Maggie Kessler

Ribosomal crystallography: a flexible nucleotide anchoring tRNA translocation, facilitates peptide-bond formation, chirality discrimination and antibiotics synergism

The linkage between internal ribosomal symmetry and transfer RNA (tRNA) positioning confirmed positional catalysis of amino‐acid polymerization. Peptide bonds are formed concurrently with tRNA‐3′end rotatory motion, in conjunction with the overall messenger RNA (mRNA)/tRNA translocation. Accurate substrate alignment, mandatory for the processivity of protein biosynthesis, is governed by remote interactions. Inherent flexibility of a conserved nucleotide, anchoring the rotatory motion, facilitates chirality discrimination and antibiotics synergism. Potential tRNA interactions explain the universality of the tRNA CCA‐end and P‐site preference of initial tRNA. The interactions of protein L2 tail with the symmetry‐related region periphery explain its conservation and its contributions to nascent chain elongation.

The identification of selected components in electron density maps of prokaryotic ribosomes at 7 Å resolution

Crystals of small and large ribosomal subunits from thermophilic and halophilic bacteria, diffracting to 3 A, are being subjected to structural analysis with synchrotron radiation. The bright beam necessary for detecting and collecting the diffraction at the higher-resolution shell causes significant decay even at 25 K. Nevertheless, data collected from native and heavy-atom-derivatized crystals led to the construction of electron density maps of both ribosomal subunits, showing recognizable morphologies and internal features similar to those observed by EM reconstructions of the corresponding ribosomal particle. The main features of these maps include elongated dense regions traceable as well separated RNA duplexes or single strands. Also seen are globular patches of lower density, readily distinguishable from the above, in which folds observed by NMR or crystallography in isolated ribosomal proteins at atomic resolution were detected. The intercomponents contacts identified so far reveal diverse modes of recognition. Metal clusters, attached at selected sites on the particles, are being exploited to facilitate unbiased map interpretation. In this way, two surface proteins were located and several surface RNA strands were targeted.

Ribosomal Crystallography and Heteropolytungstates

Heteropolytungstates play a dual role in ribosomal crystallography. Beside generating phases, one of them, (NH4)6(P2W18O62)14H2O, was found to be extremely useful in inducing post crystallization rearrangements. These led to a significant increase in the internal order of crystals of the small ribosomal subunits from Thermus thermophilus, manifested in a dramatic extension of the resolution of their diffraction patterns, from the initial 7–9 A to 3 A. The current 3.3 A electron density map of this particle, constructed using phases obtained from this W cluster together with other metal compounds, shows the recognizable overall morphology of the small ribosomal subunit. Over 96% of the nucleotides were traced and the fold of all proteins was determined fully or partially. Specific sites were determined independently by covalently bound heavy atom clusters, among them the surface of two proteins and a functional center, the gate for mRNA binding. All tungsten-cluster sites detected in this map are located in close proximity to the proteins of the particle, in positions that may have an influence on the stability and the rigidity of this rather flexible ribosomal subunit.