SummaryRNA polymerase III (Pol III) assembles together with transcription factor IIIB (TFIIIB) on different promoter types to initiate the transcription of small, structured RNAs. Here, we present structures of Pol III pre-initiation complexes comprising the 17-subunit Pol III and hetero-trimeric transcription factor TFIIIB with subunits TATA-binding protein (TBP), B-related factor 1 (Brf1) and B double prime 1 (Bdp1) bound to a natural promoter in different functional states. Electron cryo-microscopy (cryo-EM) reconstructions varying from 3.7 Å to 5.5 Å resolution include two early intermediates in which the DNA duplex is closed, an open DNA complex and an initially transcribing complex with RNA in the active site. Our structures reveal an extremely tight and multivalent interaction of TFIIIB with promoter DNA and explain how TFIIIB recruits Pol III. TFIIIB and Pol III subunit C37 together activate the intrinsic transcription factor-like activity of the Pol III-specific heterotrimer to initiate melting of double-stranded DNA in a mechanism similar as used in the Pol II system.

RNA polymerase III (Pol III) synthesises tRNAs and other short, essential RNAs. Human Pol III misregulation is linked to tumour transformation, neurodegenerative and developmental disorders, and increased sensitivity to viral infections. Here, we present cryo-EM structures at 2.8 to 3.3 Å resolution of transcribing and unbound human Pol III. We observe insertion of the TFIIS-like subunit RPC10 into the polymerase funnel, providing insights into how RPC10 triggers transcription termination. Our structures resolve elements absent from S. cerevisiae Pol III such as the winged-helix domains of RPC5 and an iron-sulphur cluster, which tethers the heterotrimer subcomplex to the core. The cancer-associated RPC7α isoform binds the polymerase clamp, potentially interfering with Pol III inhibition by tumour suppressor MAF1, which may explain why overexpressed RPC7α enhances tumour transformation. Finally, the human Pol III structure allows mapping of disease-related mutations and might contribute to developing inhibitors that selectively target Pol III for therapeutic interventions.

The majority of non-protein-coding RNAs present in eukaryotic cells comprises rRNAs, tRNAs and U6 snRNA that are involved in protein biosynthesis and are synthesized by DNA-dependent-RNA polymerase I and III. The transcription cycle (initiation, elongation and termination) has similar principles in all three nuclear RNA polymerases with specific features that are reflected back in their structures. Recently, owing to the 'resolution revolution' in electron cryo-microscopy, there has been a significant advancement in the understanding of these molecular machines. Here, we highlight the structure-function adaptation in specificity and activity of these molecular machines and present parallels and distinctions between their transcription mechanisms.

Newly made messenger RNAs are processed and packaged into ribonucleoprotein complexes (mRNPs) and recognized by the essential transcription-export complex (TREX) for nuclear export 1,2 . However, the mechanisms of mRNP recognition and three-dimensional organization are poorly understood 3 . Here, we report cryo-electron microscopy and tomography structures of reconstituted and endogenous human mRNPs bound to the two-megadalton TREX complex. We show that mRNPs are recognized through multivalent interactions between the TREX subunit ALYREF and mRNP-bound exon-junction complexes. Exon-junction complexes can multimerize through ALYREF, suggesting a mechanism for mRNP organization. Endogenous mRNPs form compact 'globules' that are coated by multiple TREX complexes. These results reveal how TREX may simultaneously recognize, compact, and protect mRNAs to promote their packaging for nuclear export. The mRNP globule organization provides a framework to understand how mRNP architecture could facilitate mRNA biogenesis and export. This work is licensed under a CC BY 4.0 International license.

Maf1 is a conserved inhibitor of RNA polymerase III (Pol III) that influences phenotypes from metabolic efficiency to lifespan. Here, we present a 3.3 Å cryo-EM structure of yeast Maf1 bound to Pol III, establishing that Maf1 sequesters Pol III elements involved in transcription initiation and binds the mobile C34 WH2 domain, sealing off the active site. The Maf1 binding site overlaps with that of TFIIIB in the pre-initiation complex.