ABSTRACT
Here, we report the design, construction, and characterization of a tRNA neochromosome, a designer chromosome that functions as an additional, de novo counterpart to the native complement of Saccharomyces cerevisiae. Intending to address one of the central design principles of the Sc2.0 project, the â¼190-kb tRNA neochromosome houses all 275 relocated nuclear tRNA genes. To maximize stability, the design incorporates orthogonal genetic elements from non-S. cerevisiae yeast species. Furthermore, the presence of 283 rox recombination sites enables an orthogonal tRNA SCRaMbLE system. Following construction in yeast, we obtained evidence of a potent selective force, manifesting as a spontaneous doubling in cell ploidy. Furthermore, tRNA sequencing, transcriptomics, proteomics, nucleosome mapping, replication profiling, FISH, and Hi-C were undertaken to investigate questions of tRNA neochromosome behavior and function. Its construction demonstrates the remarkable tractability of the yeast model and opens up opportunities to directly test hypotheses surrounding these essential non-coding RNAs.
Subject(s)
Chromosomes, Artificial, Yeast , Genome, Fungal , Saccharomyces cerevisiae , Gene Expression Profiling , Proteomics , Saccharomyces cerevisiae/genetics , Synthetic Biology , RNA, Transfer/genetics , Chromosomes, Artificial, Yeast/geneticsABSTRACT
Genetic code reprogramming has enabled us to ribosomally incorporate various nonproteinogenic amino acids (npAAs) into peptides in vitro. The repertoire of usable npAAs has been expanded to include not only l-α-amino acids with noncanonical sidechains but also those with noncanonical backbones. Despite successful single incorporation of npAAs, multiple and consecutive incorporations often suffer from low efficiency or are even unsuccessful. To overcome this stumbling block, engineering approaches have been used to modify ribosomes, EF-Tu, and tRNAs. Here, we provide an overview of these in vitro methods that are aimed at optimal expansion of the npAA repertoire and their applications for the development of de novo bioactive peptides containing various npAAs.
Subject(s)
Amino Acids , Genetic Code , Amino Acids/metabolism , Peptides , RNA, Transfer/genetics , RNA, Transfer/metabolism , Ribosomes/genetics , Ribosomes/metabolismABSTRACT
Codon usage bias, the preference for certain synonymous codons, is found in all genomes. Although synonymous mutations were previously thought to be silent, a large body of evidence has demonstrated that codon usage can play major roles in determining gene expression levels and protein structures. Codon usage influences translation elongation speed and regulates translation efficiency and accuracy. Adaptation of codon usage to tRNA expression determines the proteome landscape. In addition, codon usage biases result in nonuniform ribosome decoding rates on mRNAs, which in turn influence the cotranslational protein folding process that is critical for protein function in diverse biological processes. Conserved genome-wide correlations have also been found between codon usage and protein structures. Furthermore, codon usage is a major determinant of mRNA levels through translation-dependent effects on mRNA decay and translation-independent effects on transcriptional and posttranscriptional processes. Here, we discuss the multifaceted roles and mechanisms of codon usage in different gene regulatory processes.
Subject(s)
Codon Usage , Gene Expression , Protein Biosynthesis , Protein Folding , Animals , Eukaryota/genetics , Humans , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Transfer/genetics , RNA, Transfer/metabolism , Ribosomes/genetics , Ribosomes/metabolismABSTRACT
Codon-dependent translation underlies genetics and phylogenetic inferences, but its origins pose two challenges. Prevailing narratives cannot account for the fact that aminoacyl-tRNA synthetases (aaRSs), which translate the genetic code, must collectively enforce the rules used to assemble themselves. Nor can they explain how specific assignments arose from rudimentary differentiation between ancestral aaRSs and corresponding transfer RNAs (tRNAs). Experimental deconstruction of the two aaRS superfamilies created new experimental tools with which to analyze the emergence of the code. Amino acid and tRNA substrate recognition are linked to phase transfer free energies of amino acids and arise largely from aaRS class-specific differences in secondary structure. Sensitivity to protein folding rules endowed ancestral aaRS-tRNA pairs with the feedback necessary to rapidly compare alternative genetic codes and coding sequences. These and other experimental data suggest that the aaRS bidirectional genetic ancestry stabilized the differentiation and interdependence required to initiate and elaborate the genetic coding table.
Subject(s)
Amino Acyl-tRNA Synthetases/genetics , Amino Acyl-tRNA Synthetases/metabolism , Evolution, Molecular , Genetic Code , Selection, Genetic , Amino Acids/metabolism , Amino Acyl-tRNA Synthetases/chemistry , Catalysis , Genotype , Phenotype , Phylogeny , Protein Biosynthesis , Protein Folding , Protein Structure, Secondary , RNA, Transfer/genetics , ThermodynamicsABSTRACT
A growing class of small RNAs, known as tRNA-derived RNAs (tdRs), tRNA-derived small RNAs or tRNA-derived fragments, have long been considered mere intermediates of tRNA degradation. These small RNAs have recently been implicated in an evolutionarily conserved repertoire of biological processes. In this Review, we discuss the biogenesis and molecular functions of tdRs in mammals, including tdR-mediated gene regulation in cell metabolism, immune responses, transgenerational inheritance, development and cancer. We also discuss the accumulation of tRNA-derived stress-induced RNAs as a distinct adaptive cellular response to pathophysiological conditions. Furthermore, we highlight new conceptual advances linking RNA modifications with tdR activities and discuss challenges in studying tdR biology in health and disease.
Subject(s)
RNA, Transfer , Animals , RNA, Transfer/metabolism , RNA, Transfer/genetics , Humans , Neoplasms/genetics , Neoplasms/metabolism , Gene Expression Regulation , RNA, Small Untranslated/genetics , RNA, Small Untranslated/metabolismABSTRACT
Pancreatic ductal adenocarcinoma (PDAC) tumors have a nutrient-poor, desmoplastic, and highly innervated tumor microenvironment. Although neurons can release stimulatory factors to accelerate PDAC tumorigenesis, the metabolic contribution of peripheral axons has not been explored. We found that peripheral axons release serine (Ser) to support the growth of exogenous Ser (exSer)-dependent PDAC cells during Ser/Gly (glycine) deprivation. Ser deprivation resulted in ribosomal stalling on two of the six Ser codons, TCC and TCT, and allowed the selective translation and secretion of nerve growth factor (NGF) by PDAC cells to promote tumor innervation. Consistent with this, exSer-dependent PDAC tumors grew slower and displayed enhanced innervation in mice on a Ser/Gly-free diet. Blockade of compensatory neuronal innervation using LOXO-101, a Trk-NGF inhibitor, further decreased PDAC tumor growth. Our data indicate that axonal-cancer metabolic crosstalk is a critical adaptation to support PDAC growth in nutrient poor environments.
Subject(s)
Neurons/metabolism , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Protein Biosynthesis , Serine/metabolism , Adenocarcinoma/genetics , Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Aged , Animals , Axons/metabolism , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/metabolism , Carcinoma, Pancreatic Ductal/pathology , Cell Line, Tumor , Cell Proliferation , Codon/genetics , Female , Glycine/metabolism , Humans , Male , Mice , Middle Aged , Mitochondria/metabolism , Nerve Tissue/pathology , Oxygen Consumption , Pancreatic Neoplasms/pathology , Pyrazoles , Pyrimidines , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Transfer/genetics , RatsABSTRACT
Naive T cells undergo radical changes during the transition from dormant to hyperactive states upon activation, which necessitates de novo protein production via transcription and translation. However, the mechanism whereby T cells globally promote translation remains largely unknown. Here, we show that on exit from quiescence, T cells upregulate transfer RNA (tRNA) m1A58 'writer' proteins TRMT61A and TRMT6, which confer m1A58 RNA modification on a specific subset of early expressed tRNAs. These m1A-modified early tRNAs enhance translation efficiency, enabling rapid and necessary synthesis of MYC and of a specific group of key functional proteins. The MYC protein then guides the exit of naive T cells from a quiescent state into a proliferative state and promotes rapid T cell expansion after activation. Conditional deletion of the Trmt61a gene in mouse CD4+ T cells causes MYC protein deficiency and cell cycle arrest, disrupts T cell expansion upon cognate antigen stimulation and alleviates colitis in a mouse adoptive transfer colitis model. Our study elucidates for the first time, to our knowledge, the in vivo physiological roles of tRNA-m1A58 modification in T cell-mediated pathogenesis and reveals a new mechanism of tRNA-m1A58-controlled T cell homeostasis and signal-dependent translational control of specific key proteins.
Subject(s)
Colitis , RNA, Transfer , Adoptive Transfer , Animals , Cell Proliferation/genetics , Colitis/genetics , Mice , Protein Biosynthesis , RNA, Transfer/genetics , RNA, Transfer/metabolism , T-Lymphocytes/metabolismABSTRACT
RNA polymerase (Pol) III has a specialized role in transcribing the most abundant RNAs in eukaryotic cells, transfer RNAs (tRNAs), along with other ubiquitous small noncoding RNAs, many of which have functions related to the ribosome and protein synthesis. The high energetic cost of producing these RNAs and their central role in protein synthesis underlie the robust regulation of Pol III transcription in response to nutrients and stress by growth regulatory pathways. Downstream of Pol III, signaling impacts posttranscriptional processes affecting tRNA function in translation and tRNA cleavage into smaller fragments that are increasingly attributed with novel cellular activities. In this review, we consider how nutrients and stress control Pol III transcription via its factors and its negative regulator, Maf1. We highlight recent work showing that the composition of the tRNA population and the function of individual tRNAs is dynamically controlled and that unrestrained Pol III transcription can reprogram central metabolic pathways.
Subject(s)
RNA Polymerase III/genetics , RNA Polymerase III/metabolism , RNA, Transfer/genetics , RNA, Transfer/metabolism , Animals , Humans , Models, Biological , Models, Molecular , Neoplasms/genetics , Neoplasms/metabolism , Phosphorylation , Protein Conformation , RNA Polymerase III/chemistry , RNA Processing, Post-Transcriptional , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Signal Transduction , Stress, Physiological , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism , Transcription Factor TFIIIB/genetics , Transcription Factor TFIIIB/metabolism , Transcription Factors/chemistry , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, GeneticABSTRACT
Mitochondria are cellular organelles responsible for generation of chemical energy in the process called oxidative phosphorylation. They originate from a bacterial ancestor and maintain their own genome, which is expressed by designated, mitochondrial transcription and translation machineries that differ from those operating for nuclear gene expression. In particular, the mitochondrial protein synthesis machinery is structurally and functionally very different from that governing eukaryotic, cytosolic translation. Despite harbouring their own genetic information, mitochondria are far from being independent of the rest of the cell and, conversely, cellular fitness is closely linked to mitochondrial function. Mitochondria depend heavily on the import of nuclear-encoded proteins for gene expression and function, and hence engage in extensive inter-compartmental crosstalk to regulate their proteome. This connectivity allows mitochondria to adapt to changes in cellular conditions and also mediates responses to stress and mitochondrial dysfunction. With a focus on mammals and yeast, we review fundamental insights that have been made into the biogenesis, architecture and mechanisms of the mitochondrial translation apparatus in the past years owing to the emergence of numerous near-atomic structures and a considerable amount of biochemical work. Moreover, we discuss how cellular mitochondrial protein expression is regulated, including aspects of mRNA and tRNA maturation and stability, roles of auxiliary factors, such as translation regulators, that adapt mitochondrial translation rates, and the importance of inter-compartmental crosstalk with nuclear gene expression and cytosolic translation and how it enables integration of mitochondrial translation into the cellular context.
Subject(s)
Eukaryota/genetics , Mitochondria/genetics , Protein Biosynthesis/genetics , Transcription, Genetic , Animals , Gene Expression Regulation/genetics , Humans , Oxidative Phosphorylation , RNA, Messenger/genetics , RNA, Transfer/geneticsABSTRACT
Transfer RNA (tRNA) is an adapter molecule that links a specific codon in mRNA with its corresponding amino acid during protein synthesis. tRNAs are enzymatically modified post-transcriptionally. A wide variety of tRNA modifications are found in the tRNA anticodon, which are crucial for precise codon recognition and reading frame maintenance, thereby ensuring accurate and efficient protein synthesis. In addition, tRNA-body regions are also frequently modified and thus stabilized in the cell. Over the past two decades, 16 novel tRNA modifications were discovered in various organisms, and the chemical space of tRNA modification continues to expand. Recent studies have revealed that tRNA modifications can be dynamically altered in response to levels of cellular metabolites and environmental stresses. Importantly, we now understand that deficiencies in tRNA modification can have pathological consequences, which are termed 'RNA modopathies'. Dysregulation of tRNA modification is involved in mitochondrial diseases, neurological disorders and cancer.
Subject(s)
RNA Processing, Post-Transcriptional/genetics , RNA, Transfer/genetics , Animals , Codon/genetics , Humans , Protein Biosynthesis/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
The pool of transfer RNA (tRNA) molecules in cells allows the ribosome to decode genetic information. This repertoire of molecular decoders is positioned in the crossroad of the genome, the transcriptome, and the proteome. Omics and systems biology now allow scientists to explore the entire repertoire of tRNAs of many organisms, revealing basic exciting biology. The tRNA gene set of hundreds of species is now characterized, in addition to the tRNA genes of organelles and viruses. Genes encoding tRNAs for certain anticodon types appear in dozens of copies in a genome, while others are universally absent from any genome. Transcriptome measurement of tRNAs is challenging, but in recent years new technologies have allowed researchers to determine the dynamic expression patterns of tRNAs. These advances reveal that availability of ready-to-translate tRNA molecules is highly controlled by several transcriptional and posttranscriptional regulatory processes. This regulation shapes the proteome according to the cellular state. The tRNA pool profoundly impacts many aspects of cellular and organismal life, including protein expression level, translation accuracy, adequacy of folding, and even mRNA stability. As a result, the shape of the tRNA pool affects organismal health and may participate in causing conditions such as cancer and neurological conditions.
Subject(s)
Genome/genetics , Protein Biosynthesis , Proteomics/trends , RNA, Transfer/genetics , Anticodon/genetics , Codon/genetics , Genomics/trends , Humans , Transcriptome/geneticsABSTRACT
RNA molecules are modified post-transcriptionally to acquire their diverse functions. Transfer RNA (tRNA) has the widest variety and largest numbers of RNA modifications. tRNA modifications are pivotal for decoding the genetic code and stabilizing the tertiary structure of tRNA molecules. Alternation of tRNA modifications directly modulates the structure and function of tRNAs and regulates gene expression. Notably, thermophilic organisms exhibit characteristic tRNA modifications that are dynamically regulated in response to varying growth temperatures, thereby bolstering fitness in extreme environments. Here, we review the history and latest findings regarding the functions and biogenesis of several tRNA modifications that contribute to the cellular thermotolerance of thermophiles.
Subject(s)
Thermotolerance , Thermotolerance/genetics , RNA Processing, Post-Transcriptional , Genetic Code , RNA, Transfer/genetics , RNA/geneticsABSTRACT
Leone et al.1 reveal that Pol III transcription complexes recruit a chaperone, HSP70, to execute cotranscriptional cleavage of precursor tRNA. HSP70 binds to the polymerase and translocates to nascent precursor tRNA and then tRNA. The last complex facilitates Pol III to engage in a new, efficient transcription cycle with another HSP70.
Subject(s)
RNA, Transfer , Transcription, Genetic , RNA, Transfer/genetics , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , HSP70 Heat-Shock Proteins/genetics , HSP70 Heat-Shock Proteins/metabolism , RNA Polymerase III/metabolismABSTRACT
Molecular chaperones are critical for protein homeostasis and are implicated in several human pathologies such as neurodegeneration and cancer. While the binding of chaperones to nascent and misfolded proteins has been studied in great detail, the direct interaction between chaperones and RNA has not been systematically investigated. Here, we provide the evidence for widespread interaction between chaperones and RNA in human cells. We show that the major chaperone heat shock protein 70 (HSP70) binds to non-coding RNA transcribed by RNA polymerase III (RNA Pol III) such as tRNA and 5S rRNA. Global chromatin profiling revealed that HSP70 binds genomic sites of transcription by RNA Pol III. Detailed biochemical analyses showed that HSP70 alleviates the inhibitory effect of cognate tRNA transcript on tRNA gene transcription. Thus, our study uncovers an unexpected role of HSP70-RNA interaction in the biogenesis of a specific class of non-coding RNA with wider implications in cancer therapeutics.
Subject(s)
HSP70 Heat-Shock Proteins , Neoplasms , Humans , HSP70 Heat-Shock Proteins/genetics , HSP70 Heat-Shock Proteins/metabolism , Molecular Chaperones/metabolism , RNA , RNA Polymerase III/genetics , RNA Polymerase III/metabolism , RNA, Transfer/genetics , RNA, Untranslated/geneticsABSTRACT
Pseudouridine (Ψ), the isomer of uridine, is ubiquitously found in RNA, including tRNA, rRNA, and mRNA. Human pseudouridine synthase 3 (PUS3) catalyzes pseudouridylation of position 38/39 in tRNAs. However, the molecular mechanisms by which it recognizes its RNA targets and achieves site specificity remain elusive. Here, we determine single-particle cryo-EM structures of PUS3 in its apo form and bound to three tRNAs, showing how the symmetric PUS3 homodimer recognizes tRNAs and positions the target uridine next to its active site. Structure-guided and patient-derived mutations validate our structural findings in complementary biochemical assays. Furthermore, we deleted PUS1 and PUS3 in HEK293 cells and mapped transcriptome-wide Ψ sites by Pseudo-seq. Although PUS1-dependent sites were detectable in tRNA and mRNA, we found no evidence that human PUS3 modifies mRNAs. Our work provides the molecular basis for PUS3-mediated tRNA modification in humans and explains how its tRNA modification activity is linked to intellectual disabilities.
Subject(s)
Cryoelectron Microscopy , Hydro-Lyases , Intramolecular Transferases , Pseudouridine , RNA, Transfer , Humans , Catalytic Domain , HEK293 Cells , Hydro-Lyases/metabolism , Hydro-Lyases/genetics , Hydro-Lyases/chemistry , Intellectual Disability/genetics , Intellectual Disability/metabolism , Intellectual Disability/enzymology , Models, Molecular , Mutation , Protein Binding , Pseudouridine/metabolism , Pseudouridine/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Transfer/metabolism , RNA, Transfer/genetics , Substrate SpecificityABSTRACT
Many types of human cancers suppress the expression of argininosuccinate synthase 1 (ASS1), a rate-limiting enzyme for arginine production. Although dependency on exogenous arginine can be harnessed by arginine-deprivation therapies, the impact of ASS1 suppression on the quality of the tumor proteome is unknown. We therefore interrogated proteomes of cancer patients for arginine codon reassignments (substitutants) and surprisingly identified a strong enrichment for cysteine (R>C) in lung tumors specifically. Most R>C events did not coincide with genetically encoded R>C mutations but were likely products of tRNA misalignments. The expression of R>C substitutants was highly associated with oncogenic kelch-like epichlorohydrin (ECH)-associated protein 1 (KEAP1)-pathway mutations and suppressed by intact-KEAP1 in KEAP1-mutated cancer cells. Finally, functional interrogation indicated a key role for R>C substitutants in cell survival to cisplatin, suggesting that regulatory codon reassignments endow cancer cells with more resilience to stress. Thus, we present a mechanism for enriching lung cancer proteomes with cysteines that may affect therapeutic decisions.
Subject(s)
Arginine , Cysteine , Kelch-Like ECH-Associated Protein 1 , Lung Neoplasms , Proteome , Humans , Cysteine/metabolism , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , Kelch-Like ECH-Associated Protein 1/metabolism , Kelch-Like ECH-Associated Protein 1/genetics , Proteome/metabolism , Arginine/metabolism , Mutation , Argininosuccinate Synthase/metabolism , Argininosuccinate Synthase/genetics , Cisplatin/pharmacology , Cell Line, Tumor , Proteomics/methods , Gene Expression Regulation, Neoplastic , Cell Survival/drug effects , RNA, Transfer/metabolism , RNA, Transfer/geneticsABSTRACT
Mitochondrial ribosomes (mitoribosomes) perform protein synthesis inside mitochondria, the organelles responsible for energy conversion and adenosine triphosphate production in eukaryotic cells. Throughout evolution, mitoribosomes have become functionally specialized for synthesizing mitochondrial membrane proteins, and this has been accompanied by large changes to their structure and composition. We review recent high-resolution structural data that have provided unprecedented insight into the structure and function of mitoribosomes in mammals and fungi.
Subject(s)
DNA, Mitochondrial/genetics , Mitochondria/genetics , Mitochondrial Proteins/genetics , Mitochondrial Ribosomes/ultrastructure , Protein Biosynthesis , Ribosome Subunits/ultrastructure , Animals , Anti-Bacterial Agents/pharmacology , Biological Evolution , Carboxylic Ester Hydrolases/genetics , Carboxylic Ester Hydrolases/metabolism , DNA, Mitochondrial/metabolism , Mammals , Mitochondria/metabolism , Mitochondrial Membranes/metabolism , Mitochondrial Proteins/metabolism , Mitochondrial Ribosomes/chemistry , Mitochondrial Ribosomes/metabolism , Models, Molecular , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Transfer/genetics , RNA, Transfer/metabolism , Ribosome Subunits/chemistry , Ribosome Subunits/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolismABSTRACT
Enzymes that phosphorylate, dephosphorylate, and ligate RNA 5' and 3' ends were discovered more than half a century ago and were eventually shown to repair purposeful site-specific endonucleolytic breaks in the RNA phosphodiester backbone. The pace of discovery and characterization of new candidate RNA repair activities in taxa from all phylogenetic domains greatly exceeds our understanding of the biological pathways in which they act. The key questions anent RNA break repair in vivo are (a) identifying the triggers, agents, and targets of RNA cleavage and (b) determining whether RNA repair results in restoration of the original RNA, modification of the RNA (by loss or gain at the ends), or rearrangements of the broken RNA segments (i.e., RNA recombination). This review provides a perspective on the discovery, mechanisms, and physiology of purposeful RNA break repair, highlighting exemplary repair pathways (e.g., tRNA restriction-repair and tRNA splicing) for which genetics has figured prominently in their elucidation.
Subject(s)
RNA Ligase (ATP) , RNA , Phylogeny , RNA Ligase (ATP)/genetics , RNA Ligase (ATP)/metabolism , RNA/genetics , RNA, Transfer/genetics , RNA, Transfer/metabolism , RNA Splicing/geneticsABSTRACT
tRNAs are best known as basic modules for global regulation of protein synthesis. Goodarzi et al. now show that two tRNAs upregulated in metastatic breast cancer cells enhance stability and translation of transcripts enriched with these codons, leading to specific increase in production of pro-metastatic proteins.
Subject(s)
Codon , RNA, Transfer/genetics , Humans , Protein BiosynthesisABSTRACT
Ribosome-associated quality control (RQC) is a conserved process degrading potentially toxic truncated nascent peptides whose malfunction underlies neurodegeneration and proteostasis decline in aging. During RQC, dissociation of stalled ribosomes is followed by elongation of the nascent peptide with alanine and threonine residues, driven by Rqc2 independently of mRNA, the small ribosomal subunit and guanosine triphosphate (GTP)-hydrolyzing factors. The resulting CAT tails (carboxy-terminal tails) and ubiquitination by Ltn1 mark nascent peptides for proteasomal degradation. Here we present ten cryogenic electron microscopy (cryo-EM) structures, revealing the mechanistic basis of individual steps of the CAT tailing cycle covering initiation, decoding, peptidyl transfer, and tRNA translocation. We discovered eIF5A as a crucial eukaryotic RQC factor enabling peptidyl transfer. Moreover, we observed dynamic behavior of RQC factors and tRNAs allowing for processivity of the CAT tailing cycle without additional energy input. Together, these results elucidate key differences as well as common principles between CAT tailing and canonical translation.