Yukawa Laboratory (Nagoya City University, Japan)

Our group has been involved in developing an understanding of the mechanistic aspects of genes expression during transcription as well as translation in higher plants. Our overall aim is to identify the molecular components involved in these fundamental processes of gene expression and understand their regulation at molecular level. Development of in vitro systems that could transcribe any gene or translate any mRNA with high fidelity was central to achieve the above stated objectives. During the years, we have developed an in vitro transcription system from tobacco nuclei and in vitro translation system from tobacco chloroplasts, which are two major assets of our group and are central to most of our research activities. We hope that by developing a deeper understanding of these fundamental processes of life, our studies would have applications in solving global issues like, food, energy and climate change.

Schematic representation of in vitro transcripiton systemWe are studying various aspects of transcriptional regulation of nuclear genes under cell-free conditions. The in vitro system was developed from BY-2 tobacco cultured cells that are also known as HeLa cells of plants. As eukaryotic cells possess three different RNA polymerases, Pol I, Pol II and Pol III, our in vitro transcription system also supports all these activities. Since our knowledge of Pol III transcription is not as good as for the other two polymerases, our group’s recent efforts are concentrated towards understanding various aspects of Pol III activity. As show below, Pol III transcribed genes (class III genes) in plants have been classified into four types. This classification is different from that of animals and fungi. We therefore believe that a better understanding of plant Pol III based transcription would help in developing a comprehensive understanding of the eukaryotic transcription.

Chloroplast (left) and cytosolic (right) lysates including translation acitivityChloroplasts are plant organelles that are the site for photosynthesis in higher plants. Photosynthesis is a plant-specific biochemical process that produces organic molecules by processing atmospheric CO ₂ with the help of energy captured from sun-light. Interestingly, chloroplasts contain a small circular genome that encodes a large number of proteins involved in catalysis of photosynthetic reactions. Since proteins involved in photosynthesis have high turnover rates, chloroplasts require a robust translation system to sustain this high protein turnover. For example, chloroplast translation system is involved in the production of RuBisCO (ribulose-1, 5-bisphosphate carboxylase/oxygenase), which in molar terms is ~50% of all other plastid protein put together and also known to be the most abundant protein on the earth. Chloroplast factory project in our lab focuses on this translational activity and aims to utilize this ability to produce useful proteins in the chloroplast. For this purpose, we have established in vitro translation system from tobacco chloroplast. This system has many advantages, as this system is non-isotopic with high activity and high throughput. Using this system we are trying to study a number of basic aspects related to translation in chloroplasts.

Predicted secondary structure of AtR8 RNAAfter completion of major projects on sequencing of plant genomes, annotation of various structural components of these genomes is in progress. According to the latest estimates the protein coding genes are not so many, however, a large number of non-protein-coding RNA (ncRNA) genes have been discovered. Although the protein coding genes can be predicted based on their structural features viz., open reading frame, and features of the upstream and downstream sequences, it is difficult to predict an ncRNA gene only from its sequence information. Because, there is no open reading frame and no sequence similarity, whatsoever so far found, in the characterized genes, in silico analysis has proven to be difficult. With an aim to discover novel ncRNA in higher plants, we are using the in vitro transcription system developed from tobacco cell nuclei. Our efforts have revealed some conserved regulatory elements in Pol III transcribed genes, which are being used to scan genomic data to find new ncRNA candidates. After their amplification by genomic PCR, these candidate genes can be validated for authenticity by using the in vitro transcription system developed in our lab.

Graduate school of Natural Sciences, Professor

Dr. Yasushi Yukawa

Speciality: Plant molecular biology, RNA biology
Projects : Study of transcripution mechanism of higher plants
Profile Ph.D.

Researcher

Maki Yukawa

Projects: Production of recombinant proteins in chloroplast (Chloroplast Factory Project)
Profile Master of Science

Graduate student（2nd-year doctor's degree）

Ayaka Ido

Project: Study of light dependent transcription of gene.

Graduate student（2nd-year master's degree）

Toma Fujii

Project: Functional study of plant histone chaperones.

raduate student（2nd-year master's degree）

Sou Li

Project: Functional study of plant long non-coding RNA.

raduate student（1st-year master's degree）

Sota Asano

raduate student（1st-year master's degree）

Na Renhu

Collaborator

Toshiyuki Nagai

Project: rice non-coding RNA.

Google Scholar Citations (Yasushi Yukawa)