In this thesis, we developed and applied novel single-molecule imaging approaches to investigate how two central molecular machines of gene expression, the ribosome and the proteasome, operate inside living cells. These machines control the synthesis and degradation of proteins, two processes that together determine the composition of the cellular proteome. By examining protein synthesis and degradation at the level of individual molecules, this work reveals regulatory mechanisms and molecular heterogeneity that are not accessible through traditional ensemble measurements.

In Chapter 2, we introduced Stopless-ORF circular RNAs (socRNAs) as a new imaging platform that enables long-term visualization of translation by individual ribosomes in living cells. By combining stopless circular RNAs with the SunTag fluorescence amplification system, this method allows single ribosomes to be tracked continuously over extended periods of time. Using socRNAs, we obtained high-precision measurements of translation elongation dynamics and discovered that individual ribosomes translate at heterogeneous rates, suggesting intrinsic variability in ribosome behavior. Our system further enabled quantitative analysis of ribosome speed and pausing induced by specific mRNA sequences or pharmacological inhibitors, providing detailed insight into translation elongation dynamics under different conditions. In addition, our method allowed direct measurement of ribosome processivity, an aspect of translation that has traditionally been difficult to study, as well as highly sensitive quantification of frameshifting frequencies. Using this approach, we quantified the occurrence of rare translation errors and demonstrated that frameshifting can occur even on non repetitive RNA sequences.

In Chapter 3, we applied the socRNA imaging platform to investigate how ribosomes interact with each other during polysomal translation, where multiple ribosomes translate the same mRNA simultaneously. Through long-term tracking of individual ribosomes combined with computational modeling, we uncovered that ribosomes can help each other overcome obstacles encountered during translation elongation, a phenomenon we refer to as ribosome cooperativity. Specifically, we find that collisions between ribosomes can promote the resolution of ribosome pausing, allowing ribosomes to resume translation more efficiently. This unexpected observation reveals a previously unrecognized functional interaction between neighboring ribosomes and provides a conceptual framework for understanding how cells distinguish transient ribosome collisions that occur during normal translation from persistent collisions that activate cellular quality-control pathways. 6

In Chapter 4, we extended our single-molecule imaging strategies to the study of protein degradation by the proteasome, the central executor of regulated protein turnover in eukaryotic cells. We developed experimental approaches that allow direct visualization of how individual proteasomes engage, translocate, and degrade protein substrates in living cells. This method enabled us to quantitatively measure the kinetics of substrate processing in vivo and revealed that proteasomal activity is strongly influenced by the manner in which substrates are engaged by the degradation machinery. In addition, we found that certain, but not all, modes of substrate engagement require specific cofactors, highlighting previously unappreciated layers of regulation in proteasomal degradation. These results demonstrate that proteasomes operate as context-dependent molecular machines whose efficiency is determined by both substrate features and the mode of substrate engagement.

Together, the approaches developed in this thesis provide powerful tools to directly observe protein synthesis and degradation at the level of individual molecular machines in living cells. By uncovering new principles governing translation elongation dynamics, ribosome-ribosome interactions, and proteasomal degradation, this work provides a deeper mechanistic understanding of gene expression regulation and the maintenance of proteome homeostasis in living cells.
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The genetic information stored in mRNAs is decoded by ribosomes during mRNA translation. mRNAs are typically translated by multiple ribosomes simultaneously, but it is unclear whether and how the activity of different ribosomes on an mRNA is coordinated. Here, we develop an imaging approach based on stopless-ORF circular RNAs (socRNAs) to monitor translation of individual ribosomes in either monosomes or polysomes with very high resolution. Using experiments and simulations, we find that translating ribosomes frequently undergo transient collisions. However, unlike persistent collisions, such transient collisions escape detection by cellular quality control pathways. Rather, transient ribosome collisions promote productive translation by reducing ribosome pausing on problematic sequences, a process we term ribosome cooperativity. Ribosome cooperativity also reduces recycling of ribosomes by quality control pathways, thus enhancing processive translation. Together, our single-ribosome imaging approach reveals that ribosomes cooperate during translation to ensure fast and efficient translation. ... Read more