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Structures of the Ribosome in Intermediate States of Ratcheting Print

 

Protein synthesis is conducted by the ribosome: a megadalton sized complex responsible for making proteins from amino acids. Translation—the conversion of a three letter nucleic acid code (a codon) in a messenger RNA (mRNA) into an amino acid sequence—is essential to gene expression. For this reason, translational accuracy is imperative, and has a very low error rate of 10-3 to 10-4. Translation and other manipulations of ribosomal substrates must occur rapidly to match the speed of peptide bond formation, a process that links amino acids together to form a protein, which occurs at a rate of 20 bonds per second. One of these substrate manipulations is translocation, the movement of mRNA and transfer RNA (tRNA) through the functional sites of the ribosome making room for new substrates to enter. Using x-ray crystallography, Berkeley Lab and University of California, Berkeley researchers have solved structures of the ribosome that provide mechanistic insight into the process of mRNA/tRNA translocation. This research contributes to the understanding of how some antibiotics inhibit bacterial protein synthesis by interfering with translocation, and will hopefully aid in the design of new antibiotics in this class.

Antibiotics Target Ratcheting

Structural biologists have made significant progress during the past decade in understanding the processes of decoding DNA in the form of mRNA and of peptide bond formation. But questions concerning the last step of this cycle, tRNA/mRNA translocation, have remained unanswered. This research makes great strides toward understanding translocation, including the role of ribosome ratcheting. It also contributes to the understanding of how certain antibiotics target translocation and ratcheting mechanisms to kill infectious bacteria.

Spectinomycin is a treatment for penicillin-resistant N. gonorrhoeae infections. It inhibits movement of the ribosomal 30S head domain, a movement that is part of ratcheting, is necessary for translocation, and is ultimately required for protein synthesis. Capreomycin is an antibiotic used to treat tuberculosis. A functional equivalent to viomycin, capreomycin inhibits ribosome ratcheting and translocation, although exactly how it does this­—whether by inhibiting the transition into or out of the ratcheted state—is still controversial.

Though potent antibiotics, neither compound is an ideal drug. Understanding the biochemical and structural basis for their actions should help make them more effective and less toxic. Research may also aid in new drug design, taking advantage of bacteria’s vulnerability to protein synthesis inhibition by antibiotics through the translocation mechanism.

Overview of the structure of the prokaryotic ribosome. The 30S subunit is in blue, the 50S subunit is in grey and purple. The mRNA in the 30S channel is labeled, as well as the A-site tRNA. From this point of view 30S ratcheting occurs perpendicular to the plane of the paper.

Bacterial ribosomes are composed of ribosomal RNA and proteins, which form the 30S and 50S subunits. These two particles combine to form a 70S ribosome during the process of translating mRNA into amino acids. The 30S subunit of a translating 70S ribosome contains a channel through which the mRNA is threaded. Between the two subunits, a cavity accommodates tRNA molecules that bind mRNA base pairs on one end, and on the other end are covalently linked to whichever amino acid is coded for by the mRNA. These amino-acylated tRNAs can bind the ribosome at three sites, designated the A (aminoacyl), P (peptidyl) and E (exit) sites. The P site holds a tRNA covalently linked to a nascent peptide, while in the A site, the correct tRNA pairs with the mRNA, decoding the codon. A peptide bond is then formed linking the new amino acid into the growing protein chain, and the tRNA and mRNA translocate into the E site, making room for the next tRNA to enter the A site.

Translocation of mRNA and tRNA on the ribosome occurs through a "ratcheting" mechanism. Ratcheting is central to tRNA positioning within the ribosome and to translation. Domains of the 30S subunit perform a choreographed set of discrete movements, where first the body rotates, then the head domain swivels forward, and the subunit’s body and platform follow the rotation. The head then rotates backwards as internal contacts, or bridges, between the subunits approach their final ratcheted position.

Stepwise ratcheting of the 30S subunit. A cartoon view of the 30S subunit is shown (left) from the point of view of the subunit interface. Next (from left to right), the stepwise changes that occur as the ribosome adopts the ratcheted conformation are shown as difference vectors between either the Cα or Phosphate atoms in the protein or RNA portion of the molecule.

X-ray crystallography done at ALS Beamlines 12.3.1 and 8.3.1 revealed the structures of intermediate states of intersubunit rotation, and provided a better understanding of the implications of these conformations for the ribosomes' substrates. The intermediates observed provided insight into how tRNAs can assume partially translocated, or hybrid, states of binding preceding the final steps of translocation.

The structures were solved at atomic resolution, enabling researchers to observe the precise changes in the intersubunit bridges that occur during ratcheting. For example, the N-terminus of ribosomal protein S13 contacts protein L5 in the un-ratcheted state of the ribosome, but is moved by 20 Å during ratcheting.

Interactions between two proteins in the ribosome (L5 and S13) are shown before ratcheting (blue) and during ratcheting (red). Movement of the 30S subunit of the ribosome between the ratcheted and un-ratcheted state is inhibited by spectinomycin and capreomycin. An arrowhead in the inset (upper left) reveals the direction of view.

Furthermore, the observed motions of the 30S head domain (a forward swivel of 11°, followed by a backwards swivel of 6° to reach the fully ratcheted state) suggest that during translocation tRNA can occupy hybrid states before it reaches the fully ratcheted state. This explains previous research showing that the formation of hybrid states of tRNA binding can happen at a faster rate than ribosome ratcheting.

 


 

Research conducted by W. Zhang and J.A. Dunkle (University of California, Berkeley) and J.H.D. Cate (University of California, Berkeley, and Berkeley Lab).

Research funding: National Institutes of Heath and U.S. Department of Energy, Office of Biological and Environmental Research. Operation of the ALS is supported by the U.S. Department of Energy, Office of Basic Energy Sciences.

Publication about this research: W. Zhang, J.A. Dunkle, and J.H.D. Cate, "Structures of the Ribosome in Intermediate States of Ratcheting," Science 325, 1014 (2009).

 

ALSNews Vol. 307