Genetic information is carried by two kinds of nucleic acid molecules, DNA and RNA. The master blueprint from DNA must be copied, or "transcribed," into messenger RNA before a cell can grow or differentiate. To understand this transcription process, which is critical to all living things, scientists must study the inner structure of the RNA polymerase enzyme. Guided by helper molecules, known as transcription factors, RNA polymerase binds to the start of a gene, unwinds the DNA double helix, and manufactures an RNA message.

The eukaryotic cells of higher organisms contain three forms of RNA polymerase, made up of 10 to 15 polypeptides. These enzymes require the help of general transcription factor molecules to recognize a promoter (the region of DNA in front of a gene that binds RNA polymerase to promote gene expression) and initiate transcription. Structural similarities among RNA polymerases and general transcription factors hold the key to understanding the universal features of the transcription mechanism.

RNA Pol II, a 12-subunit protein complex, is responsible for all messenger RNA production. Previous experiments demonstrated that a mutant 10-subunit Pol II could not recognize a promoter to initiate transcription–two additional subunits, Rpb4 and Rpb7, were required. These subunits are normally found in substoichiometric amounts, a problem overcome by the researchers, who successfully crystallized a 12-subunit Pol II.

Picky Polymerase

Every cell of the human body contains the same DNA. The variation in cell types–such as blood, nerve, and liver cells–comes from the selection of genes chosen to be copied by RNA polymerase from DNA into messenger RNA. RNA polymerase, regulated by transcription factors, is made up of several protein subunits with different roles.

RNA polymerase II (Pol II) is the protein complex responsible for all messenger RNA production, the first step in gene expression. The structure of the Pol II enzyme provides the basis for understanding all gene activity in the eukaryotic cells of higher organisms, such as yeast, plants, and humans. TFIIB and TBP are transcription factors that interact with Pol II to start the transcription assembly line. The study of the structure and function of Pol II and its transcription factors is the key to translating an organism's genetic code and understanding the flow of information from gene to protein.

RNA polymerase II pre-initiation complex model.

To initiate transcription from a promoter, Pol II also requires the help of five general transcription factor molecules. Two of these factors, TFIIB and TBP, are responsible for promoter recognition and interaction with Pol II.

Crystals of Pol II–TFIIB complexes were studied at ALS Beamlines 5.0.2 and 8.2.2, and diffraction data complete to 4.5-Å resolution were collected at Stanford Synchrotron Radiation Laboratory's Beamline 9-2. These observations revealed three features that are crucial to transcription initiation: an N-terminal zinc ribbon of TFIIB that "docks" with the polymerase near the path of RNA exit from a transcribing enzyme; a "finger" of TFIIB inserted into the active center of the polymerase, possibly to slow down the transcription process so that the strands of DNA and new RNA can separate properly; and a C-terminal that orients DNA for unwinding and transcription.

The researchers also observed that TFIIB may interact with the DNA template strand to determine the location where transcription starts. TFIIB may also define the roles of other transcription factors during initiation.

The team also studied the elongation phase of transcription (the growth of a polypeptide chain) by Pol II. Previous studies of the elongating polymerase had problematic structures that failed to separate newly synthesized RNA from the template DNA strand. The researchers drew on their considerable expertise in the preparation of protein crystals to add Pol II to a preassembled DNA–RNA scaffold. These crystal complexes diffracted on ALS Beamline 5.0.2 to 3.6 Å and revealed proper separation of the RNA strand from the DNA template.

Close-up of strand separation. RNA (red) and DNA (blue) strands are separated by a network of interactions with three protein loops. The loops limit the extent of RNA separation (orange), stabilize the separated single strand, and form part of the RNA exit pore (green and pink).

Strand separation was achieved by a network of interactions between three previously unobserved protein loops in Pol II and the RNA–DNA phosphate backbone. The strand-loop network also moved the Pol II so that the binding site for an incoming nucleotide triphosphate was empty. Future work can exploit the empty binding site for studies on the mechanisms of nucleotide selection and addition.

Research conducted by D.A. Bushnell, K.D. Westover, R.E. Davis, and R.D. Kornberg (Stanford University).

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

Publications about this research: D.A. Bushnell, K.D. Westover, R.E. Davis, and R.D. Kornberg, "Structural Basis of Transcription: An RNA Polymerase II-TFIIB Cocrystal at 4.5 Angstroms," Science 303, 983 (2004); K.D. Westover, D.A. Bushnell, and R.D. Kornberg, "Structural Basis of Transcription: Separation of RNA from DNA by RNA Polymerase II," Science 303, 1014 (2004).

ALSNews Vol. 244, August 25, 2004