In:
Science, American Association for the Advancement of Science (AAAS), Vol. 377, No. 6611 ( 2022-09-09)
Abstract:
In the eukaryotic cell nucleus, genomic DNA is stored as chromatin, comprising multiple nucleosomes carrying various genetic and epigenetic information. Gene transcription by RNA polymerase II (RNAPII) intrinsically affects nucleosome structures because it requires temporary unfolding of the nucleosomes to read the DNA sequence. However, RNAPII transcribes genes while maintaining the nucleosome structures, suggesting the existence of a transcription-coupled mechanism to restore the nucleosomes. However, this mechanism has remained elusive. RATIONALE We designed nucleosomal DNA templates so that the RNAPII elongation complex (EC) would stall at the 42-, 49-, 58-, and 115-bp positions from the nucleosome entry. When EC stalls at these positions, its leading edge is near super helical locations (SHLs) –1, 0, +1, and +6, respectively, of the nucleosome. Transcription was conducted in the presence of the transcription elongation factors Spn1, Spt6, Spt4/5, Elf1, Paf1C, and TFIIS and the histone chaperone FACT. The ECs formed at these positions were analyzed by cryo–electron microscopy single-particle analysis. RESULTS We obtained six nucleosome-transcribing EC structures: EC42, EC49, EC49B, EC58 hex , EC58 oct , and EC115, where the numbers denote the DNA positions where EC stalled. The ECs contain the RNAPII-associated Spn1, Spt6, Spt4/5, Elf1, and Paf1C proteins, which constitute the EC downstream and/or upstream edge. The structures represent serial snapshots of the EC passage through the nucleosome. In EC42, the EC leading edge resides near SHL(–1) within the downstream nucleosome, in which an ~60-bp DNA segment is removed from the histone octamer surface. One of the H2A-H2B dimers is exposed and bound with the C-terminal tail of the Spt16 subunit of FACT. In EC49, the EC leading edge is just before the nucleosomal dyad [SHL(0)], and an ~70-bp DNA segment is removed from the histone octamer. As one of the H3-H4 dimers is exposed, the main body of FACT engages with the histones primarily through Spt16. An ~30-bp DNA segment is also removed from the distal end of the nucleosome, and thus only a third of the histone octamer surface is covered by DNA. This FACT-histone complex probably represents the state before its detachment from the DNA and its subsequent transfer. In EC49B, the nucleosome is shifted downstream by ~17 bp, which might have originated by the downstream transfer of the FACT-histone intermediate. By contrast, EC58 hex and EC58 oct , in which the EC leading edge has overrun the dyad of the original nucleosome, reveal a FACT-histone complex transferred upstream of the EC. In EC58 hex , a FACT-histone hexamer [H2A-H2B-(H3-H4) 2 ] complex is deposited onto the emerging DNA at the EC DNA exit site, and the resultant hexasome is wrapped by an ~40 bp DNA segment. In EC58 oct , the remaining H2A-H2B dimer is deposited onto the histone hexamer to form the histone octamer (octasome), which is wrapped by an ~75-bp DNA segment. Finally, EC115 reveals a near-complete nucleosome on the EC upstream side, with the histone octamer covered by an ~120-bp DNA segment. At the rim of the EC DNA exit, the domains of Spt4, Spt5, Spt6, Leo1, and Rtf1 form a “cradle” that flexibly adapts to the subnucleosome intermediates in EC58 hex , EC58 oct , and EC115, supporting the sequential nucleosome reassembly process upstream of the EC. CONCLUSION The obtained structures visualize key steps of nucleosome traversal by the EC accompanied by the downstream nucleosome disassembly, followed by its reassembly upstream of the EC with the aid of FACT. When the EC passes through the nucleosomal dyad, the downstream-to-upstream transfer of the nucleosomal histones occurs. These views explain the mechanism by which the nucleosome structures are maintained during transcription. Transcription over a nucleosome mediated by the EC and FACT. Cryo–electron microscopy structures of the nucleosome-transcribing ECs: EC42, EC49, EC58 hex , EC58 oct , and EC115. EC49B is omitted in this figure. The EC contains the transcription elongation factors Spn1, Spt6, Spt4/5, Elf1, and Paf1C. These structures show snapshots of the EC progression on DNA mediating downstream nucleosome disassembly, followed by reassembly upstream of the EC with the aid of FACT.
Type of Medium:
Online Resource
ISSN:
0036-8075
,
1095-9203
DOI:
10.1126/science.abp9466
Language:
English
Publisher:
American Association for the Advancement of Science (AAAS)
Publication Date:
2022
detail.hit.zdb_id:
128410-1
detail.hit.zdb_id:
2066996-3
detail.hit.zdb_id:
2060783-0
SSG:
11
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