Biology subjects

Penev, P. I.

Publications and source records attributed to Penev, P. I..

7 recordsLinked to original sources

bioRxiv2025

Engineered orthogonal translation systems from metagenomic libraries expand the genetic code

Genetic code expansion with non-canonical amino acids (ncAAs) opens new opportunities for the function and design of proteins by broadening their chemical repertoire. Unfortunately, ncAA incorporation is limited both by a small collection of orthogonal aminoacyl-tRNA synthetases (aaRSs) and tRNAs and by low-throughput methods to discover them. Here, we report the discovery, characterization, and engineering of a UGA suppressing orthogonal translation system mined from metagenomic data. We developed an integrated computational and experimental pipeline to profile the orthogonality of >200 tRNAs, test >1,250 combinations of aaRS:tRNA pairs, and identify the AP1 TrpRS:tRNATrpUCA as an orthogonal pair that natively encodes tryptophan at the UGA codon. We demonstrate that the AP1 TrpRS:tRNATrpUCA is highly active in cell-free and cellular contexts. We then use Ochre, a genomically recoded Escherichia coli strain that lacks UAG and UGA codons, to engineer an AP1 TrpRS variant capable of 5-hydroxytryptophan incorporation at an open UGA codon. We anticipate that our strategy of integrating metagenomic bioprospecting with cell-free screening and cell-based engineering will accelerate the discovery and optimization of orthogonal translation systems for genetic code expansion.

synthetic biology↗

bioRxiv2024

The active subset of grassland soil microbiomes changes with soil depth, water availability and prominently features predatory bacteria and episymbionts

Mediterranean grasslands, vital natural and agricultural ecosystems, experience seasonal variation in water content that likely affect microbial activity. We used metagenomics-informed stable isotope probing to investigate how the activities of microorganisms in Angelo Reserve (2160 mm rainfall) and Hopland (956 mm rainfall) soil change over depth and the seasons. At both sites, we find that the relative abundances of organisms in shallow soil changes relatively little but the most abundant organisms vary greatly with soil depth. Notably the highest levels of isotope incorporation, indicative of growth, occurs in deep soils. The active part of the 0-10 cm soil community varies over time, especially in Hopland soils during the fall rewetting. We defined a large, novel clade of Actinomycetota with notable capacity for thiosulfate oxidation whose representatives are prevalent and active in deep soils (>20 cm) across both ecosystems. Active Saccharibacteria unexpectedly encode nucleotide synthesis genes that enabled isotope incorporation while growing in shallow Angelo soils over all time periods. In contrast to predicted episymbiotic lifestyles of Saccharibacteria, other highly active bacteria are predicted predators. Obligately predatory Pseudobdellovibrio are active in intermediate depth Hopland soils whereas bacteria of the order Haliangiales are active in shallow Angelo soils. Supporting predatory lifestyles of Haliangiales, we used in silico structure prediction to assemble a large protein complex that we identify as a contractile injection system. Overall, the results indicate the potential for active carbon turnover in deep grassland soil and strong seasonal changes in the active members of microbial communities, despite relatively minor shifts in community composition.

bioinformatics↗

bioRxiv2024

Structure of an Archaeal Ribosome with a Divergent Active Site

The ribosome is the universal translator of the genetic code and is shared across all life. Despite divergence in ribosome structure over the course of evolution, the peptidyl transferase center (PTC), the catalytic site of the ribosome, has been thought to be nearly universally conserved. Here, we identify clades of archaea that have highly divergent ribosomal RNA sequences in the PTC. To understand how these PTC sequences fold, we determined cryo-EM structures of the Pyrobaculum calidifontis ribosome. We find that sequence variation leads to the rearrangement of key PTC base triples and differences between archaeal and bacterial ribosomal proteins also enable sequence variation in archaeal PTCs. Finally, we identify a novel archaeal ribosome hibernation factor that differs from known bacterial and eukaryotic hibernation factors and is found in multiple archaeal phyla. Overall, this work identifies factors that regulate ribosome function in archaea and reveals a larger diversity of the most ancient sequences in the ribosome.

evolutionary biology↗

bioRxiv2023

Methanotrophic Methanoperedens archaea host diverse and interacting extrachromosomal elements

Methane emissions that contribute to climate change can be mitigated by anaerobic methane-oxidizing archaea such as Methanoperedens. Some Methanoperedens have huge extrachromosomal genetic elements (ECEs) called Borgs that may modulate their activity, yet the broader diversity of Methanoperedens ECEs is little studied. Here, we report small enigmatic linear ECEs, circular viruses and unclassified ECEs, that we predict replicate within Methanoperedens. The linear ECEs have features such as inverted terminal repeats, pervasive tandem repeats, and coding patterns that are strongly reminiscent of Borgs, but they are only 52 kb to 145 kb in length. They share proteins with Borgs and Methanoperedens. Thus, we refer to them as mini-Borgs. Mini-Borgs are genetically diverse and we assign them to at least five family-level groups. We also identify eight novel families of Methanoperedens viruses, some of which encode multiheme cytochromes, and unclassified circular ECEs that encode TnpB genes. A population-heterogeneous CRISPR array is in close proximity to the TnpB and has spacers that target other Methanoperedens ECEs including previously reported plasmids. The diverse groups of ECEs exchange genetic information with each other and with Methanoperedens, likely impacting the activity and evolution of these environmentally important archaea.

microbiology↗

bioRxiv2022

Rare Ribosomal RNA Sequences from Archaea Stabilize the Bacterial Ribosome

Ribosomes serve as the universally conserved translators of the genetic code into proteins and must support life across temperatures ranging from below freezing to above the boiling point of water. Ribosomes are capable of functioning across this wide range of temperatures even though the catalytic site for peptide bond formation, the peptidyl transferase center, is nearly universally conserved. Peptide bond formation by the ribosome requires correct positioning of the 3 s-end of the aminoacylated tRNA (aa-tRNA) substrate, which is aided by an RNA hairpin in the ribosomal RNA (rRNA) of the large subunit, termed the A loop. Here we find that Thermoproteota, a phylum of thermophilic Archaea, substitute cytidine for uridine at large subunit rRNA positions 2554 and 2555 (Escherichia coli numbering) in the A loop, immediately adjacent to the binding site for the 3'-end of A-site tRNA. We show by cryo-EM that E. coli ribosomes with uridine to cytidine mutations at these positions retain the proper fold and post-transcriptional modification of the A loop. Additionally, these mutations do not exert a dominant negative effect on cellular growth, protect the large ribosomal subunit from thermal denaturation, and increase the mutational robustness of nucleotides in the peptidyl transferase center. This work identifies sequence variation in the peptidyl transferase center of the archaeal ribosome that likely confers stabilization of the ribosome at high temperatures and develops a stable mutant bacterial ribosome that can act as a scaffold for future ribosome engineering efforts.

biophysics↗

bioRxiv2019

Eukaryotic-like ribosomal RNA region in Lokiarchaeota

The ribosomes common core, comprised of ribosomal RNA (rRNA) and universal ribosomal proteins, connects all life back to a common ancestor and serves as a window to relationships among organisms. The rRNA of the common core is most similar to rRNA of extant bacteria. In eukaryotes, the rRNA of the common core is decorated by expansion segments (ESs) that vastly increase its size. Supersized ESs have not been observed previously in Archaea, and the origin of eukaryotic ESs remains enigmatic. We discovered that the large subunit (LSU) rRNA of two Asgard phyla, Lokiarchaeota and Heimdallarchaeota, considered to be the closest modern archaeal cell lineages to Eukarya, bridge the gap in size between prokaryotic and eukaryotic LSU rRNA. The elongated LSU rRNAs in Lokiarchaeota and Heimdallarchaeota stem from the presence of two supersized ESs, ES9 and ES39. We applied chemical footprinting experiments to study the structure of Lokiarchaeota ES39. Furthermore, we used covariation and sequence analysis to study the evolution of Asgard ES39s and ES9s. By defining the common eukaryotic ES39 signature fold, we found that Asgard ES39s have more and longer helices than eukaryotic ES39s. While Asgard ES39s have sequences and structures distinct from eukaryotic ES39s, we found overall conservation of a three-way junction across the Asgard species that matches eukaryotic ES39 topology, a result consistent with the accretion model of ribosomal evolution. SignificanceEukaryotes possess longer and more complex ribosomal RNA (rRNA) than Bacteria and Archaea, including eukaryotic-specific rRNA expansion segments (ESs). The origin and evolution of ESs has long remained a mystery. We show that two recently discovered Asgard archaeal phyla, Lokiarchaeota and Heimdallarchaeota, contain large subunit rRNA with ESs are "supersized" (>100 nt) compared to all other prokaryotes studied to date. Asgard ESs have distinct structures from eukaryotic ESs, but share a common three-way junction out of which the ES grew. Our findings raise the possibility that supersized ESs existed on the ribosomal surface before the last eukaryotic common ancestor, opening the question of whether ribosomal complexity is more deeply rooted than previously thought.

evolutionary biology↗

bioRxiv2018

Dynamic G-quadruplexes on the surface of the human ribosome

Profound similarities and critical differences mark ribosomes across phylogeny. The ribosomal core, approximated by the prokaryotic ribosome, is universal, yet mammalian ribosomes are nearly twice as large as those of prokaryotes. Differences in size are due in part to rRNA expansion segments. Here we show rRNA tentacles of Expansion Segment 7 (ES7) of Homo sapiens can form G-quadruplexes in vitro. G-quadruplex-forming regions are located on the most surface-exposed regions of the ribosome, near the termini of rRNA tentacles. We characterized rRNA of the large ribosomal subunit by computation, circular dichroism, gel mobility, fluorescent probes, nuclease accessibility, electrophoretic mobility shifts and blotting. We investigated ES7 and oligomers derived from ES7, intact 28S rRNA, and 80S ribosomes and polysomes. We used mass spectrometry to identify proteins that bind to rRNA G-quadruplexes in cell lysates. Proteins that associate with rRNA G-quadruplexes include helicases (DDX3, CNBP, DDX21, DDX17) and heterogeneous nuclear ribonucleoproteins (hnRNPs). And finally, by multiple sequence alignments, we observed that G-quadruplex-forming sequences appear to be a general feature LSU rRNA of the phylum Chordata but not in other phyla. It is known that G-quadruplexes form in telomeres, promoters, and untranslated regions of mRNA but, to our knowledge, they have not been reported previously in ribosomes.

biochemistry↗