The multi-subunit DNA-dependent RNA polymerase (RNAP) is the principal enzyme of transcription for gene expression. Transcription is regulated by various transcription factors. Gre factor homologue 1 (Gfh1), found in the Thermus genus, is a close homologue of the well-conserved bacterial transcription factor GreA, and inhibits transcription initiation and elongation by binding directly to RNAP. The structural basis of transcription inhibition by Gfh1 has remained elusive, although the crystal structures of RNAP and Gfh1 have been determined separately. Here we report the crystal structure of Thermus thermophilus RNAP complexed with Gfh1. The amino-terminal coiled-coil domain of Gfh1 fully occludes the channel formed between the two central modules of RNAP; this channel would normally be used for nucleotide triphosphate (NTP) entry into the catalytic site. Furthermore, the tip of the coiled-coil domain occupies the NTP β-γ phosphate-binding site. The NTP-entry channel is expanded, because the central modules are 'ratcheted' relative to each other by ∼7°, as compared with the previously reported elongation complexes. This 'ratcheted state' is an alternative structural state, defined by a newly acquired contact between the central modules. Therefore, the shape of Gfh1 is appropriate to maintain RNAP in the ratcheted state. Simultaneously, the ratcheting expands the nucleic-acid-binding channel, and kinks the bridge helix, which connects the central modules. Taken together, the present results reveal that Gfh1 inhibits transcription by preventing NTP binding and freezing RNAP in the alternative structural state. The ratcheted state might also be associated with other aspects of transcription, such as RNAP translocation and transcription termination.

The emergence of functional interactions between nucleic acids and polypeptides was a key transition in the origin of life and remains at the heart of all biology. However, how and why simple non-coded peptides could have become critical for RNA function is unclear. Here we show that putative ancient peptide segments from the cores of both ribosomal subunits, as well as derived homopolymeric peptides comprising lysine or the non-proteinogenic lysine analogues ornithine or diaminobutyric acid, potently enhance RNA polymerase ribozyme (RPR) function irrespective of chirality or chiral purity. Lysine decapeptides enhance RPR function by promoting holoenzyme assembly through primer-template docking, accelerate RPR evolution and enable RPR-catalyzed RNA synthesis at near physiological (≥1 mM) Mg 2+ concentrations enabling templated RNA synthesis within membranous protocells. Our results outline how compositionally simple, mixed chirality peptides may have augmented the functional potential of early RNAs and promoted the emergence of the first protocells.Life is widely believed to have descended from a simpler, primordial biology that lacked DNA and proteins but in which RNA played a central role1. A core component of this "RNA world" would have been an RNA polymerase ribozyme (RPR) to replicate primordial RNA genomes and become encapsulated within membranous compartments to form the first protocells2. Modern day RPR analogues have been developed3,4 some of which can synthesize ribozymes4, aptamers5 or ~200 nucleotide (nt) sequences using favorable RNA templates6, and even amplify short RNA sequences5, but their activity is strictly dependent on very high concentrations of available magnesium ions (optimal [Mg 2+ ] ~ 200 mM)7,8. Such high [Mg 2+ ] not only greatly accelerate RNA degradation9 but cause amphiphile aggregation, precipitation and membrane destabilization10,11, suggesting a fundamental incompatibility of RPR activity with the formation of membranous protocells. However, ribozyme function within the RNA world likely emerged not in isolation but in the context *

Lasso peptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) with a unique 3Dinterlocked structure, in which an N-terminal macrolactam ring is threaded by a linear C-terminal part. The unique structure of lasso peptides is introduced into ribosomally translated precursor peptides by lasso peptide synthetase encompassing proteins B and C or B1, B2, and C when the B enzyme is split into two distinct proteins. The B1 protein recognizes the leader sequence of the precursor peptide, and then the B2 protein cleaves it. The C protein catalyzes the formation of the macrolactam ring. However, the detailed mechanism of lasso peptide maturation has remained elusive, due to the lack of structural information about the responsible proteins. Here we report the crystal structure of the B1 protein from the thermophilic actinobacteria, Thermobifida f usca (TfuB1), complexed with the leader peptide (TfuA-Leader), which revealed the detailed mechanism of leader peptide recognition. The structure of TfuB1 consists of an N-terminal β-sheet and three C-terminal helices. The leader peptide is docked on one edge of the N-terminal β-sheet of TfuB1, as an additional β strand. Three conserved amino acid residues of the leader peptide fit well on the hydrophobic cleft between the β-sheet and adjacent helices. Biochemical analysis demonstrated that these conserved residues are essential for affinity between TfuB1 and the TfuA-Leader. Furthermore, we found that TfuB1 and the leader peptide jointly form a hydrophobic patch on the β-sheet, which includes the highly conserved TfuA Phe-6 and TfuB1 Tyr33. Homology modeling and mutational analysis of the B1 protein from a firmicute, Bacillus pseudomycoides (PsmB1), revealed that the hydrophobic patch is conserved in a wide range of species and involved in the cleavage activity of the B2 protein, indicating it forms the interaction surface for the B2 protein or the core part of the precursor peptide.