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TitleTranscription elongation of the plant RNA polymerase IV is prone to backtracking.
Journal, issue, pagesSci Adv, Vol. 10, Issue 34, Page eadq3087, Year 2024
Publish dateAug 23, 2024
AuthorsChengli Fang / Kun Huang / Xiaoxian Wu / Hongwei Zhang / Zhanxi Gu / Jiawei Wang / Yu Zhang /
PubMed AbstractRNA polymerase IV (Pol IV) forms a complex with RNA-directed RNA polymerase 2 (RDR2) to produce double-stranded RNA (dsRNA) precursors essential for plant gene silencing. In the "backtracking- ...RNA polymerase IV (Pol IV) forms a complex with RNA-directed RNA polymerase 2 (RDR2) to produce double-stranded RNA (dsRNA) precursors essential for plant gene silencing. In the "backtracking-triggered RNA channeling" model, Pol IV backtracks and delivers its transcript's 3' terminus to RDR2, which synthesizes dsRNA. However, the mechanisms underlying Pol IV backtracking and RNA protection from cleavage are unclear. Here, we determined cryo-electron microscopy structures of Pol IV elongation complexes at four states of its nucleotide addition cycle (NAC): posttranslocation, guanosine triphosphate-bound, pretranslocation, and backtracked states. The structures reveal that Pol IV maintains an open DNA cleft and kinked bridge helix in all NAC states, loosely interacts with the nucleoside triphosphate substrate, and barely contacts proximal backtracked nucleotides. Biochemical data indicate that Pol IV is inefficient in forward translocation and RNA cleavage. These findings suggest that Pol IV transcription elongation is prone to backtracking and incapable of RNA hydrolysis, ensuring efficient dsRNA production by Pol IV-RDR2.
External linksSci Adv / PubMed:39178250 / PubMed Central
MethodsEM (single particle)
Resolution3.1 - 3.4 Å
Structure data

38470

8xmb

EMDB-38470, PDB-8xmb:
NTP-bound Pol IV transcription elongation complex
Method: EM (single particle) / Resolution: 3.4 Å

38471

8xmc

EMDB-38471, PDB-8xmc:
Post-translocated Pol IV transcription elongation complex
Method: EM (single particle) / Resolution: 3.1 Å

38472

8xmd

EMDB-38472, PDB-8xmd:
Pre-translocated Pol IV transcription elongation complex
Method: EM (single particle) / Resolution: 3.4 Å

38473

8xme

EMDB-38473, PDB-8xme:
Backtracked Pol IV transcription elongation complex
Method: EM (single particle) / Resolution: 3.1 Å

Chemicals

ChemComp-ZN:
Unknown entry

ChemComp-MG:
Unknown entry

ChemComp-GTP:
GUANOSINE-5'-TRIPHOSPHATE / GTP, energy-carrying molecule*YM

Source
  • arabidopsis thaliana (thale cress)
  • synthetic construct (others)
KeywordsTRANSCRIPTION/DNA/RNA / Transcription / elongation / Pol IV / RNA polymerase / TRANSCRIPTION-DNA-RNA complex

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