東華大學圖書館 |

Language: English

Help

回圖書館首頁

手機版館藏查詢

Back

Switch To: Labeled | MARC Mode | ISBD

Reengineering the Uridine-54 tRNA Me...

Smith, Tyler Samuel.

Linked to FindBook

Google Book

Amazon

博客來

Reengineering the Uridine-54 tRNA Methyltransferase, TrmA, to Covalently Capture New RNA Substrates.

Record Type:	Electronic resources : Monograph/item
Title/Author:	Reengineering the Uridine-54 tRNA Methyltransferase, TrmA, to Covalently Capture New RNA Substrates./
Author:	Smith, Tyler Samuel.
Published:	Ann Arbor : ProQuest Dissertations & Theses, : 2019,
Description:	90 p.
Notes:	Source: Dissertations Abstracts International, Volume: 81-10, Section: B.
Contained By:	Dissertations Abstracts International81-10B.
Subject:	Biochemistry. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=22615902
ISBN:	9798607313784

Reengineering the Uridine-54 tRNA Methyltransferase, TrmA, to Covalently Capture New RNA Substrates.
Smith, Tyler Samuel.

Reengineering the Uridine-54 tRNA Methyltransferase, TrmA, to Covalently Capture New RNA Substrates. - Ann Arbor : ProQuest Dissertations & Theses, 2019 - 90 p.

Source: Dissertations Abstracts International, Volume: 81-10, Section: B.

Thesis (Ph.D.)--Yale University, 2019.

This item must not be sold to any third party vendors.

Cellular RNAs can be regulated by enzymes that covalently modify specific RNA substrates. Altering the RNA specificities of these enzymes can provide powerful tools to study and manipulate cellular RNAs. I studied the RNA binding properties of the uridine-54 tRNA methyltransferase, TrmA, using a mutant of the enzyme (TrmA*) that covalently binds substrate RNA. This covalent binding protein allowed me to use high-throughput sequencing methods to examine the RNA substrates of TrmA in a way that would be impossible to do using traditional methyltransferase assays. In addition to substrate screening, I employed rational design based on the crystal structure of TrmA and its substrate to reengineer the enzyme and substrate RNA specificity. This engineered interaction resulted in a substantial change in TrmA binding specificity, but not a complete change. Combining rational design of TrmA and high-throughput substrate screening, I discovered a triple mutant of the substrate RNA (C56A A58G C60U) that was bound by a TrmA* double mutant (E49R R51E) but not by the wild type enzyme.In parallel to my work exploring the specificity of TrmA, I have worked to use TrmA* to form covalent RNA-protein adducts. One possible application of this would be to tag an RNA of interest and use TrmA* to pull it down. In order to study RNA-protein interactions, researchers employ a variety of affinity tags to capture an RNA of interest. The ability to purify RNA-protein complexes are limited by low affinity binding, which limits the wash conditions that are available to remove background contamination. Because TrmA* covalently binds RNA, I am less limited in the wash conditions and have successfully pulled down tagged RNA in vivo and in vitro using TrmA* coupled with fully denaturing washes. While I have not solved the problem of capturing RNA-protein complexes from crosslinked extracts, I have laid the foundations for future researchers to use this covalent affinity tagging system. Combining my discoveries of altered TrmA specificity with covalent capture will allow researchers to biochemically purify an RNA of interest with fully denaturing washes in order to identify bound proteins with high confidence.Another application I have explored with TrmA* is to tag an RNA transcript both 5' and 3' with TrmA substrate RNA and coexpress TrmA*. This forms protein crosslinks in vivo, which could function in a manner similar to sno-lncRNAs to block exonuclease degradation and stabilize transcripts. My preliminary results expressing TrmA with a tagged luciferase transcript show that this may be possible and could be a valuable tool for stabilizing RNAs of interest. My doctoral research on reengineering TrmA provides the groundwork for future researchers to covalently bind RNA with TrmA in a variety of ways similar, but not restricted to, those described here.

ISBN: 9798607313784Subjects--Topical Terms:

518028
Biochemistry.
Subjects--Index Terms:

Covalent binding

Reengineering the Uridine-54 tRNA Methyltransferase, TrmA, to Covalently Capture New RNA Substrates.
LDR:04013nmm a2200361 4500 001 2272732
005 20201105110209.5
008 220629s2019 ||||||||||||||||| ||eng d
020 $a 9798607313784
035 $a (MiAaPQ)AAI22615902
035 $a AAI22615902
040 $a MiAaPQ $c MiAaPQ
100 1 $a Smith, Tyler Samuel. $3 3550158
245 1 0 $a Reengineering the Uridine-54 tRNA Methyltransferase, TrmA, to Covalently Capture New RNA Substrates.
260 1 $a Ann Arbor : $b ProQuest Dissertations & Theses, $c 2019
300 $a 90 p.
500 $a Source: Dissertations Abstracts International, Volume: 81-10, Section: B.
500 $a Advisor: Simon, Matthew D.
502 $a Thesis (Ph.D.)--Yale University, 2019.
506 $a This item must not be sold to any third party vendors.
520 $a Cellular RNAs can be regulated by enzymes that covalently modify specific RNA substrates. Altering the RNA specificities of these enzymes can provide powerful tools to study and manipulate cellular RNAs. I studied the RNA binding properties of the uridine-54 tRNA methyltransferase, TrmA, using a mutant of the enzyme (TrmA*) that covalently binds substrate RNA. This covalent binding protein allowed me to use high-throughput sequencing methods to examine the RNA substrates of TrmA in a way that would be impossible to do using traditional methyltransferase assays. In addition to substrate screening, I employed rational design based on the crystal structure of TrmA and its substrate to reengineer the enzyme and substrate RNA specificity. This engineered interaction resulted in a substantial change in TrmA binding specificity, but not a complete change. Combining rational design of TrmA and high-throughput substrate screening, I discovered a triple mutant of the substrate RNA (C56A A58G C60U) that was bound by a TrmA* double mutant (E49R R51E) but not by the wild type enzyme.In parallel to my work exploring the specificity of TrmA, I have worked to use TrmA* to form covalent RNA-protein adducts. One possible application of this would be to tag an RNA of interest and use TrmA* to pull it down. In order to study RNA-protein interactions, researchers employ a variety of affinity tags to capture an RNA of interest. The ability to purify RNA-protein complexes are limited by low affinity binding, which limits the wash conditions that are available to remove background contamination. Because TrmA* covalently binds RNA, I am less limited in the wash conditions and have successfully pulled down tagged RNA in vivo and in vitro using TrmA* coupled with fully denaturing washes. While I have not solved the problem of capturing RNA-protein complexes from crosslinked extracts, I have laid the foundations for future researchers to use this covalent affinity tagging system. Combining my discoveries of altered TrmA specificity with covalent capture will allow researchers to biochemically purify an RNA of interest with fully denaturing washes in order to identify bound proteins with high confidence.Another application I have explored with TrmA* is to tag an RNA transcript both 5' and 3' with TrmA substrate RNA and coexpress TrmA*. This forms protein crosslinks in vivo, which could function in a manner similar to sno-lncRNAs to block exonuclease degradation and stabilize transcripts. My preliminary results expressing TrmA with a tagged luciferase transcript show that this may be possible and could be a valuable tool for stabilizing RNAs of interest. My doctoral research on reengineering TrmA provides the groundwork for future researchers to covalently bind RNA with TrmA in a variety of ways similar, but not restricted to, those described here.
590 $a School code: 0265.
650 4 $a Biochemistry. $3 518028
650 4 $a Biophysics. $3 518360
653 $a Covalent binding
653 $a Protein engineering
653 $a RNA methyltransferase
653 $a TrmA
653 $a tRNA
690 $a 0487
690 $a 0786
710 2 $a Yale University. $b Molecular Biophysics and Biochemistry. $3 3540261
773 0 $t Dissertations Abstracts International $g 81-10B.
790 $a 0265
791 $a Ph.D.
792 $a 2019
793 $a English
856 4 0 $u http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=22615902