Biology subjects

Osuna, B. A.

Publications and source records attributed to Osuna, B. A..

3 recordsLinked to original sources

bioRxiv2018

Temperature-responsive competitive inhibition of CRISPR-Cas9

CRISPR-Cas immune systems utilize RNA-guided nucleases to protect bacteria from bacteriophage infection. Bacteriophages have in turn evolved inhibitory anti-CRISPR (Acr) proteins, including six inhibitors (AcrIIA1-6) that can block DNA cutting and genome editing by type II-A CRISPR-Cas9 enzymes. We show here that AcrIIA2 and its homologue, AcrIIA2b, prevent Cas9 binding to DNA by occluding protein residues required for DNA binding. Cryo-EM-determined structures of AcrIIA2 or AcrIIA2b bound to S. pyogenes Cas9 reveal a mode of competitive inhibition of DNA binding that is distinct from other known Acrs. Differences in the temperature dependence of Cas9 inhibition by AcrIIA2 and AcrIIA2b arise from differences in both inhibitor structure and the local inhibitor-binding environment on Cas9. These findings expand the natural toolbox for regulating CRISPR-Cas9 genome editing temporally, spatially and conditionally.

biochemistry↗

bioRxiv2018

Vms1p is a release factor for the Ribosome-associated Quality control Complex

Eukaryotic cells employ the Ribosome-associated Quality control Complex (RQC) to maintain homeostasis despite defects that cause ribosomes to stall. The RQC comprises the E3 ubiquitin ligase Ltn1p, the ATPase Cdc48p, and the novel proteins Rqc1p and Rqc2p1-3. Following recognition and subunit splitting of stalled ribosomes, the RQC detects and assembles on 60S subunits that hold incomplete polypeptides linked to a tRNA (60S:peptidyl-tRNA)4-8. Ltn1p cooperates with Rqc1p to facilitate ubiquitination of the incomplete nascent chain, marking it for degradation7,9,10. Rqc2p stabilizes Ltn1p on the 60S3-5,8 and recruits charged tRNAs to the 60S to catalyze elongation of the nascent protein with Carboxy-terminal Alanine and Threonine extensions, or CAT tails, via a mechanism that is distinct from canonical translation4,10. CAT-tailing mobilizes and exposes lysine residues in the nascent chain, especially those stalled within the exit tunnel, thereby supporting efficient ubiquitination10,11. If the ubiquitin-proteasome system is overwhelmed or unavailable, CAT-tailed nascent chains aggregate in the cytosol or within organelles like the mitochondria12-14. Here we identify Vms1p as the tRNA hydrolase that releases nascent polypeptides for extraction and degradation in the RQC pathway.

biochemistry↗

bioRxiv2018

Bacteriophage cooperation suppresses CRISPR-Cas3 and Cas9 immunity

>Bacteria utilize CRISPR-Cas adaptive immune systems for protection from bacteriophages (phages), and some phages produce anti-CRISPR (Acr) proteins that inhibit immune function. Despite thorough mechanistic and structural information for some Acr proteins, how they are deployed and utilized by a phage during infection is unknown. Here, we show that Acr production does not guarantee phage replication, but instead, infections fail when phage population numbers fall below a critical threshold. Failing infections can be rescued by related phages that act as Acr donors, demonstrating that infections succeed if a sufficient Acr dose is contributed to a single cell by multiple phage genomes. The production of Acr proteins by phage genomes that fail to replicate leave the cell immunosuppressed, which predisposes the cell for successful infection by other phages in the population. This \"cooperative\" phage mechanism for CRISPR-Cas inhibition demonstrates inter-virus cooperation that may also manifest in other host-parasite interactions.

microbiology↗