Some biologists have been using the ribosome's structure as a jumping-off point to examine what happens to newly born protein chains when they emerge from the ribosome. Good health depends on proteins getting where they're supposed to go, but no one knew precisely how new protein chains make their way across various cell membranes—or how some of these chains become lodged inside the membranes. In 2004, cell biologist Tom A. Rapoport and structural biologist Stephen Harrison, both HHMI investigators at Harvard Medical School, led a team that solved the atomic structure of a surprisingly narrow membrane channel that new proteins must squeeze through, as through a birth canal. Docking next to this channel, the ribosome extrudes limp nascent protein chains right into the channel opening and pushes them in. Once on the other side of the membrane, the chain folds into its active shape and gets to work.

The channel turned out to have a very tricky structure—it is shaped in part like a clamshell that opens and shuts, allowing a number of proteins to cross the membrane while holding back millions of others. It also directs some proteins sideways, to positions inside the membrane.

"The system is extremely ancient," Rapoport explains. Every bacterial or mammalian cell that has an outer membrane or some internal compartments must be able to transport proteins across these membranes to their destination as well as find ways to respond to its environment. Proteins such as insulin need to exit from the cell and travel to other parts of the body. All cells need certain proteins to be embedded in their membranes, to act as receptors for signals from other cells. The membrane channels that conduct such proteins in different species are amazingly similar. If they malfunction—if essential proteins are misdirected, misfolded, or destroyed—a variety of diseases can result.

Meanwhile, scientists have been pursuing the 3-D structures of several other "large molecular machines that control the birth, growth, and death of proteins," says Kuriyan. Some researchers are even studying machines as dynamic as the spliceosome, a structure in the cell nucleus that is put together very loosely from different components that keep shifting location as the machine does its work. The spliceosome acts on RNA molecules that are copied from genes; its job is to excise any noncoding intervening sequences (introns) from mRNA and stitch together coding (exon) sequences to make "mature" mRNA that is then translated to proteins encoded by the gene.

At Brandeis University, HHMI investigators Melissa J. Moore and Nikolaus Grigorieff are collaborating in an effort to map the spliceosome's structure, and the scope of the challenge is clear. The spliceosome must be exceedingly precise while splicing out introns, because a mistake that shifts even one nucleotide in a splice site will throw the entire gene-coding region "out of frame" and produce possibly dangerous mutations. Splicing errors are the basic cause of genetic diseases such as retinitis pigmentosa, some forms of dementia, cystic fibrosis, spinal muscular atrophy, and cancer. To prevent such outcomes in humans, the spliceosome must accurately identify more than 100,000 introns in diverse sequences of RNA.

Using electron microscopy, the scientists obtained a low-resolution structure of the spliceosome that showed several asymmetric sections forming an unusually large number of tunnels and bridges—an intriguing start. They could not use x-ray crystallography for these studies, Grigorieff explains, because there are relatively few spliceosomes in a cell nucleus—far too few to grow into a crystal—and because "for crystallization, all spliceosomes would have to assume essentially the same shape." Nevertheless, he says, "We have collected and averaged thousands of images of spliceosomes, which should give us a detailed structure at a higher resolution."

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