An ancestral fold reveals the evolutionary link between RNA polymerase and ribosomal proteins.

Numerous molecular machines are required to drive the central dogma of molecular biology. However, the means by which these numerous proteins emerged in the early evolutionary stage of life remains enigmatic. Many of them possess small ß-barrel folds with different topologies, represented by double-psi ß-barrels (DPBBs) conserved in DNA and RNA polymerases, and similar but topologically distinct six-stranded ß-barrel RIFT or five-stranded ß-barrel folds such as OB and SH3 in ribosomal proteins. Here, we discover that the previously reconstructed ancient DPBB sequence could also adopt a ß-barrel fold named Double-Zeta ß-barrel (DZBB), as a metamorphic protein. The DZBB fold is not found in any modern protein, although its structure shares similarities with RIFT and OB. Indeed, DZBB could be transformed into them through simple engineering experiments. Furthermore, the OB designs could be further converted into SH3 by circular-permutation as previously predicted. These results indicate that these ß-barrels diversified quickly from a common ancestor at the beginning of the central dogma evolution.

Tail-tape-fused virion and non-virion RNA polymerases of a thermophilic virus with an extremely long tail.

Thermus thermophilus bacteriophage P23-45 encodes a giant 5,002-residue tail tape measure protein (TMP) that defines the length of its extraordinarily long tail. Here, we show that the N-terminal portion of P23-45 TMP is an unusual RNA polymerase (RNAP) homologous to cellular RNAPs. The TMP-fused virion RNAP transcribes pre-early phage genes, including a gene that encodes another, non-virion RNAP, that transcribes early and some middle phage genes. We report the crystal structures of both P23-45 RNAPs. The non-virion RNAP has a crab-claw-like architecture. By contrast, the virion RNAP adopts a unique flat structure without a clamp. Structure and sequence comparisons of the P23-45 RNAPs with other RNAPs suggest that, despite the extensive functional differences, the two P23-45 RNAPs originate from an ancient gene duplication in an ancestral phage. Our findings demonstrate striking adaptability of RNAPs that can be attained within a single virus species.

The origin of life: RNA and protein co-evolution on the ancient Earth.

How life emerged from simple non-life chemicals on the ancient Earth is one of the greatest mysteries in biology. The gene expression system of extant life is based on the interdependence between multiple molecular species (DNA, RNA, and proteins). While DNA is mainly used as genetic material and proteins as functional molecules in modern biology, RNA serves as both genetic material and enzymes (ribozymes). Thus, the evolution of life may have begun with the birth of a ribozyme that replicated itself (the RNA world hypothesis), and proteins and DNA joined later. However, the complete self-replication of ribozymes from monomeric substrates has not yet been demonstrated experimentally, due to their limited activity and stability. In contrast, peptides are more chemically stable and are considered to have existed on the ancient Earth, leading to the hypothesis of RNA-peptide co-evolution from the very beginning. Our group and collaborators recently demonstrated that (1) peptides with both hydrophobic and cationic moieties (e.g., KKVVVVVV) form ß-amyloid aggregates that adsorb RNA and enhance RNA synthesis by an artificial RNA polymerase ribozyme and (2) a simple peptide with only seven amino acid types (especially rich in valine and lysine) can fold into the ancient ß-barrel conserved in various enzymes, including the core of cellular RNA polymerases. These findings, together with recent reports from other groups, suggest that simple prebiotic peptides could have supported the ancient RNA-based replication system, gradually folded into RNA-binding proteins, and eventually evolved into complex proteins like RNA polymerase.

Lasso peptide microcin J25 variant containing RGD motif as a PET probe for integrin a v ß 3 in tumor imaging.

Microcin J25 (MccJ25), a lasso peptide, has a unique 3-D interlocked structure that provides high stability under acidic conditions, at high temperatures, and in the presence of proteases. In this study, we generated a positron emission tomography (PET) probe based on MccJ25 analog with an RGD motif and investigated their pharmacokinetics and utility for integrin αvß3 imaging in tumors. The MccJ25 variant with an RGD motif in the loop region and a lysine substitution at the C-terminus (MccJ25(RGDF)GtoK) was produced in E. coli transfected with plasmid DNA containing the MccJ25 biosynthetic gene cluster (mcjABCD). [64Cu]Cu-MccJ25(RGDF)GtoK was synthesized using the C-terminal lysine labeled with copper-64 (t1/2 = 12.7 h) via a bifunctional chelator; it showed stability in 90% mouse plasma for 45 min. Using PET imaging for integrin αvß3 positive U87MG tumor bearing mice, [64Cu]Cu-MccJ25(RGDF)GtoK could clearly distinguish the tumor, and its accumulation was significantly higher than that of MccJ25(GIGT)GtoK without the binding motif for integrin αvß3. Furthermore, MccJ25(RGDF)GtoK enabled visualization of only U87MG tumors but not MCF-7 tumors with low integrin αvß3 expression in double tumor-bearing mice. In ex vivo biodistribution analysis, the integrin αvß3 non-specific accumulation of [64Cu]Cu-MccJ25(RGDF)GtoK was significantly lower in various tissues, except for the kidneys, as compared to the control probe ([64Cu]Cu-cyclic RGD peptide). These results of the present study indicate that 64Cu-labeling methods are appropriate for the synthesis of MccJ25-based PET probes, and [64Cu]Cu-MccJ25 variants are useful tools for cancer molecular imaging.

Why we are made of proteins and nucleic acids: Structural biology views on extraterrestrial life.

Is it a miracle that life exists on the Earth, or is it a common phenomenon in the universe? If extraterrestrial organisms exist, what are they like? To answer these questions, we must understand what kinds of molecules could evolve into life, or in other words, what properties are generally required to perform biological functions and store genetic information. This review summarizes recent findings on simple ancestral proteins, outlines the basic knowledge in textbooks, and discusses the generally required properties for biological molecules from structural biology viewpoints (e.g., restriction of shapes, and types of intra- and intermolecular interactions), leading to the conclusion that proteins and nucleic acids are at least one of the simplest (and perhaps very common) forms of catalytic and genetic biopolymers in the universe. This review article is an extended version of the Japanese article, On the Origin of Life: Coevolution between RNA and Peptide, published in SEIBUTSU BUTSURI Vol. 61, p. 232-235 (2021).

Hydrophobic-cationic peptides modulate RNA polymerase ribozyme activity by accretion.

Accretion and the resulting increase in local concentration is a widespread mechanism in biology to enhance biomolecular functions (for example, in liquid-liquid demixing phases). Such macromolecular aggregation phases (e.g., coacervates, amyloids) may also have played a role in the origin of life. Here, we report that a hydrophobic-cationic RNA binding peptide selected by phage display (P43: AKKVWIIMGGS) forms insoluble amyloid-containing aggregates, which reversibly accrete RNA on their surfaces in an RNA-length and Mg2+-concentration dependent manner. The aggregates formed by P43 or its sequence-simplified version (K2V6: KKVVVVVV) inhibited RNA polymerase ribozyme (RPR) activity at 25 mM MgCl2, while enhancing it significantly at 400 mM MgCl2. Our work shows that such hydrophobic-cationic peptide aggregates can reversibly concentrate RNA and enhance the RPR activity, and suggests that they could have aided the emergence and evolution of longer and functional RNAs in the fluctuating environments of the prebiotic earth.

Cell-Free Mutant Analysis Combined with Structure Prediction of a Lasso Peptide Biosynthetic Protease B2.

Biochemical and structural analyses of purified proteins are essential for the understanding of their properties. However, many proteins are unstable and difficult to purify, hindering their characterization. The B2 proteins of the lasso peptide biosynthetic pathways are cysteine proteases that cleave precursor peptides during the maturation process. The B2 proteins are poorly soluble, and no experimentally solved structures are available. Here, we performed a rapid semicomprehensive mutational analysis of the B2 protein from the thermophilic actinobacterium, Thermobifida fusca (FusB2), using a cell-free transcription/translation system, and compared the results with the structure prediction by AlphaFold2. Analysis of 34 FusB2 mutants with substitutions of hydrophobic residues confirmed the accuracy of the predicted structure, and revealed a hydrophobic patch on the protein surface, which likely serves as the binding site of the partner protein, FusB1. Our results suggest that the combination of rapid cell-free mutant analyses with precise structure predictions can greatly accelerate structure-function research of proteins for which no structures are available.

Seven Amino Acid Types Suffice to Create the Core Fold of RNA Polymerase.

The extant complex proteins must have evolved from ancient short and simple ancestors. The double-ψ ß-barrel (DPBB) is one of the oldest protein folds and conserved in various fundamental enzymes, such as the core domain of RNA polymerase. Here, by reverse engineering a modern DPBB domain, we reconstructed its plausible evolutionary pathway started by "interlacing homodimerization" of a half-size peptide, followed by gene duplication and fusion. Furthermore, by simplifying the amino acid repertoire of the peptide, we successfully created the DPBB fold with only seven amino acid types (Ala, Asp, Glu, Gly, Lys, Arg, and Val), which can be coded by only GNN and ARR (R = A or G) codons in the modern translation system. Thus, the DPBB fold could have been materialized by the early translation system and genetic code.

Development of p53 knockout U87MG cell line for unbiased drug delivery testing system using CRISPR-Cas9 and transcriptomic analysis.

Antibody-drug conjugates offers many advantages as a drug delivery platform that allows for highly specific targeting of cell types and genes. Ideally, testing the efficacy of these systems requires two cell types to be different only in the gene targeted by the drug, with the rest of the cellular machinery unchanged, in order to minimize other potential differences from obscuring the effects of the drug. In this study, we created multiple variants of U87MG cells with targeted mutation in the TP53 gene using the CRISPR-Cas9 system, and determined that their major transcriptional differences stem from the loss of p53 function. Using the transcriptome data, we predicted which mutant clones would have less divergent phenotypes from the wild type and thereby serve as the best candidates to be used as drug delivery testing platforms. Further in vitro and in vivo assays of cell morphology, proliferation rate and target antigen-mediated uptake supported our predictions. Based on the combined analysis results, we successfully selected the best qualifying mutant clone. This study serves as proof-of-principle of the approach and paves the way for extending to additional cell types and target genes.

Structural Basis of Leader Peptide Recognition in Lasso Peptide Biosynthesis Pathway.

Lasso peptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) with a unique 3D-interlocked 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 fusca (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 ß-sheet and three C-terminal helices. The leader peptide is docked on one edge of the N-terminal ß-sheet of TfuB1, as an additional ß strand. Three conserved amino acid residues of the leader peptide (TfuA Tyr-17, Pro-14, and Leu-12) fit well on the hydrophobic cleft between the ß-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 ß-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.

Simple peptides derived from the ribosomal core potentiate RNA polymerase ribozyme function.

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 enhance RNA polymerase ribozyme (RPR) function, as do derived homopolymeric peptides comprising lysine or the non-proteinogenic lysine analogues ornithine or, to a lesser extent, diaminobutyric acid, irrespective of chirality or chiral purity. Lysine decapeptides enhance RPR function by promoting holoenzyme assembly through primer-template docking, accelerate RPR evolution, and allow RPR-catalysed RNA synthesis at near physiological (≥1 mM) Mg2+ 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.

Structural basis for promoter specificity switching of RNA polymerase by a phage factor.

Transcription of DNA to RNA by DNA-dependent RNA polymerase (RNAP) is the first step of gene expression and a major regulation point. Bacteriophages hijack their host's transcription machinery and direct it to serve their needs. The gp39 protein encoded by Thermus thermophilus phage P23-45 binds the host's RNAP and inhibits transcription initiation from its major "-10/-35" class promoters. Phage promoters belonging to the minor "extended -10" class are minimally inhibited. We report the crystal structure of the T. thermophilus RNAP holoenzyme complexed with gp39, which explains the mechanism for RNAP promoter specificity switching. gp39 simultaneously binds to the RNAP ß-flap domain and the C-terminal domain of the σ subunit (region 4 of the σ subunit [σ4]), thus relocating the ß-flap tip and σ4. The ~45 Å displacement of σ4 is incompatible with its binding to the -35 promoter consensus element, thus accounting for the inhibition of transcription from -10/-35 class promoters. In contrast, this conformational change is compatible with the recognition of extended -10 class promoters. These results provide the structural bases for the conformational modulation of the host's RNAP promoter specificity to switch gene expression toward supporting phage development for gp39 and, potentially, other phage proteins, such as T4 AsiA.

Structural basis of transcription by bacterial and eukaryotic RNA polymerases.

DNA-dependent RNA polymerase (RNAP) is responsible for cellular gene transcription. Although crystallographic studies on prokaryotic and eukaryotic RNAPs have elucidated the basic RNAP architectures, the structural details of many essential events during transcription initiation, elongation, and termination are still largely unknown. Recent crystallographic studies on a bacterial RNAP and yeast RNAP II have revealed different RNAP structural states from that of the normal transcribing complex, as well as the basis of transcription factor functions, advancing our understanding of transcription. These studies have highlighted unexpected similarities in many fundamental aspects of transcription mechanisms between the bacterial and eukaryotic transcription machineries. Remarkable differences also exist between the bacterial and eukaryotic transcription systems, suggesting directions for future studies.

A novel conformation of RNA polymerase sheds light on the mechanism of transcription.

Transcription is a complicated, multistep process requiring stringent control. Its accuracy may be achieved in part by the conformational changes of RNA polymerase (RNAP). Here, we discuss the functional relevance of the recently reported conformational changes of RNAP, which may affect transcription control, RNAP translocation and transcription termination.

Crystal structure of bacterial RNA polymerase bound with a transcription inhibitor protein.

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 ß-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.

Crystallization and preliminary X-ray crystallographic analysis of Thermus thermophilus transcription elongation complex bound to Gfh1.

RNA polymerase (RNAP) elongates RNA by iterative nucleotide-addition cycles (NAC). A specific structural state (or states) of RNAP may be the target of transcription elongation factors. Gfh1, a Thermus thermophilus Gre-family protein, inhibits NAC. To elucidate which RNAP structural state Gfh1 associates with, the T. thermophilus RNAP elongation complex (EC) was cocrystallized with Gfh1. Of the 70 DNA/RNA scaffolds tested, two (for EC1 and EC2) were successfully crystallized. In the presence of Gfh1, EC1 and EC2 yielded crystals belonging to space group P2(1) with similar unit-cell parameters (crystals 1 and 2, respectively). X-ray diffraction data sets were obtained at 3.6 and 3.8 A resolution, respectively.