Mariusz Czarnocki-Cieciura
@mariuszczc.bsky.social
Structural biologist in the Nowotny lab at IIMCB Warsaw, Poland #cryoEM
The article was published in JBC and is available here:
www.jbc.org/article/S002...
I wish to thank all the authors from the Nowotny Lab @ IIMCB Warsaw and all the collaborators!
www.jbc.org/article/S002...
I wish to thank all the authors from the Nowotny Lab @ IIMCB Warsaw and all the collaborators!
Structural and biochemical characterization of the 3'-5' tRNA splicing ligases
In archaea and metazoa, tRNA exons are ligated by the RNA ligases RtcB and RTCB, respectively.
The metazoan RTCB forms a stable complex with four additional subunits, DDX1, FAM98B,
CGI99 and ASHWIN. T...
www.jbc.org
April 15, 2025 at 10:17 AM
The article was published in JBC and is available here:
www.jbc.org/article/S002...
I wish to thank all the authors from the Nowotny Lab @ IIMCB Warsaw and all the collaborators!
www.jbc.org/article/S002...
I wish to thank all the authors from the Nowotny Lab @ IIMCB Warsaw and all the collaborators!
Finally, we also performed a chemical cross-linking analysis of the archaeal RtcB protein with its RNA substrate. We observed specific protein-RNA X-links, confirming that the mode of substrate binding is very similar to the AlphaFold3 predictions and observed for the DNA mimic.
April 15, 2025 at 10:16 AM
Finally, we also performed a chemical cross-linking analysis of the archaeal RtcB protein with its RNA substrate. We observed specific protein-RNA X-links, confirming that the mode of substrate binding is very similar to the AlphaFold3 predictions and observed for the DNA mimic.
We also observed that the tRNA ligase complex with truncated DDX1 helicase showed slightly higher activity. These results suggest that the mobile DDX1 subunit has a negative effect on the ligase activity.
April 15, 2025 at 10:15 AM
We also observed that the tRNA ligase complex with truncated DDX1 helicase showed slightly higher activity. These results suggest that the mobile DDX1 subunit has a negative effect on the ligase activity.
AlphaFold 3 predictions suggest that the tRNA interacts only with a small region of the RtcB protein, which would make it quite mobile. Interestingly, the same region is also predicted to be involved in DDX1 binding.
April 15, 2025 at 10:14 AM
AlphaFold 3 predictions suggest that the tRNA interacts only with a small region of the RtcB protein, which would make it quite mobile. Interestingly, the same region is also predicted to be involved in DDX1 binding.
Despite numerous attempts, we have not been able to obtain a structure with a tRNA substrate. We also did not observe any additional density for the – presumably highly mobile – DDX1 catalytic domains.
April 15, 2025 at 10:14 AM
Despite numerous attempts, we have not been able to obtain a structure with a tRNA substrate. We also did not observe any additional density for the – presumably highly mobile – DDX1 catalytic domains.
To improve the stability and overall behavior of the sample, we reconstituted the complex with truncated variants of the ASHWIN and DDX1 subunits. We verified the functionality of such truncated complex by testing its stability, substrate binding and enzymatic activity in vitro.
April 15, 2025 at 10:13 AM
To improve the stability and overall behavior of the sample, we reconstituted the complex with truncated variants of the ASHWIN and DDX1 subunits. We verified the functionality of such truncated complex by testing its stability, substrate binding and enzymatic activity in vitro.