Birth of a Protein
This color-coded cryo-EM map of the E. coli ribosome (25 angstrom resolution) shows the interface between the small (30S) and large (50S) ribosomal subunits. The genetic message of the incoming messenger-RNA (mRNA, orange) is composed of successive three-base sequences (codons). As protein translation proceeds, each succeeding codon selects for the appropriate transfer-RNA (tRNA). One end of the tRNA (the anticodon site) binds to the mRNA codon. The other end (the acceptor site) carries the amino-acid that corresponds to the codon. As an incoming tRNA (purple) enters the ribosome at the A site, the amino-acid at its acceptor site bonds to the amino-acid of the preceding tRNA (green), which has moved to the P site, elongating the polypeptide chain (multi-colored beads) by one amino-acid. The nascent protein exits the ribosome through a tunnel in the large subunit.
Image courtesy of J. Frank.
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Producing proteins, made to the order of our DNA, is the fundamental, bottom-line business of the cell. Biologists call this process protein translation, and ribosomes are the assembly-lines where it happens. Raw materials enter (amino-acids carried by transfer-RNA), assembly plans (messenger-RNA) are deciphered and followed, and new protein chains get put together, one amino-acid at a time, until the assembly instructions say stop and the new protein rolls out the door.
Improved knowledge of protein translation gained from Frank's detailed pictures is certain to payoff in many ways impossible to predict. One promising avenue for applying this research is bacterial resistance to antibiotics, a current, pressing public-health concern. Some of the most potent, useful antibiotics interfere with the ribosome of bacterial cells, killing the germs by shutting down their protein factories. Over time, the bacterial DNA responds, mutating to change its ribosome structure so that the antibiotic is blocked or bypassed, creating a resistant strain.
"Before you have a detailed understanding of how the whole translation process works," explains Frank, "the fight against drug resistance is somewhat of a random approach. If you have detailed understanding, you know exactly what you can do -- how to develop a drug, for instance, that provides multiple interference with ribosomal function, so that a single step of circumvention by the ribosome could not by itself produce drug resistance."
Other research groups are using crystallographic and NMR methods to determine the molecular structure of ribosomal proteins, individual components of the ribosome composite. The larger-scale work of Frank's group will eventually intersect with these atomic-level efforts. The Wadsworth team has completed a 15 angstrom ribosome reconstruction and is now working toward 12 angstrom resolution. When they reach about 10 angstroms, says Frank, the EM image will begin to show individual protein helices. "At that level, it becomes possible to fit existing x-ray structures into our EM map, and we'll start to build an atomic-level map with EM as a framework."
Until atomic-level resolution is achieved, Frank's team will continue to push toward finer detail, better knowledge. His first published ribosome reconstruction came from 4,300 separate particles. In current work, relying on NCSA's computational resources, he's using tens of thousands. A related goal involves using the visualization resources of the Alliance to depict the translation process as a time sequence: computer-generated movies can show the ribosome up close in 3D from multiple angles, with start-stop motion in millisecond time-steps -- a guided tour of the protein factory at work.
References:
Roland Beckmann, Doryen Bubeck, Robert Grassucci, Pawel Penczek, Adriana Verschoor, Günter Blobel, Joachim Frank, "Alignment of Conduits for the Nascent Polypeptide Chain in the Ribosome-Sec61 Complex," Science 278, 2123-26 (1997).
Rajendra K. Agrawal, Pawel Penczek, Robert A Grassucci, Yanhong Li, ArDean Leith, Knud H. Nierhaus, Joachim Frank, "Direct Visualization of A-, P-, and E-Site Transfer RNAs in the Escherichia coli Ribosome," Science 271, 1000-1002 (1996).
Joachim Frank, Jun Zhu, Pawel Penczek, Yanhon Li, Suman Srivastava, Adriana Verschoor, Michael Radermacher, Robert Grassucci, Ramani K. Lata & Rajendra K. Agrawal, "A model of protein synthesis based on cryo-electron microscopy of the E. coli ribosome, Nature 376, 441-44 (1995).
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