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 functions of most RNA molecules are critically dependent on the distinct local dynamics that characterize secondary structure and tertiary interactions and on structural changes that occur upon binding by proteins and small molecule ligands. Measurements of RNA dynamics at nucleotide resolution set the foundation for understanding the roles of individual residues in folding, catalysis, and ligand recognition. In favorable cases, local order in small RNAs can be quantitatively analyzed by NMR in terms of a generalized order parameter, S2. Alternatively, SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) chemistry measures local nucleotide flexibility in RNAs of any size using structure-sensitive reagents that acylate the 2'-hydroxyl position. In this work, we compare per-residue RNA dynamics, analyzed by both S2 and SHAPE, for three RNAs: the HIV-1 TAR element, the U1A protein binding site, and the Tetrahymena telomerase stem loop 4. We find a very strong correlation between the two measurements: nucleotides with high SHAPE reactivities consistently have low S2 values. We conclude that SHAPE chemistry quantitatively reports local nucleotide dynamics and can be used with confidence to analyze dynamics in large RNAs, RNA-protein complexes, and RNAs in vivo.

Detailed studies of ribosomal proteins, essential components of the protein biosynthetic machinery, have been hampered by the lack of readily accessible chromosomal deletions of the corresponding genes. Here, we report the systematic genomic deletion of 41 individual ribosomal protein genes in Escherichia coli, which are not included in the Keio collection. Chromosomal copies of these genes were replaced by an antibiotic resistance gene in the presence of an inducible, easy-to-exchange plasmid-born allele. Using this knockout collection, 9 ribosomal proteins (L15, L21, L24, L27, L29, L30, L34, S9 and S17) were found nonessential for survival under induction conditions at various temperatures. Taken together with previous results, this analysis revealed that 22 of the 54 E. coli ribosomal protein genes can be individually deleted from the genome. These strains also allow expression of truncated protein variants to probe the importance of RNA-protein interactions in functional sites of the ribosome. This set of strains should enhance in vivo studies of ribosome assembly/function, and may ultimately allow systematic substitution of ribosomal proteins with RNA.

The HIV-1 TAR RNA represents a well-known paradigm to study the role of dynamics and conformational change in RNA function. This regulatory RNA changes conformation in response to binding of Tat protein and of a variety of peptidic and small molecule ligands, indicating that its conformational flexibility and intrinsic dynamics play important roles in molecular recognition. We have used 13C NMR relaxation experiments to examine changes in the motional landscape of HIV-1 TAR in the presence of three ligands of different affinity and specificity. The ligands are argininamide, a linear peptide mimic of the Tat basic domain and a cyclic peptide that potently inhibits Tat-dependent activation of transcription. All three molecules induce the same motional characteristics within the three nucleotides bulge that represents the Tat-binding site. However, the cyclic peptide has a unique motional signature in the apical loop, which represents a binding site for the essential host co-factor cyclin T1. These results suggest that all peptidic mimics of Tat induce the same dynamics in TAR within this protein binding site. However, the new cyclic peptide mimic of Tat represents a new class of ligands with a unique effect on the dynamics and the structure of the apical loop.