Assembly of bacterial ribosomes.

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.

Quantitative proteomic analysis of ribosome assembly and turnover in vivo.

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 Escherichia coli.

Quantitation of the ribosomal protein autoregulatory network using mass spectrometry.

Relative levels of ribosomal proteins were quantified in crude cell lysates using mass spectrometry. A method for quantifying cellular protein levels using macromolecular standards is presented that does not require complex sample separation, identification of high-responding peptides, affinity purification, or postgrowth modifications. Perturbations in ribosomal protein levels by overexpression of individual proteins correlate to known autoregulatory mechanisms and extend the network of ribosomal protein regulation.

A dominant negative mutant of the E. coli RNA helicase DbpA blocks assembly of the 50S ribosomal subunit.

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.

A complex assembly landscape for the 30S ribosomal subunit.

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.

Envelope: interactive software for modeling and fitting complex isotope distributions.

Background: An important aspect of proteomic mass spectrometry involves quantifying and interpreting the isotope distributions arising from mixtures of macromolecules with different isotope labeling patterns. These patterns can be quite complex, in particular with in vivo metabolic labeling experiments producing fractional atomic labeling or fractional residue labeling of peptides or other macromolecules. In general, it can be difficult to distinguish the contributions of species with different labeling patterns to an experimental spectrum and difficult to calculate a theoretical isotope distribution to fit such data.

Quantitative analysis of isotope distributions in proteomic mass spectrometry using least-squares Fourier transform convolution.

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.

Simulations of RNA base pairs in a nanodroplet reveal solvation-dependent stability.

We show that RNA base pairs have variable stability depending on their degree of solvation. This finding has far-reaching biological implications for nucleic acid structure in a partially solvated cellular environment such as inside RNA-protein complexes. Molecular dynamics simulations of partially solvated Watson-Crick RNA base pairs show that whereas water serves to destabilize a base pair by competing for and disrupting base-base hydrogen bonds, when sufficient water molecules are present, fewer hydrogen bonds are available to disrupt the base pairs and the destabilization effect is reduced.

Defined DNA/nanoparticle conjugates.

Glutathione monolayer-protected gold clusters were reacted by place exchange with 19- or 20-residue thiolated oligonucleotides. The resulting DNA/nanoparticle 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 DNA/nanoparticle synthesis and yields conjugates that are precisely defined with respect to both gold and nucleic acid content.

Describing RNA structure by libraries of clustered nucleotide doublets.

The rapidly increasing wealth of structural information on RNA and knowledge of its varying roles in biology have facilitated the study of RNA structure using computational methods. Here, we present a new method to describe RNA structure based on nucleotide doublets, where a doublet is any two nucleotides in a structure. We restrict our search to doublets that are close together in space, but not necessarily in sequence, and obtain doublet libraries of various sizes by clustering a large set of doublets taken from a data set of high-resolution RNA structures.