|Structures of the Ribosome in Intermediate States of Ratcheting|
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.
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.
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.
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).