Ribosomes are self-assembling macromolecular machines that translate DNA into proteins, and an understanding of ribosome biogenesis is central to cellular physiology. Previous studies on the E. coli 30S subunit suggest that ribosome assembly occurs via multiple, parallel pathways rather than through a single rate-limiting step, but little mechanistic information is known about this process. Discovery Single-particle Profiling (DSP), an application of time resolved electron microscopy, was used to obtain over 1 million snapshots of assembling 30S subunits, identify and visualize the structures of 14 assembly intermediates, and monitor the population flux of these intermediates over time. DSP results were integrated with mass spectrometry data to construct the first ribosome assembly mechanism that incorporates binding dependencies, rate constants, and structural characterization of populated intermediates.

The Escherichia coli 30S ribosomal subunit self-assembles in vitro in a hierarchical manner, with the RNA binding by proteins enabled by the prior binding of others under equilibrium conditions. Early 16S rRNA binding proteins also bind faster than late-binding proteins, but the specific causes for the slow binding of late proteins remain unclear. Previously, a pulse-chase monitored by quantitative mass spectrometry method was developed for monitoring 30S subunit assembly kinetics, and here a modified experimental scheme was used to probe kinetic cooperativity by including a step where subsets of ribosomal proteins bind and initiate assembly prior to the pulse-chase kinetics. In this work, 30S ribosomal subunit kinetic reconstitution experiments revealed that thermodynamic dependency does not always correlate with kinetic cooperativity. Some folding transitions that cause subsequent protein binding to be more energetically favorable do not result in faster protein binding. Although 3 0 domain primary protein S7 is required for RNA binding by both proteins S9 and S19, prior binding of S7 accelerates the binding of S9, but not S19, indicating there is an additional mechanistic step required for S19 to bind. Such data on kinetic cooperativity and the presence of multiphasic assembly kinetics reveal complexity in the assembly landscape that was previously hidden. mass spectrometry | RNA folding | RNA-protein interactions R ibosome biogenesis is a central cellular program that accounts for a significant fraction of the energy budget for rapidly growing bacteria, and is an essential process in all living cells. In eukaryotes, ribosome biogenesis in the nucleus requires hundreds of proteins (1, 2), whereas in bacteria the cytoplasmic assembly of ribosomes is facilitated by approximately 20 cofactors (3). Remarkably, the Escherichia coli 30S (4) and 50S (5) ribosomal subunits can be reconstituted in vitro, which has facilitated mechanistic studies on ribosome assembly. The 30S ribosomal subunit is a 900 kDa complex composed of 20 ribosomal proteins (r-proteins; S2, S3, ... S21) and a 1500-nucleotide rRNA (16S RNA). The 30S subunit is a well-characterized model system for studying macromolecular self-assembly in vitro (4), where protein binding occurs in a defined hierarchy and in a cooperative manner (6). The protein binding hierarchy was determined by a series of equilibrium reconstitution experiments that are summarized in the Nomura assembly map (6) (Fig. 1A). Primary proteins bind directly and independently to the 16S RNA, whereas secondary and tertiary proteins require prior binding of one or more proteins, respectively. The thermodynamic order of protein binding is generally consistent with kinetic binding data that show primary proteins binding fastest and tertiary proteins binding slowest (7,8). The assembly mechanism is also organized according to three structural domains, the 5 0 domain, the central domain, and the 3 0 domain (9), that can be reconstituted independently in vitro (10-12). Kinetic reconstitution...

Development of early embryonic stages before activation of the embryonic genome depends on sufficiently stored products of the maternal genome, adequate recruitment and degradation of mRNAs, as well as activation, deactivation, and relocation of proteins. By application of an isobaric tagging for relative and absolute quantification (iTRAQ)-based approach, the proteomes of bovine embryos at the zygote and 2-cell and 4-cell stage with MII oocytes as a reference were quantitatively analyzed. Of 1072 quantified proteins, 87 differed significantly in abundance between the four stages. The proteomes of 2-cell and 4-cell embryos differed most from the reference MII oocyte, and a considerable fraction of proteins continuously increased in abundance during the stages analyzed, despite a strongly attenuated rate of translation reported for this period. Bioinformatic analysis revealed particularly interesting proteins involved in the p53 pathway, lipid metabolism, and mitosis. Verification of iTRAQ results by targeted SRM (selected reaction monitoring) analysis revealed excellent agreement for all five proteins analyzed. By principal component analysis, SRM quantifications comprising a panel of only five proteins were shown to discriminate between all four developmental stages analyzed here. For future experiments, an expanded SRM protein panel will provide the potential to detect developmental disturbances with high sensitivity and enable first insights into the underlying molecular pathways.

Although high-resolution structures of the ribosome have been solved in a series of functional states, relatively little is known about how the ribosome assembles, particularly in vivo. Here, a general method is presented for studying the dynamics of ribosome assembly and ribosomal assembly intermediates. Since significant quantities of assembly intermediates are not present under normal growth conditions, the antibiotic neomycin is used to perturb wild type E. coli. Treatment of E. coli with the antibiotic neomycin results in the accumulation of a continuum of assembly intermediates for both the 30S and 50S subunits. The protein composition and the protein stoichiometry of these intermediates were determined by quantitative mass spectrometry using purified unlabeled and 15N-labeled wild type ribosomes as external standards. The intermediates throughout the continuum are heterogeneous and are largely depleted of late-binding proteins. Pulse labeling with 15N-labeled medium timestamps the ribosomal proteins based on their time of synthesis. The assembly intermediates contain both newly synthesized proteins and proteins that originated in previously synthesized intact subunits. This observation requires either a significant amount of ribosome degradation, or the exchange or reuse of ribosomal proteins. These specific methods can be applied to any system where ribosomal assembly intermediates accumulate, including strains with deletions or mutations of assembly factors. This general approach can be applied to study the dynamics of assembly and turnover of other macromolecular complexes that can be isolated from cells.

Embryonic development is initially controlled by maternal RNAs and proteins stored in the oocyte, until gene products gradually generated by the embryo itself take over. Major embryonic genome activation (EGA) in bovine embryos occurs at the eight- to 16-cell stage. Morphological observations, such as size of blastomeres and distribution of microvilli, suggested heterogeneity among individual cells already at this developmental stage. To address cell heterogeneity on the transcriptome level, we performed single-cell RNA sequencing of 161 blastomeres from 14 in vitro produced bovine embryos at Day 2 (n = 6) and Day 3 (n = 8) post fertilization. Complementary DNA libraries were prepared using the Single-Cell RNA-Barcoding and Sequencing protocol and sequenced. Non-supervised clustering of single-cell transcriptome profiles identified six clusters with specific sets of genes. Most embryos were comprised of cells from at least two different clusters. Sorting cells according to their transcriptome profiles resulted in a non-branched pseudo-time line, arguing against major lineage inclination events at this developmental stage. In summary, our study revealed heterogeneity of transcriptome profiles among single cells in bovine Day 2 and Day 3 embryos, suggesting asynchronous blastomere development during the phase of major EGA.

Uridine phosphates may be obtained by heating uridine with inorganic phosphates for 9 months at temperatures as low as 65 degrees C. Under similar conditions, uridine-5'-phosphate, in addition to forming uridine diphosphates, undergoes some dephosphorylation to give uridine.

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Shoot tip explants, callus and embryogenic cell suspensions of Euphorbia pulcherrima have been examined for quantitative variation in nuclear DNA content by means of cytophotometry. Increasing instability was found in calli and cell suspensions from Erlenmeyer flask and bioreactor culture. Nuclear DNA content ranged from 2C up to 32 C. Plants regenerated from embryogenic cell suspensions, however, were highly uniform with regard to phenotype and ploidy level indicating strongly impaired embryogenic potential of polyploid, aneuploid or other genetically altered cells.