Abstract
Transcription of genomic information is achieved by DNA-dependent RNA polymerase (RNAP), a huge multi-subunit protein. Transcription is a well-regulated, complex process consisting of three major stages: initiation, elongation, and termination. In each stage, RNAP interacts with various transcription factors, and undergoes large conformational changes to accomplish multiple stage-specific tasks. To comprehend the structural bases of transcription and its regulation, the speaker and his research group have been performing structural studies of the essential transcription complexes.
For transcription initiation, the bacterial RNAP (α2ββ'ω) binds the promoter-specificity σ subunit, to form a holoenzyme (α2ββ'ωσ) that can recognize the promoter sequence on DNA. The speaker and his research group solved the crystal structure of the RNAP holoenzyme from the bacterium Thermus thermophilus (Nature, 2002). The structure revealed the position and the structure of the σ subunit on the RNAP, and provided insights into promoter binding and transcription initiation. Recently, they determined the crystal structure of the holoenzyme bound with a bacteriophage-encoded protein, gp39, which strongly inhibits initiation from most of the host T. thermophilus genes, without affecting transcription from the phage genes (Genes Dev., 2014). Gp39 binds to the C-terminal domain of the σ subunit (σ4) at the RNA exit site, and significantly relocates σ4. Thus, the phage-encoded protein gp39 modifies the holoenzyme conformation to switch its promoter specificity, and this is a novel mechanism to hijack the host’s transcription machinery in favor of phage development.
RNAP changes its conformation to accomplish various tasks during transcription, such as pausing, backtracking along DNA, RNA cleavage, termination, and binding transcription factors. They solved the crystal structure of T. thermophilus RNAP bound with Gfh1, a factor that inhibits the RNAP activities (Nature, 2010). The structure revealed that Gfh1 occludes the RNAP secondary channel, which serves for the intake of the substrate NTPs. Moreover, Gfh1 stabilizes the “ratcheted” conformation of RNAP, which differs from the “tight” conformation required for nucleotide addition to RNA. Thus, Gfh1 inhibits transcription by directly interfering with the substrate binding and by trapping RNAP in the alternative structural state. To characterize this newly identified ratcheted state in detail and to understand the relationship between the RNAP conformations and their functions, the speaker and his research group are developing a method to probe the RNAP conformation in solution. They are also studying the structures of several important transcription complexes. These studies will reveal the fundamental mechanisms of transcription and its regulation.
About the speaker
Prof Shigeyuki Yokoyama received his PhD from the University of Tokyo in 1981, and had been at the faculty of the Department of Biophysics and Biochemistry until 2010. He joined RIKEN, the Institute of Physical and Chemical Research in 1993, and is currently Distinguished Senior Scientist of the Structural Biology Laboratory.
Prof Yokoyama's research group studies the cubic structure of biopolymers such as protein, DNA and RNA. Using X-ray crystallography and NMR spectroscopy, they conduct structural biology research, focusing on gene expression (duplication, transcription, translation, etc.) and on proteins involved in intracellular signal transduction. The purpose of this research is to understand three-dimensionally how a system consisting of many proteins (network of biopolymers) operates, by analyzing the protein conformation and molecular functions systematically and exhaustively. The ultimate goal is to collect protein folds and simulate a cell on the computer. Recent research has shown that diseases such as cancer are caused by the abnormality of proteins that are responsible for signal transduction and gene expression. The Yokoyama group's structural biology approach is expected to provide new insight into the molecular mechanisms of diseases and to lead to the development of new methods of treatment.
Prof Yokoyama received numerous awards including the Chemical Society of Japan. He is a Foreign Honorary Member of the American Academy of Arts and Sciences.
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