Research Notes

Introducing RNA Polymerase The long-sought crystal structure of RNA polymerase II, aka PolII, reveals a complex and tenacious machine. As the linchpin in gene expression, that machine "is arguably the most important protein in biology," says Roger Kornberg, professor of structural biology at Stanford University School of Medicine, who led the two-decade-long effort (P. Cramer et al., "Architecture of RNA polymerase II and implications for the transcription mechanism," Science, 288:640-9, April 28, 2000). Complexed with precisely bound other proteins, PolII orchestrates the differential accessing of genetic information that guides development. "The entire transcription machine is almost 60 proteins. All with very minor exceptions have been identified across species, from yeast to humans," he adds. The visualized enzyme is from yeast. PolII is a monster among molecules; however, its fragility and scarcity in cells impeded crystallization. It proved possible only in a deletion mutant that lacks two of the 12 protein subunits, but can still transcribe DNA. The researchers obtained a preliminary "fuzzy" structure using a technique that Kornberg codeveloped, called 2-D crystallography, which crystallizes sheets of peptides against lipid monolayers. Reconstruction from electron micrographs of the 2-D crystals yielded a 3-D snapshot of 15 Å resolution, which they improved to 3.5 Å by working in an oxygen-free environment. The high-resolution structure revealed the functional topography of PolII. The catalytic site, where RNA trinucleotide phosphates are polymerized, is a cleft separating two large subunits that can hold about 20 bases of template DNA. Incoming DNA is threaded through two grooves, then is held in "jaws" formed from parts of three protein subunits. A "sliding clamp" provides a hinge, holding the DNA in place while allowing it to move through the cleft. Finally, a "pore" beneath the catalytic site allows RNA bases in and transcripts out. The researchers are already figuring out how the associated protein entourage binds. "We can use information from one crystal structure to solve others, so we don't have to go back to square one. We already have preliminary information on six other proteins," Kornberg adds. --Ricki Lewis Retaining Rare Genes As genetics continues to morph into genomics, a challenge is emerging: losing evidence of genes that are expressed fleetingly in time or tissue as more abundant transcripts are analyzed. "In a given cell, [most] genes are expressed at lower levels, and they constitute only a small portion of the total transcripts, whereas a small number of genes expressed at a high level constitute a large portion of the total transcripts," says San Ming Wang, an assistant professor of medicine at the University of Chicago. These rarely utilized genes nonetheless might be vital to differentiation or development. Wang hypothesized that the "subtraction" cDNAs used to remove abundant mRNAs (or cDNA versions of them) by complementary base pairing along their entire lengths might also ferret out rare sequences nonspecifically by hybridizing with their "poly A" or "poly T" tails. Because of their rarity, removing just a few of these molecules would have a greater impact on analysis than deleting a few of the more abundant sequences. Wang, with colleagues in Chicago and at the Johns Hopkins University School of Medicine, devised a way to circumvent the still theoretical problem, then tested it--with great results. They flood cells with short sequences of 11 thymines and another base or two. These "anchored primers" hybridize to the ends of rare sequences and prevent them from being removed along with the duplexes that represent abundant sequences. The researchers tracked several rare transcripts in human colon cells and demonstrated a 1.4- to 7.8-fold increase in retention when the subtraction libraries used to weed out the abundant sequences included anchored primers (S.M. Wang et al., "Screening poly [dA/dT]-cDNAs for gene identification," Proceedings of the National Academy of Sciences, 97:4162-7, April 11, 2000). Sums up team member Janet Rowley, Blume-Reise Distinguished Service Professor at Chicago, "This could make a real difference in the increasingly difficult process of cataloging human genes. It could also simplify the postgenome process of learning what these less common genes do and when they are expressed." --Ricki Lewis Attractive Repellents If scientists could somehow marshal lymphocytes, the soldiers of the immune system, more precisely, they might be able to devise better treatments for autoimmune-related diseases. But investigators must first better understand biochemical signals that affect immune cell movement. A research team at Massachusetts General Hospital (MGH) recently found a rare phenomenon that could help: a chemical signal that facilitates the attraction and repulsion of immune cells (M.C. Poznansky et al., "Active movement of T cells away from a chemokine," Nature Medicine, 6:543-8, May 2000). While studying the cues that dictate the movement of immune cells out of the thymus, a site of differentiation, the MGH team observed in vitro the tendency of many immune cells to migrate to the edges of cell culture wells. Apparently something in the thymus tissue was repelling T cells. Understanding the effect, they reasoned, might lead to clues to immune cell movement. "I have to say I was very skeptical," says senior author David T. Scadden, director of experimental hematology at MGH. But Scadden's postdoc, Mark C. Poznansky, was convinced that the apparent "chemorepulsion," a phenomenon not nearly as well understood as "chemoattraction," was consistent and real. The focus of their search became SDF-1, a chemokine and chemoattractant for T cells that's known to be essential for life. The team discovered that sufficiently high concentrations of SDF-1 repelled T cells both in vitro and in vivo. Scadden speculates that immune cell chemorepulsion may be related to inflammatory responses or to preserving "immune sanctuaries" such as the brain and eyes. --Eugene Russo

Research Notes

Introducing RNA Polymerase

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