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The Ribosome – a Restless Molecular Machine

An ICON in Clinical Research

Isilon: Scapes Up Big Data Storage

DNA, RNA and proteins are among the most important macromolecules in a living cell. These molecules are polymerized by molecular machines. These natural nano-machines polymerize such macromolecules, adding one monomer at a time, using another linear polymer as the corresponding template. The machine utilizes input chemical energy to move along the template which also serves as a track for the movements of the machine. In the Alan Turing year 2012, it is worth pointing out that these machines are "tape-copying Turing machines". We review the operational mechanisms of the polymerizer machines and their collective behavior from the perspective of statistical physics, emphasizing their common features in spite of the crucial differences in their biological functions. We also draw the attention of the physics community to another class of modular machines that carry out a different type of template-directed polymerization. We hope this review will inspire new kinetic models for these modular machines.

Analysis of the Haloarcula marismortui large ribosomal subunit has revealed a common RNA structure that we call the kink-turn, or K-turn. The six K-turns in H.marismortui 23S rRNA superimpose with an r.m.s.d. of 1.7 Å. There are two K-turns in the structure of Thermus thermophilus 16S rRNA, and the structures of U4 snRNA and L30e mRNA fragments form K-turns. The structure has a kink in the phosphodiester backbone that causes a sharp turn in the RNA helix. Its asymmetric internal loop is flanked by C–G base pairs on one side and sheared G–A base pairs on the other, with an A-minor interaction between these two helical stems. A derived consensus secondary structure for the K-turn includes 10 consensus nucleotides out of 15, and predicts its presence in the 5′-UTR of L10 mRNA, helix 78 in Escherichia coli 23S rRNA and human RNase MRP. Five K-turns in 23S rRNA interact with nine proteins. While the observed K-turns interact with proteins of unrelated structures in different ways, they interact with L7Ae and two homologous proteins in the same way.

During the elongation cycle of protein biosynthesis, the specific amino acid coded for by the mRNA is delivered by a complex that is comprised of the cognate aminoacyl-tRNA, elongation factor Tu and GTP. As this ternary complex binds to the ribosome, the anticodon end of the tRNA reaches the decoding center in the 30S subunit. Here we present the cryo-electron microscopy (EM) study of an Escherichia coli 70S ribosome-bound ternary complex stalled with an antibiotic, kirromycin. In the cryo-EM map the anticodon arm of the tRNA presents a new conformation that appears to facilitate the initial codon–anticodon interaction. Furthermore, the elbow region of the tRNA is seen to contact the GTPase-associated center on the 50S subunit of the ribosome, suggesting an active role of the tRNA in the transmission of the signal prompting the GTP hydrolysis upon codon recognition.

In the elongation cycle of translation, translocation is the process that advances the mRNA–tRNA moiety on the ribosome, to allow the next codon to move into the decoding center. New results obtained by cryoelectron microscopy, interpreted in the light of x-ray structures and kinetic data, allow us to develop a model of the molecular events during translocation.

A number of image processing parameters in the 3D reconstruction of a ribosome complex from a cryo-EM data set were varied to test their effects on the final resolution. The parameters examined were pixel size, window size, and mode of Fourier amplitude enhancement at high spatial frequencies. In addition, the strategy of switching from large to small pixel size during angular refinement was explored. The relationship between resolution (in Fourier space) and the number of particles was observed to follow a lin-log dependence, a relationship that appears to hold for other data, as well. By optimizing the above parameters, and using a lin-log extrapolation to the full data set in the estimation of resolution from half-sets, we obtained a 3D map from 131,599 ribosome particles at 6.7 Å resolution (FSC = 0.5).

Cryo-EM analysis of a wild-type Escherichia coli pretranslocational sample has revealed the presence of previously unseen intermediate substates of the bacterial ribosome during the first phase of translocation, characterized by intermediate intersubunit rotations, L1 stalk positions, and tRNA configurations. Furthermore, we describe the domain rearrangements in quantitative terms, which has allowed us to characterize the processivity and coordination of the conformational reorganization of the ribosome, along with the associated changes in tRNA ribosome-binding configuration. The results are consistent with the view of the ribosome as a molecular machine employing Brownian motion to reach a functionally productive state via a series of substates with incremental changes in conformation.