Scholars from the Chinese Academy of Sciences published an article today in Nature: revealing the structural basis of the inherent transcription termination of bacteria

On January 12, 2023, Yu Zhang's research team from the Key Laboratory of Synthetic Biology, Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, and the team of Robert Landick of the University of Wisconsin-Madison and the team of Feng Yu of Zhejiang University in Nature A research paper entitled "Structural basis for intrinsic transcription termination" captures the intermediate state cryo-EM structure of intrinsic transcription termination in bacteria, revealing the molecular mechanism
by which bacterial RNA polymerase recognizes transcription termination sequences, stops transcription, and dissociates RNA.

Transcription is the first step in gene expression and is an important component of
the law of genetic center.
RNA polymerase's transcription using DNA as a template is divided into three stages
: transcription initiation, transcription extension, and transcription termination.
Transcriptional termination of RNA polymerase occurs through two pathways, one is transcriptional termination that relies on the stop factor, and the other is transcriptional termination that is not dependent on the stop factor, also known as intrinsic transcription termination
.
Intrinsic transcriptional termination is a conserved transcriptional termination by viral, bacterial, and eukaryotic RNA
polymerases.
Bacterial intrinsic transcription termination is a transcriptional termination method in which RNA polymerase spontaneously stops transcription when it transcribes to the stop sequence, which does not depend on transcriptional termination Rho protein, so it is also called transcriptional termination
independent of Rho factors.
The intrinsic transcriptional termination sequence of the bacteria consists of a GC-rich hairpin structure followed by a U-tract sequence (Figure 1).

At transcription termination, RNA polymerases need to brake urgently from a high-speed extended state (~50 bases/sec) to stop transcription, followed by rapid conformational changes in RNA polymerases, and RNA and DNA dissociate
from highly stable RNAP-DNA-RNA complexes.
Due to the highly dynamic and responsive nature of transcriptional termination, it is difficult
to resolve the structural and molecular mechanisms of transcriptional termination.
The research team drew on the research results of predecessors to split the transcriptional termination sequence and capture three key transcriptional termination intermediate state structures
.
First, the research team analyzed the TTC-pause structure containing only the U-tract sequence and found that the RNA-DNA heterozygous duplex containing the U-tract sequence showed an inactive conformation, preventing further binding of NTP and prompting transcription pause
.
Secondly, the research team analyzed the TTC-hairpin structure containing the U-tract sequence and part of the RNA hairpin structure, and found that RNA hairpins folded into RNA polymerase, inducing conformational changes in RNA polymerase, weakening the interaction
between RNA polymerase and RNA-DNA heterozygous duplex.
Subsequently, we demonstrated through biochemical experiments that the above conformational changes promote the closure of the two DNA single-stranded structures of the transcription bubble, further destroying the RNA-DNA heterozygous double-strand structure and promoting RNA dissociation
.
Finally, we added antisense RNA to the transcriptional pause complex to mimic the formation of a complete hairpin structure and induce transcriptional termination, resolving the TTC-release structure, which captures the intermediate state
where RNA has dissociated but DNA is still bound to RNA polymerase during transcriptional termination.

Based on the three-dimensional structure and biochemical experimental results of the key intermediate states of transcription termination described above, we propose the RNA dissociation mechanism of four reaction steps of intrinsic transcriptional termination, namely "DNA-rewinding triggered RNA release" (Figure 2):

(i) TTC-pause: The Utract sequence of the transcription terminator induces the semi-shifted state of the RNA-DNA heterozygous duplex at the catalytic center (RNA has shifted, but the DNA has not been shifted), preventing the addition of NTP and inducing transcription pause

(ii) Hairpin invasion (TTC-hairpin): RNA hairpin folds into the RNA polymerase, occupies the RNA channel, induces RNA polymerase domain conformational changes, and weakens the interaction
between RNAP and RNA-DNA heterozygous double-strands.

(iii) TTC-rewinding: The two DNA single-stranded bases of the transcriptional bubble are re-paired, further disrupting the RNA-DNA heterozygous duplex
.

(iv) RNA dissociation (TTC-release): RNA polymerase releases RNA, but can still slide itself on the genome, eventually dissociating or sliding to the promoter DNA to start the next round of transcription
.

This study reports the cryo-EM structure of the key intermediate states of bacterial intrinsic transcription termination, restores the whole process of bacterial intrinsic transcription termination, and answers the molecular mechanism
of how bacterial RNA polymerase recognizes transcription termination sequence, pauses transcription, and releases RNA.
The study expands the understanding of transcription termination, and the mechanism of "DNA-rewinding triggered RNA release" proposed in this study provides a reference
for the transcription termination mechanism of eukaryotic RNA polymerase.

You Linlin, a doctoral student at the Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, is the first author of the paper, and Professor Yu Zhang, Professor Robert Landick of the University of Wisconsin-Madison and Professor Feng Yu of Zhejiang University are co-corresponding authors
.
This project is supported
by the Leading Science and Technology Project of the Chinese Academy of Sciences, the National Key R&D Program, the Shanghai Basic Research Special Zone Program and the Shanghai Science and Technology Innovation Action Plan.

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Figure 1: Schematic diagram of bacterial gene transcription and classical bacterial transcription terminator sequence

Figure 2: Model of bacterial intrinsic transcriptional termination mechanism