Chinese scientists have revealed the extremely complex structure of PEP, which plays an essential role in controlling the development of chloroplasts and gene expression in mature chloroplasts, getting them a cover article for the international academic journal Cell. (PROVIDED TO CHINA DAILY)

Chloroplasts are the essential components of plant cells responsible for photosynthesis, a process that plays a crucial role in producing oxygen and absorbing carbon dioxide and which is thus vital to the Earth's ecosystems and atmosphere.

Additionally, chloroplasts are central to the food chain, serving as the primary producers of organic matter upon which nearly every other organism depends, directly or indirectly.

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During chloroplast biogenesis, an important enzyme, known as plastid (membrane-bound organelle) encoded RNA polymerase (PEP), plays an essential role in controlling the development of chloroplasts and also of gene expression in mature chloroplasts.

However, the structure of PEP, essentially the chloroplast's gene transcription machinery, had remained elusive, posing a globally recognized challenge to the scientific community, until Chinese scientists recently revealed its workings.

Two Chinese teams detailed PEP's extremely complex structure in a cover article for the international academic journal Cell.

Chloroplasts are the sites of photosynthesis in plants. Approximately 1.5 billion years ago, primitive eukaryotic cells engulfed cyanobacteria — also known as blue-green algae — and evolved into eukaryotic single-celled algae, before eventually evolving into higher plants. As the transcription machinery for chloroplast DNA, chloroplast PEP is responsible for the development and functioning of chloroplasts

On Earth, life exists in three forms: bacteria, archaea (single-celled organisms), and eukaryotes (any cell or organism with a nucleus and organelles), each of which has its own different genetic transcription machinery, says Zhang Yu, the researcher who led the team from the Center for Excellence in Molecular Plant Sciences at the Chinese Academy of Sciences.

Transcription is a method of reading genetic information, which is written in DNA and must first be transcribed into RNA before it can be translated into the proteins that ultimately give rise to life functions, Zhang explains.

Chloroplasts are the sites of photosynthesis in plants. Approximately 1.5 billion years ago, primitive eukaryotic cells engulfed cyanobacteria — also known as blue-green algae — and evolved into eukaryotic single-celled algae, before eventually evolving into higher plants. As the transcription machinery for chloroplast DNA, chloroplast PEP is responsible for the development and functioning of chloroplasts.

"The lengthy process of evolution made the structure of chloroplast PEP exceedingly complex, and largely unknown," Zhang says. "The successful deciphering of the chloroplast PEP structure fills in the final blank in this puzzle."

One tricky part the researchers had to overcome was separating and purifying endogenous PEP complexes with transcriptional activity, because they appear in extremely low amounts, says Zhou Fei, associate professor at the Huazhong Agricultural University, the other team on the research.

"Traditional methods are difficult to use for extraction and purification, which made it impossible to further analyze the structure," Zhou explains.

To deal with this issue, the research team used chloroplast transformation technology, which allows for site-specific insertion of DNA fragments through homologous recombination, a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA.

This enabled the researchers to obtain a peptide or protein fused and expressed together with the target protein, with a very small molecular weight, for the detection and purification of the target protein.

"To put it more simply, we can add a DNA sequence as a tag to the gene sequence of PEP. Then, through purification, we can 'pull' PEP out from the complex components, obtaining the chloroplast gene transcription protein complex," Zhang explains.

After obtaining chloroplast-transformed plants with tags, it was necessary to establish a stable purification process.

An illustration of the evolution of a chloroplast. (PROVIDED TO CHINA DAILY)

"A single cell can contain thousands of different proteins, which may further form large protein complexes, some of which are abundant, while others are present in low amounts. In order to study a specific protein or protein complex, it must first be separated and purified from other proteins and nonprotein molecules," Zhou explains.

She adds that in this study, purification was achieved through multiple steps, including through exchange and molecular-exclusion chromatography, an isolation method that creates a kind of filter out of beads with tiny "tunnels" in them. Molecules above a certain molecular weight will not fit into the tunnels and pass through the filter relatively quickly by making their way between the beads. Smaller molecules take a longer path and pass through more slowly.

This method allows for the separation of molecules by size, ultimately yielding an extremely pure PEP mega-complex with transcriptional catalytic activity. Zhou likens the process to fishing for a particular type of fish from the ocean.

"We need to identify the specific type of 'fish' (using the tagged proteins), and then use specific tools to attract them," Zhou says.

Molecular-exclusion chromatography can be imagined as using a fishing net with a specific-sized mesh to catch the fish, she adds.

It wasn't until 2022 that the bottleneck in obtaining PEP proteins was finally overcome, according to Wu Xiaoxian, the first author of the study, who is attached to the Center for Excellence in Molecular Plant Sciences.

Afterward, single-particle cryoelectron microscopy (cryo-EM) technology was used. This involves rapidly freezing large biological molecules and imaging identical structurally homogeneous and dispersed particle samples at low temperatures using transmission electron microscopy. Through subsequent image processing and reconstruction calculations, a three-dimensional structure of the sample is obtained.

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"Its role is analogous to a tool for analyzing the 3D structure of the chloroplast gene transcription machinery and for understanding its architecture," Wu says.

It turns out the PEP-centered transcription apparatus comprises a bacterial-origin PEP core and more than a dozen eukaryotic-origin PEP-associated proteins (PAPs) encoded in its nucleus.

"Here, we determined the cryo-EM structures of a Nicotiana tabacum (tobacco) PEP-PAP apoenzyme (the protein part of an enzyme) and PEP-PAP transcription elongation complexes at near-atomic resolution," Zhou says.

The data show the PEP core adopts the typical fold of bacterial RNA polymerase. Fifteen PAPs bind at the periphery of the PEP core, which the experts say facilitates the assembling of the PEP-PAP super-complex, protecting it from oxidation damage, and likely coupling gene transcription with RNA processing.

"Our results report the high-resolution architecture of the chloroplast transcription apparatus and provide the structural basis for the mechanistic and functional study of transcription regulation in chloroplasts," Zhou says.

The elucidation of the structure and function of PEPs has lagged behind other polymerase complexes, partly because of the greater technical challenges of isolating transcriptionally active protein complexes from plants compared to other systems, according to F.Vanessa Loiacono and Ralph Bock, two experts from Germany's Max Planck Institute of Molecular Plant Physiology.

"Moreover, PEP is significantly larger than bacterial RNA polymerases due to the addition of numerous plant-specific proteins (PEP-associated proteins, PAPs) at the periphery of the catalytic core," they wrote in Cell.

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They point out that for the first time, the high-resolution cryo-EM structures show the precise localization of all known PAPs within the transcribing complex, enabling the assignment of roles to these proteins in the transcription cycle, and finally resolving some of the longstanding uncertainties about the unusual features of chloroplast RNA polymerase.

They state that "the studies represent a significant breakthrough in the field of organellar transcription".

At the fundamental research level, this study lays the groundwork for further exploration of the working mode of the chloroplast gene transcription machinery, and for understanding and redesigning the regulation of gene expression in chloroplasts, experts say.

In terms of the application of synthetic biology — a field of research in which the main objective is to create fully operational biological systems from the smallest constituent parts possible, including DNA, proteins, and other organic molecules — this research provides a starting point for improving the efficiency of plant chloroplast bioreactors, thereby facilitating the production of recombinant vaccines, recombinant protein drugs and natural products, Zhou says.

In terms of China's goals of reaching peak carbon emissions by 2030 and achieving carbon neutrality by 2060, the research provides new ideas for improving the gene expression levels of photosynthetic systems, helping plants become more efficient carbon sinks that accumulate and store carbon-containing chemical compounds, thereby removing more carbon dioxide from the atmosphere, Zhou says.

Contact the writers at yangfeiyue@chinadaily.com.cn