The assembly of ribosomes from a discrete set of components is a key aspect of the highly coordinated process of ribosome biogenesis. In this review, we present a brief history of the early work on ribosome assembly in Escherichia coli, including a description of in vivo and in vitro intermediates. The assembly process is believed to progress through an alternating series of RNA conformational changes and protein-binding events; we explore the effects of ribosomal proteins in driving these events. Ribosome assembly in vivo proceeds much faster than in vitro, and we outline the contributions of several of the assembly cofactors involved, including Era, RbfA, RimJ, RimM, RimP, and RsgA, which associate with the 30S subunit, and CsdA, DbpA, Der, and SrmB, which associate with the 50S subunit.

The deuterium-labeled standards [(2)H(3)]-guaiacol and [(2)H(3)]-4-methylguaiacol were synthesized and utilized in a method employing gas chromatography-mass spectrometry to determine the concentration of guaiacol and 4-methylguaiacol in wine or extracts of oak shavings. The method was combined with previously published methods for 4-ethylphenol, 4-ethylguaiacol, cis- and trans-oak lactone and vanillin, so that all these compounds could be quantified in a single analysis. The method can employ either liquid-liquid extraction or headspace solid-phase microextraction (SPME) and is rapid, robust, precise, and accurate. Under certain conditions, there was artifactual generation, to varying degrees, of guaiacol, 4-methylguaiacol, cis-oak lactone, and vanillin during the analysis of oak extracts, especially when diethyl ether extraction and injector block temperatures at or above 225 degrees C were employed. The most substantial effects were observed for guaiacol, in which results could be exaggerated by over 10 times. These artifacts could be avoided by using headspace SPME or by preparing liquid-liquid extracts with pentane or pentane/diethyl ether (2:1) injected at 200 degrees C providing spot checks using headspace SPME were performed. Data obtained for previously published quantitative determination of guaiacol in oak extracts should be reexamined carefully, with special attention paid to their respective methods of sample preparation and analysis.

The ribosome is a complex macromolecular machine responsible for protein synthesis in the cell. It consists of two subunits, each of which contains both RNA and protein components. Ribosome assembly is subject to intricate regulatory control and is aided by a multitude of assembly factors in vivo, but can also be carried out in vitro. The details of the assembly process remain unknown even in the face of atomic structures of the entire ribosome and after more than three decades of research. Some of the earliest research on ribosome assembly produced the Nomura assembly map of the small subunit, revealing a hierarchy of protein binding dependencies for the 20 proteins involved and suggesting the possibility of a single intermediate. Recent work using a combination of RNA footprinting and pulse-chase quantitative mass spectrometry paints a picture of small subunit assembly as a dynamic and varied landscape, with sequential and hierarchical RNA folding and protein binding events finally converging on complete subunits. Proteins generally lock tightly into place in a 5′ to 3′ direction along the ribosomal RNA, stabilizing transient RNA conformations, while RNA folding and the early stages of protein binding are initiated from multiple locations along the length of the RNA.

Quantitative proteomic mass spectrometry involves comparison of the amplitudes of peaks resulting from different isotope labeling patterns, including fractional atomic labeling and fractional residue labeling. We have developed a general and flexible analytical treatment of the complex isotope distributions that arise in these experiments, using Fourier transform convolution to calculate labeled isotope distributions and least-squares for quantitative comparison with experimental peaks. The degree of fractional atomic and fractional residue labeling can be determined from experimental peaks at the same time as the integrated intensity of all of the isotopomers in the isotope distribution. The approach is illustrated using data with fractional 15 N-labeling and fractional 13 C-isoleucine labeling. The least-squares Fourier transform convolution approach can be applied to many types of quantitive proteomic data, including data from stable isotope labeling by amino acids in cell culture and pulse labeling experiments.Stable isotope labeling in cells coupled with mass spectrometry has many important applications in the analysis of protein expression, modification, turnover, and metabolism. 1 Cells and organisms can be isotopically labeled by supplying labeled precursors in the form of nutrients such as amino acids, glucose, and ammonia. These labeled precursors are incorporated into proteins, whose resulting isotope labeling patterns reflect their abundance and the dynamics of protein synthesis and turnover. The era of quantitative proteomics began with experiments where mixtures of unlabeled control cells and 15 N-labeled or isotope depleted test cells were quantitatively analyzed to determine protein expression and phosphorylation levels. 2,3 More recently, the SILAC technique was developed based on specific amino acid labeling of cells for quantitative analysis of protein expression, modification, and turnover. 4-6The two basic classes of quantitative metabolic labeling approaches are the stable isotope labeling by amino acids in cell culture (SILAC) experiments and pulse labeling experiments, both of which have been implemented in a variety of ways. 7 In the SILAC method, independent samples are prepared with different labeling patterns that are mixed prior to mass spectrometry analysis. For pulse labeling experiments, labels that are added to the growth medium are taken up to metabolically label the proteome, which generates fractionally enriched proteins from a

Escherichia coli DbpA is an ATP-dependent RNA helicase with specificity for hairpin 92 of 23S ribosomal RNA, an important part of the peptidyl transferase center. The R331A active site mutant of DbpA confers a dominant slow growth and cold sensitive phenotype when overexpressed in E. coli containing endogenous DbpA. Ribosome profiles from cells overexpressing DbpA R331A display increased levels of 50S and 30S subunits and decreased levels 70S ribosomes. Profiles run at low Mg2+ exhibit fewer 50S subunits and accumulate a 45S particle that contains incompletely processed and undermodified 23S rRNA in addition to reduced levels of several ribosomal proteins that bind late in the assembly pathway. Unlike mature 50S subunits, these 45S particles can stimulate the ATPase activity of DbpA, indicating that hairpin 92 has not yet been sequestered within the 50S subunit. Overexpression of the inactive DbpA R331A mutant appears to block assembly at a late stage when the peptidyl transferase center is formed, indicating a possible role for DbpA promoting this conformational change.

Glutathione monolayer-protected gold clusters were reacted by place exchange with 19-or 20-residue thiolated oligonucleotides. The resulting DNA͞nanoparticle conjugates could be separated on the basis of the number of bound oligonucleotides by gel electrophoresis and assembled with one another by DNA-DNA hybridization. This approach overcomes previous limitations of DNA͞ nanoparticle synthesis and yields conjugates that are precisely defined with respect to both gold and nucleic acid content.DNA-DNA hybridization ͉ monolayer-protected gold clusters ͉ nanostructure D NA-DNA hybridization has been exploited in the assembly of nanostructures (1-3), including nanomechanical (4, 5) and nanoelectronic (6) devices, molecular computation devices (7), biosensors, and DNA scaffolds (8). Many of these applications involve the use of DNA oligonucleotides tethered to gold nanoparticles. Additional oligonucleotides may be hybridized to these DNA͞nanoparticle conjugates, or nanoparticles may be hybridized with one another.Two types of DNA͞nanoparticle conjugates have been developed for these purposes. Both types entail the coupling of oligonucleotides through terminal thiol groups to colloidal gold particles. In one case, the oligonucleotides formed the entire monolayer coating the particles (3), whereas in the other case, the oligonucleotides were incorporated in a phosphine monolayer, and particles containing discrete numbers of oligonucleotides were separated by gel electrophoresis (2, 9). A minimal length of Ϸ50 residues was required, both for separation by electrophoresis and hybridization with complementary DNA sequences. These limitations of shorter oligonucleotides were attributed to interaction between the DNA and the gold surface (10, 11), with the DNA ''intrinsically bent'' (10) and wrapped around a colloidal particle (11), and with greatest affinity for C and G residues and least for A and T residues (12). Materials and MethodsOligonucleotides, synthesized by MWG Biotech, High Point, NC (www.mwgbiotech.com), were as follows: 20 residues, 5ЈSH-ACAACTTTCAACAGTCTAAC-3Ј; 19 residues, 5Ј-AGGC-CGCACCTAGGACGGT-3ЈSH; and 39 residues, complementary to 19 and 20 residues, TGTTGAAAGTTGTCAGATT-GTCCGGCGTGGATCCTGCCA. Glutathione monolayerprotected clusters (MPCs) were synthesized as described (13). Band 3, with a core of average mass 9 kDa, corresponding to 46 gold atoms, and diameter of 1.2 nm, was used. Oligonucleotides (10 l of 500 M) were reduced by treatment with 1 l of 50 mM Tris(2-caroxyethyl)phosphine (Sigma 646547) for 30 min at room temperature. Glutathione MPC (10 l of 1 mM) was added, followed by incubation for 1 h at 50°C.All gels were 15T͞5C acrylamide in TBE, run at a constant 100 V.A model of double-helical DNA with the sequence used here was generated with the use of NUCLEIC ACID BUILDER (14). The C6 5Ј-thiol and C3 3Ј-thiol linker regions were added to the models with PYMOL (15). Gold clusters were approximated as spheres. The DNA models were allowed to rotate about the axis of the thiol bond and attached to th...

The concentrations of the important oak aroma volatiles guaiacol, 4‐methylguaiacol cis‐ and trans‐oak lactone and vanillin in extracts of French and American oak heated under various conditions were measured using stable isotope dilution analyses coupled with gas chromatography‐mass spectrometry. Heating resulted in marked increases in the concentrations of guaiacol, 4‐methylguaiacol and vanillin, with more formed at higher temperature. Approximately twice as much guaiacol and 4‐methylguaiacol and two to five times as much vanillin were formed by heating in the presence of air compared to heating under argon. Oak lactone concentration was less affected by heating. The effects of heating different sized oak pieces were investigated for French and American oak samples. Compared to heating larger oak pieces, heating smaller fragments of oak generated up to twice as much guaiacol and 4‐methylguaiacol and two to four times as much vanillin at 235oC for both French and American oak. This effect is ascribed to the exposure of a greater surface area of oak to air when smaller fragments are heated. Variable effects were observed for cis‐ and trans‐oak lactone. Variation in chip size, as well as heating time and temperature, is clearly one way of obtaining different aroma profiles from oak products.

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.