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April 9, 2003 New Insights into Transcription Initiation in BacteriaK.S. Murakami, E.A. Campbell, S. Masuda, O. Muzzin, M. Chlenov, J.L. Sun, C.A. Olson, O. Weinman, M.L. Trester-Zedlitz, and S. A. Darst Every cell in a living being contains an instruction manual, the genome, encoded in the form of messages called genes within a double helical molecule made of deoxyribonucleic acid (DNA). This information is read and interpreted in two steps, called transcription, in which the DNA is converted into ribonucleic acid (RNA), and translation, in which the RNA is converted into proteins, used by cells to function properly. Scientists from Rockefeller University have provided structural insights into the key machinery for controlling the initiation of transcription in bacteria.
The conversion of DNA into RNA is performed by a protein called RNA polymerase, which slides along the DNA helix, unzipping the two strands as it goes, synthesizing complementary strands of RNA corresponding to packets of DNA information called genes. In bacteria, the RNA polymerase has the shape of a 150-angstrom-long crab-claw, with a large cleft between the claws in which the RNA is produced. The RNA polymerase starts synthesizing RNA at defined DNA sequences called promoters, which are recognized by the combination of the RNA polymerase with a protein called the sigma factor. The RNA polymerase with sigma is called the holoenzyme. The sigma factor plays a central role in locating the promoter sequence and in separating the DNA strands, which is required for the RNA polymerase to decode the genetic messages.
Three domains of sigma, labeled σ2, σ3, and σ4, are connected by flexible linkers and are positioned across one face of the RNA polymerase, as shown in figure 1. One of the linkers connecting the σ3 and σ4 domains is particularly long, snaking into the cleft of the RNA polymerase active site and then out again through another channel, the RNA exit channel, that is occupied by the RNA during elongation (figure 1). The linker suggests how sigma may assist in initiating transcription from nucleoside triphosphate (NTP) substrates – the basic units of RNA – by forming part of the initiating substrate-binding site. The structures mentioned above also provide insights into the curious phenomenon of abortive initiation, in which the initiating RNA polymerase generates large amounts of short RNA fragments while positioned at the promoter, but only rarely moves forward to elongate the entire RNA chain. During initiation, the elongating RNA must displace the sigma linker from the RNA exit channel. Only once in a while, when the RNA manages to push the linker completely out of the RNA exit channel, does abortive initiation cease and the transition into the elongation phase (which follows the initiation phase) can occur.
The two structures above also provide a basis for modeling complexes with the entire promoter (figure 2). Models of the initial ‘closed’ complex (where the DNA is not melted) and the final ‘open’ complex (where 14 base-pairs of DNA have been melted) provide a framework for designing further experiments to understand how the RNA polymerase operates. BEAMLINE FUNDING PUBLICATIONS FOR MORE INFORMATION |
The National Synchrotron Light Source at Brookhaven National Laboratory in New York, is a national user research
facility funded by the U.S. Department of Energy's Office of Basic Energy Science. The NSLS operates two electron
storage rings: an X-Ray ring and a Vacuum UltraViolet (VUV) ring which provide intense light spanning the electromagnetic
spectrum from the infrared through x-rays. Each year over 2300 scientists from universities, industries and government
labs perform research at the NSLS.
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A genome is the complete "recipe" or set of instructions to make
an organism. These instructions are stored in each cell of the
organism in the form of a double helical molecule of
deoxyribonucleic acid (DNA). The DNA information is read and decoded
in two processes, called transcription, in which the DNA is
converted into ribonucleic acid (RNA), and translation, in which the
RNA is converted into proteins, necessary for the normal functioning
of cells. 
Using data from the National Synchrotron Light Source, the
Cornell High Energy Synchrotron Source, as well as the Advanced
Photon Source, the Rockefeller researchers have provided
high-resolution views of the sigma factor structure as well as of
the entire holoenzyme bound to a promoter DNA fragment. 