2026 Theses Doctoral

The Mechanism of Translational Reading-Frame Maintenance by tRNA Modifications

Maiti, Meghna

The ribosome rapidly and accurately translates the consecutive triplet-nucleotide codon sequence of a messenger RNA (mRNA), incorporating aminoacyl-transfer RNAs (tRNAs) in the order dictated by the mRNA codons to synthesize the encoded protein. During this process, the ribosome advances along the mRNA in precise three-nucleotide steps through a dynamic process known as translocation, which requires coordinated, large-scale conformational rearrangements of the ribosome and its bound ligands. Despite the inherent accuracy of this process, naturally occurring frameshift-suppressor tRNAs have been identified in bacteria that efficiently shift the translational reading frame, enabling decoding of mRNAs containing single-nucleotide insertions, especially when lacking vital post-transcriptional modifications in their anticodon stem loop (ASL). In this work, a highly purified Escherichia coli in vitro translation system is combined with single-molecule fluorescence resonance energy transfer (smFRET) to dissect the mechanistic basis of this process.

Chapter 1 provides a comprehensive overview of the structural and functional architecture of the translation machinery and outlines the molecular interactions that govern deviations from canonical translation. It describes the ribosome as the central catalytic complex responsible for protein synthesis and describes the roles of messenger RNA (mRNA), transfer RNAs (tRNAs), and key translation factors in decoding genetic information. It introduces the frameshift-suppressor tRNASufB2 which contains an extended ASL and is capable of efficiently shifting the translational reading frame when lacking the vital m1G37 modification in its ASL. The chapter further reviews the biochemical and structural basis of the m¹G37 modification, emphasizing its conserved role in stabilizing codon-anticodon interactions and maintaining the reading-frame during translation. Collectively, this chapter establishes the conceptual and mechanistic framework for understanding how alterations in tRNA structure and modification can modulate ribosome dynamics and influence translational fidelity.

Chapter 2 describes a comprehensive investigation of translational reading-frame maintenance mediated by the m¹G37 modification. This chapter examines how the presence of the additional nucleotide G37a influences the dynamics of tRNA accommodation and translocation for tRNASufB2 compared to its non-frameshifting counterpart tRNAProL. It also investigates the effect of m¹G37 modification. By monitoring translation elongation in real time, the study reveals how the m¹G37 modification alters key kinetic steps during elongation. In addition, the chapter explores the interaction of Elongation Factor G (EF-G) with ribosomal complexes, providing insight into how EF-G engagement is modulated under frameshift-prone conditions. Together, these results elucidate the mechanistic role of m¹G37 in maintaining the translational reading frame.

Chapter 3 describes the development and implementation of three novel tRNA labeling strategies for single-molecule fluorescence resonance energy transfer (smFRET) studies. These approaches include RtcB-mediated ligation, deoxyribozyme-based labeling, and T4 RNA ligase 2-mediated ligation, each designed to enable site-specific incorporation of fluorophores into tRNAs with high efficiency and minimal perturbation to tRNA structure and function. The chapter outlines the principles underlying each method and demonstrates their applicability for generating labeled tRNAs suitable for probing ribosome dynamics. Collectively, these strategies expand the experimental toolkit for investigating translation at the single-molecule level.

Chapter 4 details the materials and methods used throughout this work, including the purification and fluorophore-labeling of ribosomes, messenger RNAs (mRNAs), transfer RNAs (tRNAs), and essential translation factors. It describes the protocols for assembling in vitro translation systems and biochemical activity assays. In addition, the chapter outlines the experimental design and instrumentation for smFRET measurements, along with data acquisition and analysis workflows.

This work provides mechanistic insight into the molecular basis of translational frame maintenance, with a particular focus on how tRNA sequence features and post-transcriptional modifications regulate ribosome dynamics. By integrating single-molecule approaches with biochemical and structural perspectives, it elucidates how specific elements, such as ASL architecture and nucleotide modifications, influence tRNA accommodation, translocation, and interactions with translation factors. Beyond fundamental understanding, the study also contributes to the development of tRNA engineering strategies, offering new approaches for site-specific labeling and functional manipulation. Collectively, these advances have broader implications for emerging applications in synthetic biology and tRNA-based therapeutics.