“Title: Biochemical and Structural Characterization of the Elongator, TINTIN and PI3K Gamma Complexes”Abstract: In eukaryotes, protein complexes are often composed of catalytic enzymes along with non-catalytic subunits that can modulate the stability, interaction patterns, and activities of the catalytic subunits. In this thesis, three such complexes are biochemically and structurally characterized to uncover how their activities are regulated: the the tRNA modifying Elongator complex, the transcription associated TINTIN complex, and the lipid kinase PI3Kg complex. 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. How the different human Elongator subunits coordinate with one another to catalyze tRNA modification are not fully understood. 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 counterparts, and the accessory proteins serve to stabilize ELP3 and improve the binding of substrate tRNAs. 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 the affinity of Eaf3 towards nucleosomes trimethylated at Lys 36 of histone H3 (H3K36me3). Negative stain single-particle electron microscopy (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.
Monday, November 22, 2021 at 2:30 pm at LSC #3 and or join by Zoom.
Hosted by: Dr. Calvin Yip