SPring-8, the large synchrotron radiation facility

Release Date

30 Apr, 2004

30 April 2004
RIKEN

1. Introduction
   The genetic information stored in a DNA molecule is transcribed and/or translated to be expressed. An RNA molecule is synthesized by transcribing a gene encoded in a DNA, and then its sequence information is translated into a protein sequence. This flow of genetic information is essential for all the living organisms. RNA polymerase is an essential protein responsible for the transcription. RNAP is a huge multi-subunit enzyme of molecular weight > 450kDa, and its basic structure and function are conserved from bacteria to higher eukaryote including human.
   The transcription by RNAP is well controlled by a lot of transcription factors in a cell. In bacteria, a phenomenon so-called “stringent control” is known. In response to the amino-acid starvation, bacterial cells produce a compound “guanosine tetraphosphate (ppGpp)” to control the RNAP activity. Transcription of components of translation machinery is inhibited, while transcription of genes involved in amino acid biosynthesis and transport is enhanced. Unlike numerous other mechanisms of transcription regulation relying on protein transcription factors, the stringent control strongly depends on the action of a small nucleotide, ppGpp. However, the ppGpp binding site of the RNAP and structural basis of the ppGpp dependent control of the RNAP activity has not been uncovered. To address these questions, the research group attempted X-ray crystallography of a complex of bacterial RNAP and ppGpp, and succeeded in determining the structure.

2. Methods and Results
   The research group purified RNAP holoenzyme from Thermus thermophilus, and obtained cocrystals of RNAP and ppGpp. They obtained a diffraction data set by using the synchrotron radiation of the RIKEN beam line at SPring-8, and determined the structure at 2.7A resolution.
   It was found that ppGpp binds to a close vicinity of the RNAP active center. There are two RNAP/ppGpp complexes in the crystallographic asymmetric unit. It is surprising that the ppGpp binding manners are different between the two complexes in the asymmetric unit. The ppGpp orientations in the two complexes are almost in a reversed symmetry. The difference in the ppGpp binding mode are due to a subtle but significant difference in the RNAP active site configurations. This implies a possibility that the ppGpp molecule might modulate the RNAP active site structure to control its enzymatic activity.
   To clarify the mechanism of transcription regulation by ppGpp, the research group built a model of the RNAP/ppGpp complex bound to DNA. According to the model, the guanine base of the ppGpp bound to RNAP can interact with a cytidine base on the non-template strand of the DNA (-1 position). It is known that GC-rich sequences are conserved upstream of the transcription start site of genes that are inhibited by ppGpp. This implies that the guanine base of the ppGpp may play a role in recognizing and selecting the target genes. Actually, they showed that a mutation of the cytidine just upstream of the transcription start site eliminated the sensitivity toward ppGpp.

3. Perspectives
   Mechanism of the transcription control by ppGpp was elucidated. This achievement is quite important as it first explains the structural basis of the transcription regulation by a small alarmone. The research group is now trying to determine structures of RNAP in complex with nucleic aicds and/or several transcription factors to obtain a comprehensive view for the mechanisms of transcription and its regulation.