Puglisi

Lab

I am using bulk biochemical and single-molecule fluorescence techniques to investigate non-canonical mechanisms of translation initiation in humans.
Repeat associated non-AUG (RAN) translation: Repeat expansion disorders are a group of complex neurological diseases caused by the expansion of short, repetitive, intragenic elements. The discovery that expanded nucleotide repeats, in the context of a mRNA, initiate protein synthesis in the absence of an AUG start codon and produce toxic proteins has further elucidated the complex pathology of these diseases. I am investigating the molecular mechanism(s) of RAN translation initiation by the G4C2 hexanucleotide repeat expansion from gene C9orf72, which is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).
Hepatitis C virus (HCV) internal ribosome entry site (IRES) mediated initiation: The HCV IRES recruits and assembles a translationally competent 80S ribosome-mRNA complex using only a subset of host initiation factors. Bulk biochemical and cell biology data indicate HCV IRES-mediated initiation is a heterogenous process. I am interested in understanding the reaction network that defines HCV IRES-mediated initiation and how the interplay between conformational and compositional dynamics of the IRES, ribosome and initiation factors guide this process to yield an elongation-competent system.

I am broadly interested in using structural and biophysical tools, namely x-ray crystallography and cryo-electron microscopy, to study how RNA structures can regulate viral and cellular processes. My current work involves investigating how the structures of viral RNA and cellular tRNA relate to the regulation and timing of HIV reverse transcription in cells.

Ribosomes are special butterflies. Yet their purification traditionally involves washing them with non-physiologically high levels of salt to reduce heterogeneity. This procedure effectively cuts off the ribosomes' wings by displacing numerous factors that control the dynamics of protein synthesis. I am using biochemical reconstitution to stitch back together the wings of human ribosomes and then watching them fly in real time with high-throughput single molecule spectroscopy.

Mark Capece is using NMR to study how mRNA structure regulates translation. He is currently focusing on reinitiation promoting elements in the yeast gene GCN4 and translation bypassing hairpins in the T4 bacteriophage gene 60.

My background is in Physics and Electrical Engineering, with a dilettante interest in information theory and signal processing. My current research focuses on: 1) Understanding protein synthesis and involved control mechanisms using Escherichia coli in vitro translation as a model system, 2) using this model system to expand the current tools, and 3) applying developed techniques to different biological systems to observe complex phenomena at the single-molecule level and speculate their mechanisms.

The end of translation is signified by the the timely release of the synthesized protein from the ribosome and subsequent disassembly of the ribosomal complex. The ribosome achieves these tasks efficiently with the help of numerous protein factors during stages of termination and recycling. I am interested in how the factors' interactions with the ribosome mediate ribosome conformation temporally to effect disassembly. To reveal these dynamics, I have expanded our bacterial single-molecule translation system by developing fluorescently labeled protein factors to track termination and recycling in real time. With these new tools I hope to understand how these latter stages bring translation to a full circle in the cell.

My goal is to uncover how the synthesis of proteins (translation) is controlled by regulatory elements housed within an mRNA. Many of the required steps for translation have been identified, but the interplay between translation factors, the mRNA, tRNAs, the ribosome, and regulatory proteins remain unclear. In particular, control elements housed near both ends of mRNAs often work together to control how much protein is synthesized from it at a given time. The mechanisms for how translation is increased or decreased by these control elements has long remained elusive. This is because it involves many components, multiple parallel pathways that lead to control, and very brief or weak interactions spread over long distances. To overcome these long-standing challenges, I employ single-molecule strategies to analyze how control elements within mRNAs directly impact recruitment of the ribosome. Our analyses should reveal new insight into general mechanisms used to control the synthesis of human proteins, which could lead to new therapeutics for disease.

Protein synthesis must occur with temporal and spatial precision to maintain normal cell physiology. Initiation of translation is the focus of translational control in eukaryotes. Mis-regulation at this stage is linked to numerous diseases including cancer. I will apply in vitro single molecule fluorescence methods to yeast translation initiation to determine key compositional events and conformational dynamics during the initiation process. The outcome of this project will contribute to a better understanding of the mechanism of eukaryotic translation initiation and its regulation.