Structure and function of virion RNA polymerase of crAss-like phage

Abstract

25CrAss-like phages are a recently described family-level group of viruses that includes the most 26 abundant virus in the human gut 1,2 . Genomes of all crAss-like phages encode a large virion-27 packaged protein 2,3 that contains a DFDxD sequence motif, which forms the catalytic site in 28 cellular multisubunit RNA polymerases (RNAPs) 4 . Using Cellulophaga baltica crAss-like phage 29 phi14:2 as a model system, we show that this protein is a novel DNA-dependent RNAP that is 30 translocated into the host cell along with the phage DNA and transcribes early phage genes. We 31 determined the crystal structure of this 2,180-residue enzyme in a self-inhibited, likely pre-virion-32 packaged state. This conformation is attained with the help of a Cleft-blocking domain that 33 interacts with the active site motif and occupies the RNA-DNA hybrid binding grove. Structurally, 34 phi14:2 RNAP is most similar to eukaryotic RNAPs involved in RNA interference 5,6 , although most 35 of phi14:2 RNAP structure (nearly 1,600 residues) maps to a new region of protein folding space. 36Considering the structural similarity, we propose that eukaryal RNA interference polymerases 37 take their origin in a phage, which parallels the emergence of the mitochondrial transcription 38 apparatus 7 . 39 40 Transcription of bacterial, archaeal, and nuclear eukaryal genes is performed by multisubunit 41 DNA-dependent RNA polymerases (RNAPs), complex molecular machines that have a common 42 ancestor 4,8-10 . Their active site is located at the interface of two double-psi β-barrel (DPBB) 43 domains that belong to two different polypeptide chains. One of the DPBB domains carries the 44 universally conserved amino acid motif DFDGD, where the three aspartates coordinate Mg 2+ ions 45 required for catalysis 11,12 . Gene g066 of Cellulophaga baltica crAss-like phage phi14:2 encodes 46 a 2,180-residue protein that shows a limited sequence similarity to one of the two DPBB domains 47 of cellular RNAPs and contains a motif ( 1361 DFDID 1365 ) that is conserved in orthologs of this protein 48 across the crAss-like phage family 2 . Gp66 protein has been identified as a component of the 49 phage particle 3 . We hypothesized that gp66 is an evolutionarily divergent virion-packaged RNAP 50 of phi14:2 that is delivered into the host cell early in the infection process where it transcribes the 51 early phi14:2 genes. To test this hypothesis, we examined the in vitro and in vivo activity of gp66 52 and solved its crystal structure. 53 RNAP gp66 transcribes single-stranded and denatured double-stranded DNA in vitro 54We expressed recombinant gp66 in Escherichia coli, purified it (Extended data Fig. 1), and tested 55 its RNA synthesis activity in a diverse set of assays. 56First, we tested whether gp66 could extend the RNA primer of an 8-nucleotide long RNA-DNA 57 hybrid in the presence of ribonucleoside triphosphates (rNTPs). This hybrid molecule mimics the 58 nucleic acid structure in the transcription elongation complex 13 . Gp66 was inactive in this assay 59whe...

The replication machinery of LUCA: common origin of DNA replication and transcription

“…1a) [27,35]. Unexpectedly, the eRdRPs appear to be most closely related to a particular group of bacteriophage single subunit 2xDPBB RNAPs [27,36]. The two large RNAP subunits are also fused in linear cytoplasmic plasmids from plants and fungi [37], suggesting that such fusions occur repeatedly in the course of RNAP evolution.…”

Long-term persistence of crAss-like phage crAss001 is associated with phase variation inBacteroides intestinalis

“…The predicted products of these genes include a transcriptional regulator, two conserved domain proteins with unknown function and a TROVE domain protein (possible ribonucleoprotein component). A recent study in a distantly related marine crAss-like phage Φ14:2 infecting Cellulophaga baltica indicated that a similarly organised operon of early genes is transcribed by a giant virion-associated multi-subunit RNA polymerase [RNAP, (Drobysheva et al, 2020)], ejected into the cell in the course of infection (Holmfeldt et al, 2013). A homologue of this giant polymerase is present in all crAss-like phages and is also encoded by three large ORFs located in the central part of ΦcrAss001 genome (Guerin et al, 2018;Shkoporov et al, 2018;Yutin et al, 2018).…”

“…Proteomic analysis of the crAss001 virion also indicated the presence of the subunits of the giant polymerase in the assembled phage particle (Shkoporov et al, 2018). Transcription of the middle and late genes is initiated between 10-30 and 30-60 minutes, respectively, and is likely to depend on the action of the host RNAP [as suggested for Φ14:2 in (Drobysheva et al, 2020)]. The middle genes are largely composed of functions associated with DNA replication and recombination, as well as nucleotide metabolism.…”

Unique genomic features of crAss-like phages, the dominant component of the human gut virome

“…The predicted RNAPs of crAss-like phages are extremely divergent in sequence from all known RNAPs and from each other, suggestive of unusually high evolutionary rates. The recent structural and functional study of the RNAP of Cellulophaga baltica phage phi 14:2 of confirms the involvement of this large protein in the transcription of the phage early genes and shows that it contains two double-psi beta-barrel (DPBB) domains within a single polypeptide, unlike cellular RNAPs in which the two DPBB domains belong to the two largest subunits 12 .…”

“…The gene complements of the crAss-like phages shows a number of distinct features, in particular, a predicted complex transcription machinery centered around an RNA polymerase (RNAP) with an unusual strtucture related to the structure of eukaryotic RNA-dependent RNA polymerases involved in RNA inteference. Recently, the prediction of this unique RNAP has been validated by structural and functional analysis 12 . A phylogenomic analysis of 250 genomes of crAss-like phages led to the proposal of 4 distinct subfamilies and 10 genera 13 .…”