東華大學圖書館 |

語系: 繁體中文

說明(常見問題)

回圖書館首頁

手機版館藏查詢

登入

回首頁

切換: 標籤 | MARC模式 | ISBD

Mammalian Translation Termination In...

Dadhwal, Prikshat,

FindBook

Google Book

Amazon

博客來

Mammalian Translation Termination Intermediates Captured Using PDMS Microfluidics-Based Time-Resolved Cryo-EM (TRCEM) /

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	Mammalian Translation Termination Intermediates Captured Using PDMS Microfluidics-Based Time-Resolved Cryo-EM (TRCEM) // Prikshat Dadhwal.
作者:	Dadhwal, Prikshat,
面頁冊數:	1 electronic resource (104 pages)
附註:	Source: Dissertations Abstracts International, Volume: 85-06, Section: B.
Contained By:	Dissertations Abstracts International85-06B.
標題:	Biology. -
電子資源:	https://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=30812219
ISBN:	9798381184815

Mammalian Translation Termination Intermediates Captured Using PDMS Microfluidics-Based Time-Resolved Cryo-EM (TRCEM) /
Dadhwal, Prikshat,

Mammalian Translation Termination Intermediates Captured Using PDMS Microfluidics-Based Time-Resolved Cryo-EM (TRCEM) /Prikshat Dadhwal. - 1 electronic resource (104 pages)

Source: Dissertations Abstracts International, Volume: 85-06, Section: B.

Termination of translation in eukaryotes occurs when a translating ribosome encounters a stop codon (UAA, UAG, or UGA) in its A site. This triggers the recruitment of translation termination factors eRF1, a tRNA-mimicking protein responsible for decoding the stop codon and catalyzing peptide release, and eRF3, a translational GTPase that stimulates peptide release in a GTP-hydrolysis-dependent manner. Upon successful stop codon decoding by eRF1, eRF3 carries out GTP hydrolysis and dissociates from the ribosome. eRF1 subsequently gets accommodated into the peptidyl transferase center (PTC) and catalyzes the release of the nascent peptide. The structures for the pre-accommodated eRF1 with eRF3 trapped on ribosome using non-hydrolysable GTP analogs as well as for the PTC-accommodated eRF1 have been solved using cryogenic electron-microscopy (cryo-EM). The structures reveal the binding mode and interactions between the release factors and the pre-termination complex. However, the mechanism of eRF3 GTPase activation and subsequent eRF1 accommodation into the PTC remains poorly understood. To address this knowledge gap, we used single-particle time-resolved cryo-EM (TRCEM) to capture the structures of intermediates formed during the termination process.For our TRCEM experiments, we first developed a Polydimethylsiloxane (PDMS)-based modular microfluidic mixing-spraying device with a SiO2 internal coating. The device has a SiO2-coated PDMS-based 3D splitting-and-recombination (SAR) micro-mixer capable of mixing two fluids within 0.5 ms with more than 90% efficiency. The SiO2 coating strengthens the PDMS channels and acts as a hydrophilic barrier preventing sample adsorption to the PDMS surface. The micro-mixer is connected to a glass microcapillary that acts as the reaction channel. Channels of different lengths can be used to vary the overall reaction time between 10 ms and 1000 ms. The microcapillary is connected to a 3D micro-sprayer for generating a 3D plume of sprayed droplets. A cryo-EM grid is passed through the spray cone to collect droplets and is rapidly plunged into liquid cryogen for vitrification. By using TRCEM as well as the conventional blotting method for cryo-EM sample preparation, we captured the reaction between a pre-termination (pre-TC) mimic and the ternary complex of eRF1, eRF3, and GTP at reaction times of 450 ms, 900 ms, 15 s, and 10 min. Classification of the cryo-EM data resulted in maps for five distinct factor-bound classes. Four maps belonged to intermediates with densities for eRF1 and eRF3 bound to the pre-TC in varying conformations. The fifth map had a density matching the PTC-accommodated eRF1. Population analysis allowed us to arrange the classes chronologically and track the events leading to GTPase activation during the termination process. Atomic model building and refinement allowed us to determine the hydrolysis state of the eRF3-bound GTP and revealed the catalytic mechanism for GTP-hydrolysis. The models revealed a potential mechanism for GDP dissociation post-GTP-hydrolysis.

English

ISBN: 9798381184815Subjects--Topical Terms:

522710
Biology.
Subjects--Index Terms:

Peptidyl transferase center

Mammalian Translation Termination Intermediates Captured Using PDMS Microfluidics-Based Time-Resolved Cryo-EM (TRCEM) /
LDR:04497nmm a22004213i 4500 001 2400505
005 20250522084141.5
006 m o d
007 cr|nu||||||||
008 251215s2024 miu||||||m |||||||eng d
020 $a 9798381184815
035 $a (MiAaPQD)AAI30812219
035 $a AAI30812219
040 $a MiAaPQD $b eng $c MiAaPQD $e rda
100 1 $a Dadhwal, Prikshat, $e author. $3 3770524
245 1 0 $a Mammalian Translation Termination Intermediates Captured Using PDMS Microfluidics-Based Time-Resolved Cryo-EM (TRCEM) / $c Prikshat Dadhwal.
264 1 $a Ann Arbor : $b ProQuest Dissertations & Theses, $c 2024
300 $a 1 electronic resource (104 pages)
336 $a text $b txt $2 rdacontent
337 $a computer $b c $2 rdamedia
338 $a online resource $b cr $2 rdacarrier
500 $a Source: Dissertations Abstracts International, Volume: 85-06, Section: B.
500 $a Advisors: Frank, Joachim Committee members: Tong, Liang; Hunt, John F.
502 $b Ph.D. $c Columbia University $d 2024.
520 $a Termination of translation in eukaryotes occurs when a translating ribosome encounters a stop codon (UAA, UAG, or UGA) in its A site. This triggers the recruitment of translation termination factors eRF1, a tRNA-mimicking protein responsible for decoding the stop codon and catalyzing peptide release, and eRF3, a translational GTPase that stimulates peptide release in a GTP-hydrolysis-dependent manner. Upon successful stop codon decoding by eRF1, eRF3 carries out GTP hydrolysis and dissociates from the ribosome. eRF1 subsequently gets accommodated into the peptidyl transferase center (PTC) and catalyzes the release of the nascent peptide. The structures for the pre-accommodated eRF1 with eRF3 trapped on ribosome using non-hydrolysable GTP analogs as well as for the PTC-accommodated eRF1 have been solved using cryogenic electron-microscopy (cryo-EM). The structures reveal the binding mode and interactions between the release factors and the pre-termination complex. However, the mechanism of eRF3 GTPase activation and subsequent eRF1 accommodation into the PTC remains poorly understood. To address this knowledge gap, we used single-particle time-resolved cryo-EM (TRCEM) to capture the structures of intermediates formed during the termination process.For our TRCEM experiments, we first developed a Polydimethylsiloxane (PDMS)-based modular microfluidic mixing-spraying device with a SiO2 internal coating. The device has a SiO2-coated PDMS-based 3D splitting-and-recombination (SAR) micro-mixer capable of mixing two fluids within 0.5 ms with more than 90% efficiency. The SiO2 coating strengthens the PDMS channels and acts as a hydrophilic barrier preventing sample adsorption to the PDMS surface. The micro-mixer is connected to a glass microcapillary that acts as the reaction channel. Channels of different lengths can be used to vary the overall reaction time between 10 ms and 1000 ms. The microcapillary is connected to a 3D micro-sprayer for generating a 3D plume of sprayed droplets. A cryo-EM grid is passed through the spray cone to collect droplets and is rapidly plunged into liquid cryogen for vitrification. By using TRCEM as well as the conventional blotting method for cryo-EM sample preparation, we captured the reaction between a pre-termination (pre-TC) mimic and the ternary complex of eRF1, eRF3, and GTP at reaction times of 450 ms, 900 ms, 15 s, and 10 min. Classification of the cryo-EM data resulted in maps for five distinct factor-bound classes. Four maps belonged to intermediates with densities for eRF1 and eRF3 bound to the pre-TC in varying conformations. The fifth map had a density matching the PTC-accommodated eRF1. Population analysis allowed us to arrange the classes chronologically and track the events leading to GTPase activation during the termination process. Atomic model building and refinement allowed us to determine the hydrolysis state of the eRF3-bound GTP and revealed the catalytic mechanism for GTP-hydrolysis. The models revealed a potential mechanism for GDP dissociation post-GTP-hydrolysis.
546 $a English
590 $a School code: 0054
650 4 $a Biology. $3 522710
650 4 $a Biochemistry. $3 518028
650 4 $a Biophysics. $3 518360
653 $a Peptidyl transferase center
653 $a Eukaryotes occurs
653 $a Mammalian
653 $a Microfluidics
690 $a 0306
690 $a 0487
690 $a 0786
710 2 $a Columbia University. $b Biological Sciences. $e degree granting institution. $3 3770495
720 1 $a Frank, Joachim $e degree supervisor.
773 0 $t Dissertations Abstracts International $g 85-06B.
790 $a 0054
791 $a Ph.D.
792 $a 2024
856 4 0 $u https://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=30812219