About

Jamie H. D. Cate is a Professor of Chemistry, and of Biochemistry, Biophysics, and Structural Biology at the University of California, Berkeley. His research focuses on structural biology, biophysical chemistry, and systems biology, specifically probing the mechanisms of regulation of human translation and bacterial protein synthesis through techniques such as cryo-electron microscopy, genomics, molecular biology, and synthetic biology. His lab explores how genes are activated by translation, the universal process of protein synthesis where the ribosome translates messenger RNA into proteins. Cate's work includes strategies for engineering ribosomes to produce sequence-defined polymers and understanding the regulation of gene expression at the level of translation initiation, particularly involving eukaryotic translation initiation factors like eIF3, which is targeted by viruses such as hepatitis C. His research aims to elucidate how eIF3 binds to specific mRNAs to control cell growth and division, with implications for understanding cancer. Additionally, he is involved in engineering bacterial ribosomes to create new polymers, studying how ribosome structure and function can be altered for novel chemical synthesis.

Research topics

• Genetics
• Biology
• Computational biology
• Cell biology
• Evolutionary biology
• Biophysics

Selected publications

• miRNA-Mediated Regulation of Gene Expression During Early Activation in Jurkat Cells

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-06-17

  preprintOpen accessSenior authorCorresponding

  Abstract Background T cell activation induces substantial changes in gene expression by rapidly increasing transcription and translation. Additionally, microRNAs play a crucial role in regulating protein expression in T cell physiology, adding a layer of complexity by fine-tuning protein levels. While various miRNAs have been implicated in T cell function, a systematic analysis of differentially expressed miRNAs during early T cell activation and identification of their mRNA targets remains mostly unknown. Results We investigated dynamic changes in global gene expression during early T cell activation using a multi-omics approach combining small RNA-seq, mRNA-seq and ribosome profiling. Our results show that most differential expression changes occur by 5 hours post-activation, with translational upregulation predominating over downregulation. From 5 to 12 hours, we observed modest transcriptional and translational reprogramming. We identified 9 miRNAs that are differentially expressed (DE) during early activation, with most changes occurring as early as 5 hours. We calculated translation efficiency (TE) and classified genes based on changes in both mRNA abundance and ribosome-protected fragments (RPFs). By integrating TE and miRNA expression data, we examined the relationship between TE group-specific regulation patterns and the number of miRNA binding sites. Interestingly, rather than observing a uniform downregulation of targets with 4 or more predicted DE miRNA binding sites, we found distinct regulatory patterns that varied with both activation time point and TE category. Conclusions Our data provide new insights into how genes associated with key events in T cell activation such as translation, cell proliferation, and immune signaling are regulated at both the transcriptional and translational levels. The observation that most regulatory changes occur within the first 5 hours post-activation highlights the rapid and coordinated nature of T cell responses. The differential patterns of target regulation, based on translation efficiency groups and miRNA binding site density, suggest a context-dependent role for miRNAs in shaping protein output. Future experiments will be required to functionally validate specific miRNA–target interactions and to explore their relevance in primary T cells in vivo . This study also lays the groundwork for identifying miRNA-based regulatory circuits for therapeutic modulation of T cell activity.

  PublisherDOI

• Structure and evolution-guided design of minimal RNA-guided nucleases

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-12-08

  articleOpen access

  The design of RNA-guided nucleases with properties not limited by evolution can expand programmable genome editing capabilities. However, generating diverse multi-domain proteins with robust enzymatic properties remains challenging. Here we use an artificial intelligence-driven strategy that couples structure-guided inverse protein folding with evolution-informed residue constraints to generate active, divergent variants of TnpB, a minimal CRISPR-Cas12-like nuclease. High-throughput functional screening of AI-generated variants yielded editors that retained or exceeded wild-type activity in bacterial, plant and human cells. Cryo-EM-based structure determination of the most divergent active variant revealed new stabilizing contacts in the RNA/DNA interfaces across conformational states, demonstrating the design potential of this approach. Together these results establish a strategy for creating non-natural RNA-guided nucleases and conformationally active nucleic acid binders, enlarging the designable protein space. One-sentence abstract: An evolution- and structure-conditioned model enables design of active RNA-guided nucleases with new nucleic acid contacts resolved by cryo-EM.

  PublisherDOI

• Abstract 1528 A mutation to the exit tunnel of the E. coli ribosome improves translation of polyproline sequences with implications for incorporation of cyclic β-amino acids

  Journal of Biological Chemistry · 2025-05-01

  articleOpen accessSenior author

  Incorporation of β-amino acids into peptides imparts proteolytic stability, unique architectures, and membrane permeability as demonstrated by β-amino acid containing natural products. Additionally, cyclic β-amino acids can act as turn inducers, and multiple incorporations can generate foldamers with rigid helical structures. However, incorporation efficiency and number of residues incorporated is highly dependent on the stereochemistry of the cyclic β-amino acid. We sought to improve incorporation of cyclic β-amino acids through structure guided mutations of the E.

  PublisherOA PDFDOI

• Role of Ribosomal Protein bS1 in Orthogonal mRNA Start Codon Selection

  Biochemistry · 2025-01-24

  articleOpen accessSenior authorCorresponding

  In many bacteria, the location of the mRNA start codon is determined by a short ribosome binding site sequence that base pairs with the 3′-end of 16S rRNA (rRNA) in the 30S subunit. Many groups have changed these short sequences, termed the Shine–Dalgarno (SD) sequence in the mRNA and the anti-Shine–Dalgarno (ASD) sequence in 16S rRNA, to create “orthogonal” ribosomes to enable the synthesis of orthogonal polymers in the presence of the endogenous translation machinery. However, orthogonal ribosomes are prone to SD-independent translation. Ribosomal protein bS1, which binds to the 30S ribosomal subunit, is thought to promote translation initiation by shuttling the mRNA to the ribosome. Thus, a better understanding of how the SD and bS1 contribute to start codon selection could help efforts to improve the orthogonality of ribosomes. Here, we engineered the Escherichia coli ribosome to prevent binding of bS1 to the 30S subunit and separate the activity of bS1 binding to the ribosome from the role of the mRNA SD sequence in start codon selection. We find that ribosomes lacking bS1 are slightly less active than wild-type ribosomes in vitro. Furthermore, orthogonal 30S subunits lacking bS1 do not have an improved orthogonality. Our findings suggest that mRNA features outside the SD sequence and independent of binding of bS1 to the ribosome likely contribute to start codon selection and the lack of orthogonality of present orthogonal ribosomes.

  PublisherDOI

• Improving RNA Secondary Structure Prediction Through Expanded Training Data

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-05-03 · 1 citations

  preprintOpen accessSenior authorCorresponding

  In recent years, deep learning has revolutionized protein structure prediction, achieving remarkable speed and accuracy. RNA structure prediction, however, has lagged behind. Although several methods have shown some success in predicting RNA secondary and tertiary structures, none have reached the accuracy observed with contemporary protein models. The lack of success of these RNA structure prediction models has been proposed to be due to limited high-quality structural information that can be used as training data. To probe this proposed limitation, we developed a large and diverse dataset comprising paired RNA sequences and their corresponding secondary structures. We assess the utility of this enhanced dataset by retraining on a deep learning model, SincFold. We find that SincFold exhibited improved generalization to some previously unseen RNA families, enhancing its capability to predict accurate de novo RNA secondary structures. The RNASSTR dataset provides a substantial advance for RNA structure modeling, laying a strong foundation for the development of future RNA secondary structure prediction algorithms.

  PublisherOA PDFDOI

• Thioesters Support Efficient Protein Biosynthesis by the Ribosome

  ACS Central Science · 2025-01-30 · 4 citations

  articleOpen accessSenior authorCorresponding

  Thioesters are critical chemical intermediates in numerous extant biochemical reactions and are invoked as key reagents during prebiotic peptide synthesis on an evolving Earth. Here we asked if a thioester could replace the native oxo-ester in acyl-tRNA substrates during protein biosynthesis by the ribosome. We prepared 3′-thio-3′-deoxyadenosine triphosphate in 10 steps from xylose and demonstrated that it is an effective substrate for the Escherichia coli CCA-adding enzyme, which appends 3′-thio-3′-deoxyadenosine to truncated tRNAs ending with 3′-CC. Using a variety of aminoacyl-tRNA synthetases, flexizymes, or a direct thioester exchange reaction, we prepared a suite of 3′-thio-tRNAs acylated with α- and non-α-amino acids. All were recognized and utilized by wild-type E. coli ribosomes during in vitro translation reactions to generate oligopeptides in yields commensurate with native oxo-ester tRNAs. These results indicate that thioester intermediates widely used in Nature can be co-opted to support the incorporation of natural α-amino acids as well as noncanonical monomers by the extant translational machinery for sequence-defined polymer synthesis.

  PublisherDOI

• Overcoming the eIF2α Brake in Human Cell-Derived Translation Systems

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-11-16

  preprintOpen accessSenior authorCorresponding

  Abstract Cell-free translation from human cells is a powerful platform for studying mammalian gene expression and building synthetic biology tools, but productivity is often curtailed by inhibitory phosphorylation of eIF2α on residue Ser52. Here we systematically explored complementary strategies to bypass this initiation block across editable and hard-to-edit human cell types. In Expi293F suspension cells, precise genome editing of EIF2S1 to block Ser52 phosphorylation (eIF2α-S52A) produced high-activity extracts. Genetic knockout of EIF2AK2 (PKR)–the principal eIF2α kinase engaged in eIF2α phosphorylation in Expi293F lysates–also improved translation, further establishing eIF2α phosphorylation as the dominant bottleneck in Expi293F translation extracts. Because genome editing is impractical in many contexts including primary human cells, we also implemented expression-based rescue of eIF2α phosphorylation: stable piggyBac integration of truncated GADD34 ( PPP1R15A ) and K3L, a viral eIF2α decoy, under control of a Tet-inducible promotor in induced pluripotent stem cells (iPSCs) and primary human fibroblasts. After differentiating engineered KOLF2.1J iPSCs into cardiomyocytes, we found that stable GADD34/K3L expression increased translation output in cardiomyocyte translation extracts. Using the piggyBac expression system in primary fibroblasts also resulted in improved translational output. Together these data pinpoint eIF2α phosphorylation as the key barrier to robust translation in human cell translation extracts. They also show that editing eIF2α or removing PKR is optimal where genome editing is feasible, while providing a portable GADD34/K3L expression cassette enables production of translationally active lysates from systems where genome editing is challenging or not possible.

  PublisherOA PDFDOI

• Circular 23S rRNA within archaeal ribosomes

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-04-27 · 2 citations

  preprintOpen access

  Abstract The ribosomal RNAs (rRNAs) that form the core of ribosomes are believed to occur as linear molecules. Here, we investigated rRNAs from diverse and mostly uncultivated archaea and found evidence that, in at least eight phylum-level archaeal lineages, the 23S rRNAs within mature ribosomes are circular. Sequencing of total cellular RNA indicates that the transcriptional abundances of archaeal circular 23S rRNAs vastly exceed those of linear counterparts, and linear versions are often undetectable. As the majority of rRNAs derive from mature ribosomes, the data suggested that the circular molecules are within the ribosomes. Thus, we directly sequenced RNA extracted from isolated ribosomes of a model archaeon, Methanosarcina acetivorans , and confirmed that the assembled 23S rRNAs are circular. Structural modeling places the 5’ and 3’ ends of the linear precursors of archaeal 23S rRNAs in close proximity to form a GNRA tetraloop (in which N is A, C, G, or U and R is A or G), consistent with their further circularization within ribosomes. We confirm the existence of circular 16S rRNA intermediates in transcriptomes of most archaea, yet a circular form is not evident in some distinct archaeal groups. Overall, the results uncover unexpected variations in the processing required to generate mature rRNAs and the conformation of functional molecules in archaeal ribosomes.

  PublisherOA PDFDOI

• Escherichia coli ribosomes support translation of (R) and (S) β2-hydroxyacids in vitro: a structural and biochemical study

  ChemRxiv · 2025-05-08

  preprintOpen accessSenior author

  The ribosomal incorporation of backbone-modified amino acid analogs into peptides and proteins enables the programmed synthesis of sequence-defined biopolymers with tunable properties. However, the successful use of backbone-modified monomers as substrates by the ribosome requires coordination across multiple parts of the translation machinery, including aminoacyl-tRNA synthetases, translation factors, and finally the ribosome itself. β-hydroxyacids are particularly interesting monomers because they have potential to support the programmed biosynthesis of both polyesters (plastics) and depsipeptides (therapeutics). Previous work has reported that both enantiomers of β2-hydroxy-Nε-Boc-Lysine (β2-OH-BocK) are in vitro substrates for the orthogonal M. alvi PylRS/tRNA pair, but only one enantiomer is introduced into protein in vivo and with substantially lower yield than expected. We sought to determine whether there is a structural basis for the diminished yield as well as the preferential incorporation of one β2-OH-BocK enantiomer over the other. Here we report high-resolution cryo-EM structures of the Escherichia coli (E. coli) ribosome complexed with either (R)- or (S)-β2-OH-BocK. These structures reveal that both enantiomers are well positioned to undergo bond formation within the ribosome active site and are likely equally reactive. In vitro translation experiments confirm that orthogonal tRNAs acylated with (R)- or (S)-β2-OH-BocK are ribosome substrates, implying that the preferential incorporation of one enantiomer over the other in vivo results from deficiencies in other translation steps, such as tRNA acylation efficiency in cells or delivery to the ribosome by elongation factor Tu (EF-Tu). Taken together, this work demonstrates the plasticity of the E. coli ribosome and its tolerance for diverse substrates.

  PublisherOA PDFDOI

• eIF3 engages with 3’-UTR termini of highly translated mRNAs

  eLife · 2025-01-29 · 1 citations

  articleOpen accessSenior author

  Stem cell differentiation involves a global increase in protein synthesis to meet the demands of specialized cell types. However, the molecular mechanisms underlying this translational burst and the involvement of initiation factors remains largely unknown. Here, we investigate the role of eukaryotic initiation factor 3 (eIF3) in early differentiation of human pluripotent stem cell (hPSC)-derived neural progenitor cells (NPCs). Using Quick-irCLIP and alternative polyadenylation (APA) Seq, we show eIF3 crosslinks predominantly with 3’ untranslated region (3’-UTR) termini of multiple mRNA isoforms, adjacent to the poly(A) tail. Furthermore, we find that eIF3 engagement at 3’-UTR ends is dependent on polyadenylation. High eIF3 crosslinking at 3’-UTR termini of mRNAs correlates with high translational activity, as determined by ribosome profiling, but not with translational efficiency. The results presented here show that eIF3 engages with 3’-UTR termini of highly translated mRNAs, likely reflecting a general rather than specific regulatory function of eIF3, and supporting a role of mRNA circularization in the mechanisms governing mRNA translation.

  PublisherDOI

Recent grants

• Atomic-Resolution Analysis of eIF3-Mediated Translation Control

  NIH · $7.4M · 2001–2022

• Selective Stalling of Human Translation by Small Molecules

  NIH · $1.2M · 2019–2023

• Mechanisms of Translation Control in Humans

  NIH · $2.3M · 2023–2028

• NIH Grant P50GM102706

  NIH · $19.1M · 2017

• Improved High-resolution cryo-EM Methodology

  NIH · $63.4M · 2021

Frequent coauthors

• Jennifer A. Doudna

  University of California, Berkeley

  119 shared

• Jillian F. Banfield

  University of California, Berkeley

  83 shared

• Nicholas T. Ingolia

  QB3

  69 shared

• Alexander Marson

  University of California, San Francisco

  66 shared

• Zoe L. Watson

  University of California, Berkeley

  56 shared

• Alanna Schepartz

  Arc Research Institute

  49 shared

• Yong‐Su Jin

  University of Illinois Urbana-Champaign

  41 shared

• Robert M. Glaeser

  University of California, Berkeley

  40 shared

Awards & honors

• Searle Scholar (2000-2003)
• Sloan Research Fellow (2006-2007)
• Member, American Academy of Arts and Sciences (2017)