransferase complex as well as independently. TINTIN is targeted to chromatin through Eaf3, a chromodomain-containing protein that is shared with the Rpd3S histone deacetylase complex. A combination of co-immunoprecipitation and hydrogen deuterium exchange mass spectrometry (HDX-MS) revealed that upon binding Eaf5 and Eaf7, Eaf3 undergoes conformational changes which improves its affinity towards nucleosomes trimethylated at Lys 36 of histone H3 (H3K36me3). Negative stain EM analysis of TINTIN in complex with nucleosomes revealed that TINTIN binds to the disc edge of nucleosomes with increased specificity in the presence of H3K36me3. Together, this work provides molecular insights into the dynamics of TINTIN and the mechanism by which its interactions with chromatin are regulated.The class IB phosphoinositide 3-kinase (PI3K), PI3Kγ, is a master regulator of immune cell function and a promising drug target for both cancer and inflammatory diseases. Critical to PI3Kγ function is the association of the p110γ catalytic subunit to either a p101 or p84 regulatory subunit, which mediates activation by G protein–coupled receptors. The cryo–electron microscopy structure of p110γ-p101 reveals the novel architecture of the p101 regulatory subunit and demonstrates a unique assembly that is distinct from other class I PI3K complexes.

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Macroautophagy, often referred to as autophagy, is a non-selective degradation mechanism used by eukaryotic cells to recycle cytoplasmic material and maintain homeostasis. Upregulated under starvation to generate molecular building blocks for ongoing cellular processes, this pathway requires the coordinated action of six multi-protein complexes, the Atg1/ULK1 complex being the first. Although, the Atg1 complex has been extensively studied in Saccharomyces cerevisiae, far less is known about the biochemical and structural properties of its mammalian counterpart, the ULK1 complex. Unlike the S. cerevisiae Atg1 complex which contains five subunits (Atg1, Atg13, Atg17, Atg29, and Atg31), the ULK1 complex consists of four proteins (ULK1, FIP200/RB1CC1, ATG13, and ATG101) that are technically more challenging to study. In this thesis, I characterized the Atg1 complex from fission yeast, Schizosaccharomyces pombe, as the composition of proteins resembles the mammalian ULK1 complex but is more amenable to biochemical analyses. The Atg1 complex in S. pombe is composed of Atg1 (ULK1 counterpart), Atg13, Atg17 (FIP200 counterpart) and Atg101. We found that the interactions between Atg1, Atg17, and Atg13 are conserved while Atg101 does not replace Atg29 and Atg31. Instead, Atg101 binds to Atg1 and the HORMA domain of Atg13. Furthermore, Atg101 was previously shown to contain a conserved loop, termed the WF finger, postulated to bind and recruit downstream autophagy-related proteins and effectors. Using affinity purification mass spectrometry, we further investigated the potential interacting partners of S. pombe Atg101 under autophagy-inducing and non-inducing conditions. We obtained 625 proteins that co-purified with Atg101-GFP from cells grown in defined media. We used in vitro pairwise studies to confirm the interaction between Atg101 and prey proteins. 9 of the 16 proteins tested were confirmed including Fkh1, an FKBP-type peptidyl-prolyl cis-trans isomerase. We further explored the interaction interface between Atg101 and Fkh1 and found that the WF finger is required for the interaction in vitro. Although the S. cerevisiae Fkh1 homologue, FKBP12, interacts with rapamycin; Fkh1 it is not thought to be directly involved in autophagy. Collectively, our results give new insights into an Atg101-containing Atg1/ULK1 complex and reveals that Atg101’s function may span beyond autophagy.

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Post-translational modification of histones, such as the addition of acetyl groups, is a major regulatory mechanism for gene expression. Histone acetylation is catalyzed by highly conserved lysine acetyltransferase (KAT) enzymes that are often part of large, modular, and multifunctional complexes. Despite their fundamental importance, the reasons behind the tendency of these enzymes to form large complexes remain unclear. We investigated the organization of these complexes by elucidating the molecular architecture of three yeast KAT complexes: Spt-Ada-Gcn5 Acetyltransferase (SAGA), nucleosomal acetyltransferase of histone H4 (NuA4), and Elongator. The yeast SAGA complex is the largest KAT complex in yeast, and activates the expression of many stress response genes. Mutations of its human homologues have been implicated in spinocerebellar ataxia and oncogenesis. Using single particle electron microscopy and crosslinking coupled to mass spectrometry, we show that the catalytic module of SAGA resides within a highly flexible tail adjacent to numerous chromatin-binding subunits. We propose that the flexible SAGA tail is the nucleosome-interacting surface, and its plasticity serves to accommodate the various configurations of the chromatin substrate. NuA4 is another KAT complex whose catalytic subunit, Esa1, is the only essential KAT in yeast. NuA4 has highly conserved roles in the expression of housekeeping genes and the DNA damage repair pathway. Its subunits organize into modules that act independently of the complex. We show that these moonlighting modules form distinct globular structures that are peripherally associated with NuA4, which likely facilitates their dynamic nature. Similar to SAGA, NuA4 subunits that bind chromatin surrounds its catalytic subunit, possibly positioning its substrate nucleosome for efficient acetylation. Yeast Elongator, consisting of two copies each of six unique subunits, was initially characterized as a component of the elongating RNA polymerase II holoenzyme with histone acetyltransferase activity. However, further research has revealed a prominent role for the complex in modifying the wobble base pair of tRNAs. We generated the first three-dimensional reconstruction of Elongator and show that it organizes asymmetrically, with the two copies of its catalytic subunit residing in very different environments. Our structural investigations represent the first steps towards understanding the molecular mechanisms of these enigmatic complexes.

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