Publications by authors named "Jamie H D Cate"

103 Publications

Genome-resolved metagenomics reveals site-specific diversity of episymbiotic CPR bacteria and DPANN archaea in groundwater ecosystems.

Nat Microbiol 2021 Mar 25;6(3):354-365. Epub 2021 Jan 25.

Innovative Genomics Institute, University of California, Berkeley, CA, USA.

Candidate phyla radiation (CPR) bacteria and DPANN archaea are unisolated, small-celled symbionts that are often detected in groundwater. The effects of groundwater geochemistry on the abundance, distribution, taxonomic diversity and host association of CPR bacteria and DPANN archaea has not been studied. Here, we performed genome-resolved metagenomic analysis of one agricultural and seven pristine groundwater microbial communities and recovered 746 CPR and DPANN genomes in total. The pristine sites, which serve as local sources of drinking water, contained up to 31% CPR bacteria and 4% DPANN archaea. We observed little species-level overlap of metagenome-assembled genomes (MAGs) across the groundwater sites, indicating that CPR and DPANN communities may be differentiated according to physicochemical conditions and host populations. Cryogenic transmission electron microscopy imaging and genomic analyses enabled us to identify CPR and DPANN lineages that reproducibly attach to host cells and showed that the growth of CPR bacteria seems to be stimulated by attachment to host-cell surfaces. Our analysis reveals site-specific diversity of CPR bacteria and DPANN archaea that coexist with diverse hosts in groundwater aquifers. Given that CPR and DPANN organisms have been identified in human microbiomes and their presence is correlated with diseases such as periodontitis, our findings are relevant to considerations of drinking water quality and human health.
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http://dx.doi.org/10.1038/s41564-020-00840-5DOI Listing
March 2021

Efficient isolation of protoplasts from rice calli with pause points and its application in transient gene expression and genome editing assays.

Plant Methods 2020 Nov 12;16(1):151. Epub 2020 Nov 12.

Innovative Genomics Institute, University of California, Berkeley, CA, 94720, USA.

Background: An efficient in vivo transient transfection system using protoplasts is an important tool to study gene expression, metabolic pathways, and multiple mutagenesis parameters in plants. Although rice protoplasts can be isolated from germinated seedlings or cell suspension culture, preparation of those donor tissues can be inefficient, time-consuming, and laborious. Additionally, the lengthy process of protoplast isolation and transfection needs to be completed in a single day.

Results: Here we report a protocol for the isolation of protoplasts directly from rice calli, without using seedlings or suspension culture. The method is developed to employ discretionary pause points during protoplast isolation and before transfection. Protoplasts maintained within a sucrose cushion partway through isolation, for completion on a subsequent day, per the first pause point, are referred to as S protoplasts. Fully isolated protoplasts maintained in MMG solution for transfection on a subsequent day, per the second pause point, are referred to as M protoplasts. Both S and M protoplasts, 1 day after initiation of protoplast isolation, had minimal loss of viability and transfection efficiency compared to protoplasts 0 days after isolation. S protoplast viability decreases at a lower rate over time than that of M protoplasts and can be used with added flexibility for transient transfection assays and time-course experiments. The protoplasts produced by this method are competent for transfection of both plasmids and ribonucleoproteins (RNPs). Cas9 RNPs were used to demonstrate the utility of these protoplasts to assay genome editing in vivo.

Conclusion: The current study describes a highly effective and accessible method to isolate protoplasts from callus tissue induced from rice seeds. This method utilizes donor materials that are resource-efficient and easy to propagate, permits convenience via pause points, and allows for flexible transfection days after protoplast isolation. It provides an advantageous and useful platform for a variety of in vivo transient transfection studies in rice.
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http://dx.doi.org/10.1186/s13007-020-00692-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7663885PMC
November 2020

Selective inhibition of human translation termination by a drug-like compound.

Nat Commun 2020 10 2;11(1):4941. Epub 2020 Oct 2.

Department of Molecular & Cell Biology, University of California, Berkeley, CA, 94720, USA.

Methods to directly inhibit gene expression using small molecules hold promise for the development of new therapeutics targeting proteins that have evaded previous attempts at drug discovery. Among these, small molecules including the drug-like compound PF-06446846 (PF846) selectively inhibit the synthesis of specific proteins, by stalling translation elongation. These molecules also inhibit translation termination by an unknown mechanism. Using cryo-electron microscopy (cryo-EM) and biochemical approaches, we show that PF846 inhibits translation termination by arresting the nascent chain (NC) in the ribosome exit tunnel. The arrested NC adopts a compact α-helical conformation that induces 28 S rRNA nucleotide rearrangements that suppress the peptidyl transferase center (PTC) catalytic activity stimulated by eukaryotic release factor 1 (eRF1). These data support a mechanism of action for a small molecule targeting translation that suppresses peptidyl-tRNA hydrolysis promoted by eRF1, revealing principles of eukaryotic translation termination and laying the foundation for new therapeutic strategies.
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http://dx.doi.org/10.1038/s41467-020-18765-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7532171PMC
October 2020

Clades of huge phages from across Earth's ecosystems.

Nature 2020 02 12;578(7795):425-431. Epub 2020 Feb 12.

Innovative Genomics Institute, University of California Berkeley, Berkeley, CA, USA.

Bacteriophages typically have small genomes and depend on their bacterial hosts for replication. Here we sequenced DNA from diverse ecosystems and found hundreds of phage genomes with lengths of more than 200 kilobases (kb), including a genome of 735 kb, which is-to our knowledge-the largest phage genome to be described to date. Thirty-five genomes were manually curated to completion (circular and no gaps). Expanded genetic repertoires include diverse and previously undescribed CRISPR-Cas systems, transfer RNAs (tRNAs), tRNA synthetases, tRNA-modification enzymes, translation-initiation and elongation factors, and ribosomal proteins. The CRISPR-Cas systems of phages have the capacity to silence host transcription factors and translational genes, potentially as part of a larger interaction network that intercepts translation to redirect biosynthesis to phage-encoded functions. In addition, some phages may repurpose bacterial CRISPR-Cas systems to eliminate competing phages. We phylogenetically define the major clades of huge phages from human and other animal microbiomes, as well as from oceans, lakes, sediments, soils and the built environment. We conclude that the large gene inventories of huge phages reflect a conserved biological strategy, and that the phages are distributed across a broad bacterial host range and across Earth's ecosystems.
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http://dx.doi.org/10.1038/s41586-020-2007-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7162821PMC
February 2020

Initiation of Protein Synthesis with Non-Canonical Amino Acids In Vivo.

Angew Chem Int Ed Engl 2020 02 21;59(8):3122-3126. Epub 2020 Jan 21.

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, 06520, USA.

By transplanting identity elements into E. coli tRNA , we have engineered an orthogonal initiator tRNA (itRNA ) that is a substrate for Methanocaldococcus jannaschii TyrRS. We demonstrate that itRNA can initiate translation in vivo with aromatic non-canonical amino acids (ncAAs) bearing diverse sidechains. Although the initial system suffered from low yields, deleting redundant copies of tRNA from the genome afforded an E. coli strain in which the efficiency of non-canonical initiation equals elongation. With this improved system we produced a protein containing two distinct ncAAs at the first and second positions, an initial step towards producing completely unnatural polypeptides in vivo. This work provides a valuable tool to synthetic biology and demonstrates remarkable versatility of the E. coli translational machinery for initiation with ncAAs in vivo.
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http://dx.doi.org/10.1002/anie.201914671DOI Listing
February 2020

Defects in the Assembly of Ribosomes Selected for β-Amino Acid Incorporation.

Biochemistry 2019 11 28;58(45):4494-4504. Epub 2019 Oct 28.

Department of Molecular and Cell Biology , University of California-Berkeley , Berkeley , California 94720 , United States.

Ribosome engineering has emerged as a promising field in synthetic biology, particularly concerning the production of new sequence-defined polymers. Mutant ribosomes have been developed that improve the incorporation of several nonstandard monomers including d-amino acids, dipeptides, and β-amino acids into polypeptide chains. However, there remains little mechanistic understanding of how these ribosomes catalyze incorporation of these new substrates. Here, we probed the properties of a mutant ribosome-P7A7-evolved for better β-amino acid incorporation through biochemistry and cryo-electron microscopy. Although P7A7 is a functional ribosome , it is inactive , and assembles poorly into 70S ribosome complexes. Structural characterization revealed large regions of disorder in the peptidyltransferase center and nearby features, suggesting a defect in assembly. Comparison of RNA helix and ribosomal protein occupancy with other assembly intermediates revealed that P7A7 is stalled at a late stage in ribosome assembly, explaining its weak activity. These results highlight the importance of ensuring efficient ribosome assembly during ribosome engineering toward new catalytic abilities.
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http://dx.doi.org/10.1021/acs.biochem.9b00746DOI Listing
November 2019

Structure of ribosome-bound azole-modified peptide phazolicin rationalizes its species-specific mode of bacterial translation inhibition.

Nat Commun 2019 10 8;10(1):4563. Epub 2019 Oct 8.

Center of Life Sciences, Skolkovo Institute of Science and Technology, Moscow, Russia.

Ribosome-synthesized post-translationally modified peptides (RiPPs) represent a rapidly expanding class of natural products with various biological activities. Linear azol(in)e-containing peptides (LAPs) comprise a subclass of RiPPs that display outstanding diversity of mechanisms of action while sharing common structural features. Here, we report the discovery of a new LAP biosynthetic gene cluster in the genome of Rhizobium Pop5, which encodes the precursor peptide and modification machinery of phazolicin (PHZ) - an extensively modified peptide exhibiting narrow-spectrum antibacterial activity against some symbiotic bacteria of leguminous plants. The cryo-EM structure of the Escherichia coli 70S-PHZ complex reveals that the drug interacts with the 23S rRNA and uL4/uL22 proteins and obstructs ribosomal exit tunnel in a way that is distinct from other compounds. We show that the uL4 loop sequence determines the species-specificity of antibiotic action. PHZ expands the known diversity of LAPs and may be used in the future as biocontrol agent for agricultural needs.
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http://dx.doi.org/10.1038/s41467-019-12589-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6783444PMC
October 2019

mRNA isoforms and regulation of their translation.

RNA 2019 10 1;25(10):1324-1336. Epub 2019 Jul 1.

Graduate Study in Comparative Biochemistry, University of California, Berkeley, California 94720, USA.

Polypyrimidine tract-binding proteins (PTBPs) are RNA binding proteins that regulate a number of posttranscriptional events. Human PTBP1 transits between the nucleus and cytoplasm and is thought to regulate RNA processes in both. However, information about mRNA isoforms and regulation of PTPB1 expression remains incomplete. Here we mapped the major mRNA isoforms in HEK293T cells and identified alternative 5' and 3' untranslated regions (5'-UTRs, 3'-UTRs), as well as alternative splicing patterns in the protein coding region. We also assessed how the observed mRNA isoforms contribute to PTBP1 expression in different phases of the cell cycle. Previously, mRNAs were shown to crosslink to eukaryotic translation initiation factor 3 (eIF3). We find that eIF3 binds differently to each mRNA isoform in a cell cycle dependent manner. We also observe a strong correlation between eIF3 binding to mRNAs and repression of PTBP1 levels during the S phase of the cell cycle. Our results provide evidence of translational regulation of PTBP1 protein levels during the cell cycle, which may affect downstream regulation of alternative splicing and translation mediated by PTBP1 protein isoforms.
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http://dx.doi.org/10.1261/rna.070193.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6800477PMC
October 2019

Structural basis for selective stalling of human ribosome nascent chain complexes by a drug-like molecule.

Nat Struct Mol Biol 2019 06 3;26(6):501-509. Epub 2019 Jun 3.

Department of Molecular & Cell Biology, University of California, Berkeley, Berkeley, CA, USA.

The drug-like molecule PF-06446846 (PF846) binds the human ribosome and selectively blocks the translation of a small number of proteins by an unknown mechanism. In structures of PF846-stalled human ribosome nascent chain complexes, PF846 binds in the ribosome exit tunnel in a eukaryotic-specific pocket formed by 28S ribosomal RNA, and alters the path of the nascent polypeptide chain. PF846 arrests the translating ribosome in the rotated state of translocation, in which the peptidyl-transfer RNA 3'-CCA end is improperly docked in the peptidyl transferase center. Selections of messenger RNAs from mRNA libraries using translation extracts reveal that PF846 can stall translation elongation, arrest termination or even enhance translation, depending on nascent chain sequence context. These results illuminate how a small molecule selectively targets translation by the human ribosome, and provides a foundation for developing small molecules that modulate the production of proteins of therapeutic interest.
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http://dx.doi.org/10.1038/s41594-019-0236-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6919564PMC
June 2019

Overcoming the thermodynamic equilibrium of an isomerization reaction through oxidoreductive reactions for biotransformation.

Nat Commun 2019 03 22;10(1):1356. Epub 2019 Mar 22.

Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA.

Isomerases perform biotransformations without cofactors but often cause an undesirable mixture of substrate and product due to unfavorable thermodynamic equilibria. We demonstrate the feasibility of using an engineered yeast strain harboring oxidoreductase reactions to overcome the thermodynamic limit of an isomerization reaction. Specifically, a yeast strain capable of consuming lactose intracellularly is engineered to produce tagatose from lactose through three layers of manipulations. First, GAL1 coding for galactose kinase is deleted to eliminate galactose utilization. Second, heterologous xylose reductase (XR) and galactitol dehydrogenase (GDH) are introduced into the ∆gal1 strain. Third, the expression levels of XR and GDH are adjusted to maximize tagatose production. The resulting engineered yeast produces 37.69 g/L of tagatose from lactose with a tagatose and galactose ratio of 9:1 in the reaction broth. These results suggest that in vivo oxidoreaductase reactions can be employed to replace isomerases in vitro for biotransformation.
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http://dx.doi.org/10.1038/s41467-019-09288-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6430769PMC
March 2019

Cellular response to small molecules that selectively stall protein synthesis by the ribosome.

PLoS Genet 2019 03 15;15(3):e1008057. Epub 2019 Mar 15.

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, United States of America.

Identifying small molecules that inhibit protein synthesis by selectively stalling the ribosome constitutes a new strategy for therapeutic development. Compounds that inhibit the translation of PCSK9, a major regulator of low-density lipoprotein cholesterol, have been identified that reduce LDL cholesterol in preclinical models and that affect the translation of only a few off-target proteins. Although some of these compounds hold potential for future therapeutic development, it is not known how they impact the physiology of cells or ribosome quality control pathways. Here we used a genome-wide CRISPRi screen to identify proteins and pathways that modulate cell growth in the presence of high doses of a selective PCSK9 translational inhibitor, PF-06378503 (PF8503). The two most potent genetic modifiers of cell fitness in the presence of PF8503, the ubiquitin binding protein ASCC2 and helicase ASCC3, bind to the ribosome and protect cells from toxic effects of high concentrations of the compound. Surprisingly, translation quality control proteins Pelota (PELO) and HBS1L sensitize cells to PF8503 treatment. In genetic interaction experiments, ASCC3 acts together with ASCC2, and functions downstream of HBS1L. Taken together, these results identify new connections between ribosome quality control pathways, and provide new insights into the selectivity of compounds that stall human translation that will aid the development of next-generation selective translation stalling compounds to treat disease.
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http://dx.doi.org/10.1371/journal.pgen.1008057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6436758PMC
March 2019

Differences in the path to exit the ribosome across the three domains of life.

Nucleic Acids Res 2019 05;47(8):4198-4210

Computer Science Division, University of California, Berkeley, CA 94720, USA.

The ribosome exit tunnel is an important structure involved in the regulation of translation and other essential functions such as protein folding. By comparing 20 recently obtained cryo-EM and X-ray crystallography structures of the ribosome from all three domains of life, we here characterize the key similarities and differences of the tunnel across species. We first show that a hierarchical clustering of tunnel shapes closely reflects the species phylogeny. Then, by analyzing the ribosomal RNAs and proteins, we explain the observed geometric variations and show direct association between the conservations of the geometry, structure and sequence. We find that the tunnel is more conserved in the upper part close to the polypeptide transferase center, while in the lower part, it is substantially narrower in eukaryotes than in bacteria. Furthermore, we provide evidence for the existence of a second constriction site in eukaryotic exit tunnels. Overall, these results have several evolutionary and functional implications, which explain certain differences between eukaryotes and prokaryotes in their translation mechanisms. In particular, they suggest that major co-translational functions of bacterial tunnels were externalized in eukaryotes, while reducing the tunnel size provided some other advantages, such as facilitating the nascent chain elongation and enabling antibiotic resistance.
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http://dx.doi.org/10.1093/nar/gkz106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6486554PMC
May 2019

Bacterial ribosome heterogeneity: Changes in ribosomal protein composition during transition into stationary growth phase.

Biochimie 2019 Jan 23;156:169-180. Epub 2018 Oct 23.

Institute of Molecular and Cell Biology, University of Tartu, Riia 23B, Tartu, 51010, Estonia. Electronic address:

Ribosomes consist of many small proteins and few large RNA molecules. Both components are necessary for ribosome functioning during translation. According to widely accepted view, bacterial ribosomes contain always the same complement of ribosomal proteins. Comparative bacterial genomics data indicates that several ribosomal proteins are encoded by multiple paralogous genes suggesting structural heterogeneity of ribosomes. In E. coli, two r-proteins bL31 and bL36 are encoded by two genes: rpmE and ykgM encode bL31 protein paralogs bL31A and bL31B, and rpmJ and ykgO encode bL36 protein paralogs bL36A and bL36B respectively. We have found several similarities and differences between ribosomes of exponential and stationary growth phases by using quantitative mass spectrometry and X-ray crystallography. First, composition of ribosome associating proteins changes profoundly as cells transition from exponential to stationary growth phase. Ribosomal core proteins bL31A and bL36A are replaced by bL31B and bL36B, respectively. Second, our X-ray structure of the 70S ribosome demonstrates that bL31B and bL36B proteins have similar ribosome binding sites to their A counterparts. Third, ribosome subpopulations containing A or B paralogs existed simultaneously demonstrating that E. coli ribosomes are heterogeneous with respect to their paralogous ribosomal protein composition that changes via protein exchange.
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http://dx.doi.org/10.1016/j.biochi.2018.10.013DOI Listing
January 2019

Modulating long-range energetics via helix stabilization: A case study using T4 lysozyme.

Protein Sci 2018 12;27(12):2084-2093

California Institute for Quantitative Biosciences, University of California, Berkeley, California, 94720.

Cooperative protein folding requires distant regions of a protein to interact and provide mutual stabilization. The mechanism of this long-distance coupling remains poorly understood. Here, we use T4 lysozyme (T4L*) as a model to investigate long-range communications across two subdomains of a globular protein. T4L* is composed of two structurally distinct subdomains, although it behaves in a two-state manner at equilibrium. The subdomains of T4L* are connected via two topological connections: the N-terminal helix that is structurally part of the C-terminal subdomain (the A-helix) and a long helix that spans both subdomains (the C-helix). To understand the role that the C-helix plays in cooperative folding, we analyzed a circularly permuted version of T4L* (CP13*), whose subdomains are connected only by the C-helix. We demonstrate that when isolated as individual fragments, both subdomains of CP13* can fold autonomously into marginally stable conformations. The energetics of the N-terminal subdomain depend on the formation of a salt bridge known to be important for stability in the full-length protein. We show that the energetic contribution of the salt bridge to the stability of the N-terminal fragment increases when the C-helix is stabilized, such as occurs upon folding of the C-terminal subdomain. These results suggest a model where long-range energetic coupling is mediated by helix stabilization and not specific tertiary interactions.
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http://dx.doi.org/10.1002/pro.3521DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237701PMC
December 2018

Engineering Kluyveromyces marxianus as a Robust Synthetic Biology Platform Host.

mBio 2018 09 25;9(5). Epub 2018 Sep 25.

Department of Molecular and Cell Biology, University of California, Berkeley, California, USA

Throughout history, the yeast has played a central role in human society due to its use in food production and more recently as a major industrial and model microorganism, because of the many genetic and genomic tools available to probe its biology. However, has proven difficult to engineer to expand the carbon sources it can utilize, the products it can make, and the harsh conditions it can tolerate in industrial applications. Other yeasts that could solve many of these problems remain difficult to manipulate genetically. Here, we engineered the thermotolerant yeast to create a new synthetic biology platform. Using CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats with Cas9)-mediated genome editing, we show that wild isolates of can be made heterothallic for sexual crossing. By breeding two of these mating-type engineered strains, we combined three complex traits-thermotolerance, lipid production, and facile transformation with exogenous DNA-into a single host. The ability to cross strains with relative ease, together with CRISPR-Cas9 genome editing, should enable engineering of isolates with promising lipid production at temperatures far exceeding those of other fungi under development for industrial applications. These results establish as a synthetic biology platform comparable to , with naturally more robust traits that hold potential for the industrial production of renewable chemicals. The yeast grows at high temperatures and on a wide range of carbon sources, making it a promising host for industrial biotechnology to produce renewable chemicals from plant biomass feedstocks. However, major genetic engineering limitations have kept this yeast from replacing the commonly used yeast in industrial applications. Here, we describe genetic tools for genome editing and breeding strains, which we use to create a new thermotolerant strain with promising fatty acid production. These results open the door to using as a versatile synthetic biology platform organism for industrial applications.
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http://dx.doi.org/10.1128/mBio.01410-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6156195PMC
September 2018

Small Molecule Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Inhibitors: Hit to Lead Optimization of Systemic Agents.

J Med Chem 2018 07 25;61(13):5704-5718. Epub 2018 Jun 25.

Pfizer Medicinal Chemistry , Groton , Connecticut 06340 , United States.

The optimization of a new class of small molecule PCSK9 mRNA translation inhibitors is described. The potency, physicochemical properties, and off-target pharmacology associated with the hit compound (1) were improved by changes to two regions of the molecule. The last step in the synthesis of the congested amide center was enabled by three different routes. Subtle structural changes yielded significant changes in pharmacology and off-target margins. These efforts led to the identification of 7l and 7n with overall profiles suitable for in vivo evaluation. In a 14-day toxicology study, 7l demonstrated an improved safety profile vs lead 7f. We hypothesize that the improved safety profile is related to diminished binding of 7l to nontranslating ribosomes and an apparent improvement in transcript selectivity due to the lower strength of 7l stalling of off-target proteins.
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http://dx.doi.org/10.1021/acs.jmedchem.8b00650DOI Listing
July 2018

Enhanced cellobiose fermentation by engineered Saccharomyces cerevisiae expressing a mutant cellodextrin facilitator and cellobiose phosphorylase.

J Biotechnol 2018 Jun 13;275:53-59. Epub 2018 Apr 13.

Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA; Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA. Electronic address:

To efficiently ferment intermediate cellodextrins released during cellulose hydrolysis, Saccharomyces cerevisiae has been engineered by introduction of a heterologous cellodextrin utilizing pathway consisting of a cellodextrin transporter and either an intracellular β-glucosidase or a cellobiose phosphorylase. Among two types of cellodextrin transporters, the passive facilitator CDT-2 has not enabled better cellobiose fermentation than the active transporter CDT-1, which suggests that the CDT-2 might be engineered to provide energetic benefits over the active transporter in cellobiose fermentation. We attempted to improve cellobiose transporting activity of CDT-2 through laboratory evolution. Nine rounds of a serial subculture of S. cerevisiae expressing CDT-2 and cellobiose phosphorylase on cellobiose led to the isolation of an evolved strain capable of fermenting cellobiose to ethanol 10-fold faster than the original strain. After sequence analysis of the isolated CDT-2, a single point mutation on CDT-2 (N306I) was revealed to be responsible for enhanced cellobiose fermentation. Also, the engineered strain expressing the mutant CDT-2 with cellobiose phosphorylase showed a higher ethanol yield than the engineered strain expressing CDT-1 and intracellular β-glucosidase under anaerobic conditions, suggesting that CDT-2 coupled with cellobiose phosphorylase may be better choices for efficient production of cellulosic ethanol with the engineered yeast.
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http://dx.doi.org/10.1016/j.jbiotec.2018.04.008DOI Listing
June 2018

Evolutionary engineering improves tolerance for medium-chain alcohols in .

Biotechnol Biofuels 2018 2;11:90. Epub 2018 Apr 2.

2Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Tech E-136, Evanston, IL 60208-3109 USA.

Background: Yeast-based chemical production is an environmentally friendly alternative to petroleum-based production or processes that involve harsh chemicals. However, many potential alcohol biofuels, such as -butanol, isobutanol and -hexanol, are toxic to production organisms, lowering the efficiency and cost-effectiveness of these processes. We set out to improve the tolerance of toward these alcohols.

Results: We evolved the laboratory strain of BY4741 to be more tolerant toward -hexanol and show that the mutations which confer tolerance occur in proteins of the translation initiation complex. We found that -hexanol inhibits initiation of translation and evolved mutations in the α subunit of eIF2 and the γ subunit of its guanine exchange factor eIF2B rescue this inhibition. We further demonstrate that translation initiation is affected by other alcohols such as -pentanol and -heptanol, and that mutations in the eIF2 and eIF2B complexes greatly improve tolerance to these medium-chain alcohols.

Conclusions: We successfully generated strains that have improved tolerance toward medium-chain alcohols and have demonstrated that the causative mutations overcome inhibition of translation initiation by these alcohols.
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http://dx.doi.org/10.1186/s13068-018-1089-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5880003PMC
April 2018

Programmable RNA recognition using a CRISPR-associated Argonaute.

Proc Natl Acad Sci U S A 2018 03 12;115(13):3368-3373. Epub 2018 Mar 12.

Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720;

Argonaute proteins (Agos) are present in all domains of life. Although the physiological function of eukaryotic Agos in regulating gene expression is well documented, the biological roles of many of their prokaryotic counterparts remain enigmatic. In some bacteria, Agos are associated with CRISPR (clustered regularly interspaced short palindromic repeats) loci and use noncanonical 5'-hydroxylated guide RNAs (gRNAs) for nucleic acid targeting. Here we show that using 5-bromo-2'-deoxyuridine (BrdU) as the 5' nucleotide of gRNAs stabilizes in vitro reconstituted CRISPR-associated Argonaute-gRNA complexes (MpAgo RNPs) and significantly improves their specificity and affinity for RNA targets. Using reconstituted MpAgo RNPs with 5'-BrdU-modified gRNAs, we mapped the seed region of the gRNA and identified the nucleotides of the gRNA that play the most significant role in targeting specificity. We also show that these MpAgo RNPs can be programmed to distinguish between substrates that differ by a single nucleotide, using permutations at the sixth and seventh positions in the gRNA. Using these specificity features, we employed MpAgo RNPs to detect specific adenosine-to-inosine-edited RNAs in a complex mixture. These findings broaden our mechanistic understanding of the interactions of Argonautes with guide and substrate RNAs, and demonstrate that MpAgo RNPs with 5'-BrdU-modified gRNAs can be used as a highly specific RNA-targeting platform to probe RNA biology.
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http://dx.doi.org/10.1073/pnas.1717725115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5879674PMC
March 2018

Role for ribosome-associated complex and stress-seventy subfamily B (RAC-Ssb) in integral membrane protein translation.

J Biol Chem 2017 12 2;292(48):19610-19627. Epub 2017 Oct 2.

From the Departments of Molecular and Cell Biology and

Targeting of most integral membrane proteins to the endoplasmic reticulum is controlled by the signal recognition particle, which recognizes a hydrophobic signal sequence near the protein N terminus. Proper folding of these proteins is monitored by the unfolded protein response and involves protein degradation pathways to ensure quality control. Here, we identify a new pathway for quality control of major facilitator superfamily transporters that occurs before the first transmembrane helix, the signal sequence recognized by the signal recognition particle, is made by the ribosome. Increased rates of translation elongation of the N-terminal sequence of these integral membrane proteins can divert the nascent protein chains to the ribosome-associated complex and stress-seventy subfamily B chaperones. We also show that quality control of integral membrane proteins by ribosome-associated complex-stress-seventy subfamily B couples translation rate to the unfolded protein response, which has implications for understanding mechanisms underlying human disease and protein production in biotechnology.
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http://dx.doi.org/10.1074/jbc.M117.813857DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5712606PMC
December 2017

Screening of transporters to improve xylodextrin utilization in the yeast Saccharomyces cerevisiae.

PLoS One 2017 8;12(9):e0184730. Epub 2017 Sep 8.

Department of Molecular and Cell Biology, University of California, Berkeley, California, United States of America.

The economic production of cellulosic biofuel requires efficient and full utilization of all abundant carbohydrates naturally released from plant biomass by enzyme cocktails. Recently, we reconstituted the Neurospora crassa xylodextrin transport and consumption system in Saccharomyces cerevisiae, enabling growth of yeast on xylodextrins aerobically. However, the consumption rate of xylodextrin requires improvement for industrial applications, including consumption in anaerobic conditions. As a first step in this improvement, we report analysis of orthologues of the N. crassa transporters CDT-1 and CDT-2. Transporter ST16 from Trichoderma virens enables faster aerobic growth of S. cerevisiae on xylodextrins compared to CDT-2. ST16 is a xylodextrin-specific transporter, and the xylobiose transport activity of ST16 is not inhibited by cellobiose. Other transporters identified in the screen also enable growth on xylodextrins including xylotriose. Taken together, these results indicate that multiple transporters might prove useful to improve xylodextrin utilization in S. cerevisiae. Efforts to use directed evolution to improve ST16 from a chromosomally-integrated copy were not successful, due to background growth of yeast on other carbon sources present in the selection medium. Future experiments will require increasing the baseline growth rate of the yeast population on xylodextrins, to ensure that the selective pressure exerted on xylodextrin transport can lead to isolation of improved xylodextrin transporters.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0184730PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5591001PMC
October 2017

Cellobiose Consumption Uncouples Extracellular Glucose Sensing and Glucose Metabolism in .

mBio 2017 08 8;8(4). Epub 2017 Aug 8.

Department of Molecular and Cell Biology, University of California, Berkeley, California, USA

Glycolysis is central to energy metabolism in most organisms and is highly regulated to enable optimal growth. In the yeast , feedback mechanisms that control flux through glycolysis span transcriptional control to metabolite levels in the cell. Using a cellobiose consumption pathway, we decoupled glucose sensing from carbon utilization, revealing new modular layers of control that induce ATP consumption to drive rapid carbon fermentation. Alterations of the beta subunit of phosphofructokinase-1 (), H-plasma membrane ATPase (), and glucose sensors ( and ) revealed the importance of coupling extracellular glucose sensing to manage ATP levels in the cell. Controlling the upper bound of cellular ATP levels may be a general mechanism used to regulate energy levels in cells, via a regulatory network that can be uncoupled from ATP concentrations under perceived starvation conditions. Living cells are fine-tuned through evolution to thrive in their native environments. Genome alterations to create organisms for specific biotechnological applications may result in unexpected and undesired phenotypes. We used a minimal synthetic biological system in the yeast as a platform to reveal novel connections between carbon sensing, starvation conditions, and energy homeostasis.
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http://dx.doi.org/10.1128/mBio.00855-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5550752PMC
August 2017

An tag-and-modify protein sample generation method for single-molecule fluorescence resonance energy transfer.

J Biol Chem 2017 09 28;292(38):15636-15648. Epub 2017 Jul 28.

From the California Institute for Quantitative Biosciences and

Biomolecular systems exhibit many dynamic and biologically relevant properties, such as conformational fluctuations, multistep catalysis, transient interactions, folding, and allosteric structural transitions. These properties are challenging to detect and engineer using standard ensemble-based techniques. To address this drawback, single-molecule methods offer a way to access conformational distributions, transient states, and asynchronous dynamics inaccessible to these standard techniques. Fluorescence-based single-molecule approaches are parallelizable and compatible with multiplexed detection; to date, however, they have remained limited to serial screens of small protein libraries. This stems from the current absence of methods for generating either individual dual-labeled protein samples at high throughputs or protein libraries compatible with multiplexed screening platforms. Here, we demonstrate that by combining purified and reconstituted translation, quantitative unnatural amino acid incorporation via AUG codon reassignment, and copper-catalyzed azide-alkyne cycloaddition, we can overcome these challenges for target proteins that are, or can be, methionine-depleted. We present an parallelizable approach that does not require laborious target-specific purification to generate dual-labeled proteins and ribosome-nascent chain libraries suitable for single-molecule FRET-based conformational phenotyping. We demonstrate the power of this approach by tracking the effects of mutations, C-terminal extensions, and ribosomal tethering on the structure and stability of three protein model systems: barnase, spectrin, and T4 lysozyme. Importantly, dual-labeled ribosome-nascent chain libraries enable single-molecule co-localization of genotypes with phenotypes, are well suited for multiplexed single-molecule screening of protein libraries, and should enable the directed evolution of proteins with designer single-molecule conformational phenotypes of interest.
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http://dx.doi.org/10.1074/jbc.M117.791723DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5612098PMC
September 2017

Selective stalling of human translation through small-molecule engagement of the ribosome nascent chain.

PLoS Biol 2017 03 21;15(3):e2001882. Epub 2017 Mar 21.

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, United States of America.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a key role in regulating the levels of plasma low-density lipoprotein cholesterol (LDL-C). Here, we demonstrate that the compound PF-06446846 inhibits translation of PCSK9 by inducing the ribosome to stall around codon 34, mediated by the sequence of the nascent chain within the exit tunnel. We further show that PF-06446846 reduces plasma PCSK9 and total cholesterol levels in rats following oral dosing. Using ribosome profiling, we demonstrate that PF-06446846 is highly selective for the inhibition of PCSK9 translation. The mechanism of action employed by PF-06446846 reveals a previously unexpected tunability of the human ribosome that allows small molecules to specifically block translation of individual transcripts.
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http://dx.doi.org/10.1371/journal.pbio.2001882DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5360235PMC
March 2017

Monolayer-crystal streptavidin support films provide an internal standard of cryo-EM image quality.

J Struct Biol 2017 12 1;200(3):307-313. Epub 2017 Mar 1.

Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720, USA. Electronic address:

Analysis of images of biotinylated Escherichia coli 70S ribosome particles, bound to streptavidin affinity grids, demonstrates that the image-quality of particles can be predicted by the image-quality of the monolayer crystalline support film. The quality of the Thon rings is also a good predictor of the image-quality of particles, but only when images of the streptavidin crystals extend to relatively high resolution. When the estimated resolution of streptavidin was 5Å or worse, for example, the ribosomal density map obtained from 22,697 particles went to only 9.5Å, while the resolution of the map reached 4.0Å for the same number of particles, when the estimated resolution of streptavidin crystal was 4Å or better. It thus is easy to tell which images in a data set ought to be retained for further work, based on the highest resolution seen for Bragg peaks in the computed Fourier transforms of the streptavidin component. The refined density map obtained from 57,826 particles obtained in this way extended to 3.6Å, a marked improvement over the value of 3.9Å obtained previously from a subset of 52,433 particles obtained from the same initial data set of 101,213 particles after 3-D classification. These results are consistent with the hypothesis that interaction with the air-water interface can damage particles when the sample becomes too thin. Streptavidin monolayer crystals appear to provide a good indication of when that is the case.
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http://dx.doi.org/10.1016/j.jsb.2017.02.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5581310PMC
December 2017

Human eIF3: from 'blobology' to biological insight.

Authors:
Jamie H D Cate

Philos Trans R Soc Lond B Biol Sci 2017 03;372(1716)

Departments of Chemistry and Molecular and Cell Biology, University of California, Berkeley, CA 94720-3220, USA

Translation in eukaryotes is highly regulated during initiation, a process impacted by numerous readouts of a cell's state. There are many cases in which cellular messenger RNAs likely do not follow the canonical 'scanning' mechanism of translation initiation, but the molecular mechanisms underlying these pathways are still being uncovered. Some RNA viruses such as the hepatitis C virus use highly structured RNA elements termed internal ribosome entry sites (IRESs) that commandeer eukaryotic translation initiation, by using specific interactions with the general eukaryotic translation initiation factor eIF3. Here, I present evidence that, in addition to its general role in translation, eIF3 in humans and likely in all multicellular eukaryotes also acts as a translational activator or repressor by binding RNA structures in the 5'-untranslated regions of specific mRNAs, analogous to the role of the mediator complex in transcription. Furthermore, eIF3 in multicellular eukaryotes also harbours a 5' 7-methylguanosine cap-binding subunit-eIF3d-which replaces the general cap-binding initiation factor eIF4E in the translation of select mRNAs. Based on results from cell biological, biochemical and structural studies of eIF3, it is likely that human translation initiation proceeds through dozens of different molecular pathways, the vast majority of which remain to be explored.This article is part of the themed issue 'Perspectives on the ribosome'.
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http://dx.doi.org/10.1098/rstb.2016.0176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5311922PMC
March 2017

Quantitative determination of ribosome nascent chain stability.

Proc Natl Acad Sci U S A 2016 11 7;113(47):13402-13407. Epub 2016 Nov 7.

Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720;

Accurate protein folding is essential for proper cellular and organismal function. In the cell, protein folding is carefully regulated; changes in folding homeostasis (proteostasis) can disrupt many cellular processes and have been implicated in various neurodegenerative diseases and other pathologies. For many proteins, the initial folding process begins during translation while the protein is still tethered to the ribosome; however, most biophysical studies of a protein's energy landscape are carried out in isolation under idealized, dilute conditions and may not accurately report on the energy landscape in vivo. Thus, the energy landscape of ribosome nascent chains and the effect of the tethered ribosome on nascent chain folding remain unclear. Here we have developed a general assay for quantitatively measuring the folding stability of ribosome nascent chains, and find that the ribosome exerts a destabilizing effect on the polypeptide chain. This destabilization decreases as a function of the distance away from the peptidyl transferase center. Thus, the ribosome may add an additional layer of robustness to the protein-folding process by avoiding the formation of stable partially folded states before the protein has completely emerged from the ribosome.
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http://dx.doi.org/10.1073/pnas.1610272113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5127326PMC
November 2016

Internalization of Heterologous Sugar Transporters by Endogenous α-Arrestins in the Yeast Saccharomyces cerevisiae.

Appl Environ Microbiol 2016 12 21;82(24):7074-7085. Epub 2016 Nov 21.

Department of Molecular and Cell Biology, University of California, Berkeley, California, USA

When expressed in Saccharomyces cerevisiae using either of two constitutive yeast promoters (PGK1 and CCW12), the transporters CDT-1 and CDT-2 from the filamentous fungus Neurospora crassa are able to catalyze, respectively, active transport and facilitated diffusion of cellobiose (and, for CDT-2, also xylan and its derivatives). In S. cerevisiae, endogenous permeases are removed from the plasma membrane by clathrin-mediated endocytosis and are marked for internalization through ubiquitinylation catalyzed by Rsp5, a HECT class ubiquitin:protein ligase (E3). Recruitment of Rsp5 to specific targets is mediated by a 14-member family of endocytic adaptor proteins, termed α-arrestins. Here we demonstrate that CDT-1 and CDT-2 are subject to α-arrestin-mediated endocytosis, that four α-arrestins (Rod1, Rog3, Aly1, and Aly2) are primarily responsible for this internalization, that the presence of the transport substrate promotes transporter endocytosis, and that, at least for CDT-2, residues located in its C-terminal cytosolic domain are necessary for its efficient endocytosis. Both α-arrestin-deficient cells expressing CDT-2 and otherwise wild-type cells expressing CDT-2 mutants unresponsive to α-arrestin-driven internalization exhibit an increased level of plasma membrane-localized transporter compared to that of wild-type cells, and they grow, utilize the transport substrate, and generate ethanol anaerobically better than control cells.

Importance: Ethanolic fermentation of the breakdown products of plant biomass by budding yeast Saccharomyces cerevisiae remains an attractive biofuel source. To achieve this end, genes for heterologous sugar transporters and the requisite enzyme(s) for subsequent metabolism have been successfully expressed in this yeast. For one of the heterologous transporters examined in this study, we found that the amount of this protein residing in the plasma membrane was the rate-limiting factor for utilization of the cognate carbon source (cellobiose) and its conversion to ethanol.
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http://dx.doi.org/10.1128/AEM.02148-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5118918PMC
December 2016

Relief of Xylose Binding to Cellobiose Phosphorylase by a Single Distal Mutation.

ACS Synth Biol 2017 02 3;6(2):206-210. Epub 2016 Oct 3.

Department of Chemistry, University of California , Berkeley, California 94720, United States.

Cellobiose phosphorylase (CBP) cleaves cellobiose-abundant in plant biomass-to glucose and glucose 1-phosphate. However, the pentose sugar xylose, also abundant in plant biomass, acts as a mixed-inhibitor and a substrate for the reverse reaction, limiting the industrial potential of CBP. Preventing xylose, which lacks only a single hydroxymethyl group relative to glucose, from binding to the CBP active site poses a spatial challenge for protein engineering, since simple steric occlusion cannot be used to block xylose binding without also preventing glucose binding. Using CRISPR-based chromosomal library selection, we identified a distal mutation in CBP, Y47H, responsible for improved cellobiose consumption in the presence of xylose. In silico analysis suggests this mutation may alter the conformation of the cellobiose phosphorylase dimer complex to reduce xylose binding to the active site. These results may aid in engineering carbohydrate phosphorylases for improved specificity in biofuel production, and also in the production of industrially important oligosaccharides.
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http://dx.doi.org/10.1021/acssynbio.6b00211DOI Listing
February 2017