JAPAN NANONET BULLETIN - 92nd Issue - March 22, 2007

RNA polymerase molecules sliding along DNA
—Dynamics of biomolecules—

“In molecular biology, biological phenomena used to be studied mainly from functional aspects, but are now studied from mechanistic aspects to solve the mechanisms by using the static structures of molecular machines. I started researching nanobiology because of my interest in the dynamic aspects of biological phenomena, especially those in which physiological changes are governed by movements of one or two biological molecules,” Prof. Shimamoto says. He picked a transcription system among such molecular machines and focused on the dynamic and mechanical aspects of RNA polymerases on DNA. In other words, he tried to clarify how transcription, which is the transfer of genetic information from DNA into RNA, is described in terms of the motions of molecules and molecular domains using newly emerging technology, such as single molecule measurements.

RNA polymerase works in the form of a single molecule that transcribes a gene existing as one or several copies, and each molecular machine provides a different blueprint for protein synthesis according to the gene. It has been known that all RNA polymerases synthesize short RNAs in addition to the full-length RNAs that act as a blueprint. Researchers had thought that these shorter RNAs were failed or aborted precursors of the full-length RNA, assuming a sequential pathway of transcription initiation composed of the chemical steps that were believed to be essential at that time. However, when Shimamoto’s team compared the time courses of the synthesis of short and full-length RNAs by using E. coli RNA polymerase, which is the only fully reproduced artificial system, composed of purified proteins at present, they found that the shorter RNAs are continuously synthesized even after completion of the full-length RNA synthesis. This means that there is a distinct transcription machine that only produces short RNAs and that the initiation of RNA synthesis is branched into two or more pathways according to different molecular machines. One branch leads to normal synthesis of the full-length RNA, and the other leads to an arrest accompanied with the synthesis of the shorter RNAs. The team named the newly found machine “moribund complex,” and the different machines are produced from homogeneous preparations of RNA polymerase and DNA. They experimentally proved that there is indeed a branched initiation mechanism and showed that different machines have different conformations of proteins and DNA (Fig.1).

Prof. Shimamoto says, “The moribund complex may be thought to be useless, but in fact, it regulates transcription. Proteins, called GreA and GreB, which have been found as transcriptional activators working in elongation of RNA, activate initiation by converting the moribund complex into a productive complex.” Historically, we tend to assume that a biological reaction is a sequence of chemically essential processes, but this study shows that a seemingly useless process, i.e., abortive synthesis in this case, can have an essential role for life. He says, “Regulation is the core heart of biology, and living organisms even utilize a process that we superficially considered useless according to our limited knowledge. This study was a good lesson for me.”

Prof. Shimamoto also addressed the mechanism in which RNA polymerases identify the DNA segment to initiate transcription, namely, a promoter. How does RNA polymerase find a promoter that is merely a small part of huge chromosomal DNA? He observed how the molecules of DNA-binding proteins move on DNA by using single molecule imaging and proved that E. coli RNA polymerase can slide along DNA. He fixed extended T7 phage DNA in parallel on a slide glass, where both ends of the DNA are fixed, but the middle is free for the protein to access, and then, a solution containing fluorescently-labeled RNA polymerase molecules is added to the slide at an angle to the fixed DNA (Fig.2).

Prof. Shimamoto says, “When the single RNA polymerase molecules that drift with the bulk flow reach the DNA fixed in parallel, we observed that their movements were parallel to the DNA, and such movements would not be possible if the protein molecule were not sliding.” This is the first direct evidence of a protein sliding along DNA by single-molecule dynamics. His team also experimentally proved that RNA polymerase can track a helical groove of DNA during sliding by rotating around the DNA double helix, which is the proof that RNA polymerase can read a DNA sequence as a human does, by introducing a nano-tachometer (Fig.3). He says, “This is an excellent mechanism by which RNA polymerase selects the double-stranded DNA from huge excess of RNA that does not have the groove in a cell.”

The longer a DNA molecule, the more frequently a protein meets the DNA to start sliding, if the protein can slide along DNA. Such a mechanism allows the protein to reach its target sequence faster. Prof. Shimamoto focused on E. coli tryptophan repressor (TrpR) and P. putida CamR, which are typical bacterial repressors that inhibit transcription by binding to their target sequences. Since most sequences on DNA weakly catch these DNA binding proteins, such weak binding sites should compete for catching proteins against the target sequences. This means that there must be a critical number of protein molecules in a cell to bind to the target sequence. According to previous reports, the number of TrpR molecules is 100-fold less than the critical number, and thus, how can it inhibit transcription? He experimentally showed that these repressors slide along DNA in their association process to their targeted sequences, while they dissociate from their targets without sliding after binding to the targets. Consequently, as the DNA becomes longer, the stability of TrpR-target complex becomes higher. He named the effect of DNA length on the stability of a DNA-protein complex “antenna effect” because an antenna either catches a weak signal from air or senses objects in insects (Fig.4). Sliding is one of the mechanisms that exert an antenna effect; although this mechanism is sometimes misunderstood as a violation of a thermodynamic rule called “detailed balancing,” careful consideration of time ranges of the involved physical processes showed that this antenna effect does not contradict thermodynamics.

Prof. Shimamoto says, “Nanobiology is a discipline to find out how motions of molecules in cells generate macroscopic phenomena, while nanobiotechnology is a discipline to create molecular machinery by utilizing biomolecules. So, collaboration between nanobiology and nanobiotechnology is required.” However, he is strongly against so-called commercialization-oriented basic research. “In principle, basic research can be promoted only by small but widely distributed funds, and commercialization cannot be promoted by such funds. For commercialization, product-oriented projects must be essential, a large sum of money must be invested in the projects, and then, they must be strategically carried out in consideration of cost-performance. This is why basic research and commercialization cannot be directly combined. They require different strategies. We must train specialists for combining these two different components, which is more trans-disciplinary than the field called translational research (Fig.5).” He adds, “To receive research grants, some may push their research as a work that can be commercialized, but neither basic scientists nor people involved in commercialization are able to notice, if the research is original. Only specially trained people are able to notice such possibilities. If nanobiology is used for commercialization-oriented basic research, it will go nowhere. So, the correct way of funding and the need for specialists are essential, and I have to keep saying this opinion out loud.”