Biology subjects

Chaban, A.

Publications and source records attributed to Chaban, A..

3 recordsLinked to original sources

bioRxiv2024

Structural and kinetic insights into tRNA promoter engagement by yeast general transcription factor TFIIIC

Transcription of tRNA genes by RNA polymerase III requires the general transcription factor IIIC (TFIIIC), which recognizes intragenic A-box and B-box DNA motifs of type II gene promoters. However, the underlying mechanism has remained elusive, in part due to missing structural information for A-box recognition. In this study, we use single-particle cryo-EM and single-molecule FRET (smFRET) to reveal structural and real-time kinetic insights into how the 520 kDa yeast TFIIIC complex engages A- and B-box DNA motifs in the context of a tRNA gene promoter. Cryo-EM structures of {tau}A and {tau}B subcomplexes bound to the A- and B-box were obtained at 3.7 and 2.5 [A] resolution, respectively, while cryo-EM single particle mapping determined the specific distance and relative orientation of the {tau}A and {tau}B subcomplexes revealing a fully engaged state of TFIIIC. smFRET experiments show that overall recruitment and residence times of TFIIIC on a tRNA gene are primarily governed by B-box recognition, while footprinting experiments suggest a key role of {tau}A and the A-box in TFIIIB and Pol III recruitment following TFIIIC recognition of type II promoters.

biochemistry↗

bioRxiv2024

A genome-wide genetic screen identified targets for destabilizing the parasitophorous vacuole of Chlamydia trachomatis

The bacterial pathogen Chlamydia trachomatis employs the effector CpoS to suppress a host defense response that aborts intracellular bacterial growth by inducing host cell death. While conducting a CRISPR knock-out screen for genes contributing to this response, we uncovered a mutant deficient for CpoS to display a markedly increased reliance on host cellular ceramide synthesis, compensating for its diminished ability to acquire sphingolipids via modulating membrane trafficking. Employing the power of the just recently established molecular genetic toolbox for Chlamydia, we developed an innovative microscopic reporter system that revealed the mutant to thrive in unstable parasitophorous vacuoles (inclusions), characterized initially by the release of individual bacteria from otherwise intact-appearing vacuoles. CpoS-deficient inclusions were further destabilized by disruptions in ceramide synthesis, while supplementation of sphingoid bases stabilized them, also preventing the defensive host cell death response. Notably, early inclusion destabilization, achieved by simultaneous disruption of two transport routes, caused infection clearance without damaging the host cells. Overall, this study highlights the inclusions role as a refuge, demonstrates CpoS to maintain inclusion integrity by ensuring sphingolipid supply, and provides directions for a future therapeutic exploitation. SIGNIFICANCEA wide range of clinically significant microbes evolved to hide from the intrinsic defenses of their host cells by thriving within membrane-enclosed pathogen-containing vacuoles. This raises the intriguing possibility that such vacuoles could be targeted therapeutically. The bacterial pathogen Chlamydia trachomatis could be an exceptionally well-suited target for such innovative medicines given its medical importance and strict dependence on host cells. However, progress has been stalled by the lack of sensitive tools for detecting inclusion damage. Here, we resolved this major technical roadblock and uncovered the pathogen to employ the secreted effector CpoS, a modulator of membrane trafficking, to stabilize its vacuole by ensuring adequate sphingolipid supply. These methodological advances and mechanistic insights should promote the development of vacuole-destabilizing therapeutics.

microbiology↗

bioRxiv2022

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)1 that defines the length of its extraordinarily long 800 nm tail2,3. We found that the N-terminal portion of P23-45 TMP is an unusual RNA polymerase (RNAP) homologous to cellular and viral two-barrel 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 determined the crystal structures of both P23-45 RNAPs. The non-virion RNAP has a crab claw-like architecture similar to previously reported two-barrel RNAPs. The virion RNAP adopts a unique flat structure without a clamp, which likely reflects the requirement for its extrusion through the narrow channel in the phage tail for delivery into the cell. Structure and sequence comparisons of the P23-45 RNAPs with other phage and cellular 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 remarkable adaptability of two-barrel RNAPs that can be attained within a single virus species.

molecular biology↗