NEWSROOM

RNA polymerases (RNAPs) are the core enzymes of transcription, responsible for transcribing genetic information from DNA into RNA. In mammalian nuclei, RNAPs have evolved into three functionally distinct forms: Pol I, Pol II, and Pol III. Pol II, transcribing protein-coding mRNAs, has long been the focus of transcription research. Pol III—responsible for producing short non-coding RNAs like   5SrRNA, tRNAs, and U6 snRNA—plays critical roles in protein synthesis, RNA splicing, and cell cycle regulation.

Over the past few years, the research team led by Xu Yanhui has made significant breakthroughs in understanding Pol III transcription mechanisms. They have resolved the structures of the human Pol III elongation complex (Cell Research, 2021), uncovered its transcription termination mechanism (Nature Communications, 2021), and revealed the assembly of Pol III pre-initiation complex (PIC) (Cell Research, 2023). However, the dynamic regulation of Pol III transcription initiation—particularly the transition from initiation to elongation—remained poorly understood.

Transcription initiation involves multiple steps, including promoter recognition, PIC assembly, DNA unwinding, transcription bubble formation, and transition to elongation. While these steps are similar across RNA polymerases, their regulatory factors and dynamic conformational changes differ significantly. Understanding these transitions is key to deciphering transcriptional regulation.

In 2006, Richard H. Ebright’s team at Rutgers University used single-molecule methods to uncover the "scrunching" mechanism in bacterial transcription initiation, where DNA compression drives promoter escape. In 2023, Xu Yanhui’s team at Fudan University visualized Pol II transcription initiation and transition from initiation to elongation at single-nucleotide resolution (Science, 2023). Meanwhile, a collaborative study by Belgian and American researchers revealed how yeast mitochondrial RNA polymerase (mtRNAP) undergoes transcription bubble collapse during promoter escape (Nature, 2023). Despite these advances, Pol III transcription initiation dynamics remained elusive.

On June 4, 2025, Xu Yanhui’s team published a groundbreaking study in Nature, titled "Structural insights into human Pol III transcription initiation in action." The study revealed the dynamic process of Pol III transcription initiation, and how general transcription factors drive the transition from initiation to elongation.

Using in vitro transcription reaction system with a modified U6-1 promoter, the team captured key intermediate states by stalling transcription at specific positions after the transcription start site (TSS) (Figure 1). Cryo-EM analysis of these complexes showed that Pol III transitions to elongation after synthesizing just 6 nucleotides—shorter than Pol II (10 nt) or mtRNAP (8 nt) (Figure 2). This difference implies Pol III’s role in efficiently producing short non-coding RNAs.

A major discovery was the structural evidence for transcription re-initiation. After Pol III enters elongation, GTFs remain bound to the promoter, allowing rapid re-initiation—a strategy likely enabling Pol III’s high transcription frequency (Figure 3).

Additionally, the team developed a novel, non-radioactive transcription activity assay, replacing traditional radioisotope labeling with fluorescent tagging (pCp-AF647). This safer, more accessible method could revolutionize transcription research.

This study not only fills a critical gap in understanding eukaryotic transcription but also provides new perspectives on RNA polymerase evolution and function.

Authors: Qianmin Wang (first author), Yanhui Xu, and Xizi Chen (corresponding authors).

Figure 1. Assembly of the Pol III initiation complex and in vitro transcription assay.

Figure 2. Pol III transcription initiation: seven Pol III transcription complex structures.

Figure 3. Pol III transcription initiation model.

Video 1. Conformational transitions during Pol III transcription initiation.

Left: Displays the overall structure of Pol III transcription complex.
Middle: To clearly observe the formation of the transcription bubble and the interaction interfaces between DNA and proteins, portions of the protein structure obscuring the promoter DNA have been hidden.
Right: Conformation of the promoter DNA. The template strand and non-template strand are shown in yellow and blue, respectively, with the TSS highlighted in red.