Biochemical characterizations of DNA packaging enzyme gp16 of bacterial virus phi29

Biochemical characterizations of DNA packaging enzyme gp16 of bacterial virus phi29

Bibliographic Details
Main Author: Lee, Tae Jin
Language:English
Published: University of Cincinnati / OhioLINK 2009
Subjects:
Biochemistry
Biology
Molecular Biology
DNA packaging
Online Access:http://rave.ohiolink.edu/etdc/view?acc_num=ucin1250542532
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spelling ndltd-OhioLink-oai-etd.ohiolink.edu-ucin12505425322021-08-03T06:13:37Z Biochemical characterizations of DNA packaging enzyme gp16 of bacterial virus phi29 Lee, Tae Jin Biochemistry Biology Molecular Biology DNA packaging <p>Bacterial virus phi29 translocates its genomic DNA into a pre-self-assembled protein shell, known as a procapsid, to near-crystalline density. Due to the limitation of the spatial capacity of procapsids, phi29 developed a unique ATP-driven viral DNA packaging motor to complete such an entropically unfavorable DNA packaging process. The phi29 packaging motor obtains mechanical energy via ATP hydrolysis by packaging enzyme gp16, and another key component, packaging RNA (pRNA), binds to the N-terminus of the connector that is a portal channel of procapsid for DNA entry. However, the molecular mechanism of how these components interact with each other and with DNA substrate is still unclear.This study revealed that gp16 binds DNA nonspecifically, and to the 5’/3’ paired helical region of pRNA bound to procapsid connector. It suggests that gp16 serves as a linkage between pRNA and DNA, and as an essential DNA-contacting component during DNA translocation. Further kinetic studies on the ATPase activity of gp16 showed that the presence of DNA or pRNA greatly boosted the catalytic activity and the affinity of gp16 for ATP. The stimulation order of ATPase activity is poly d(pyrimidine) > dsDNA > poly d(purine), which agreed with the order of the DNA binding to gp16, as revealed by single molecule fluorescence microscopy. Interestingly, pRNA showed also stimulatory effect on the ATPase activity of gp16 with a similar extent of poly d(pyrimidine). The results suggest that gp16 itself is a slow-ATPase with a low affinity for substrate, and that interaction with both pRNA and genomic DNA affects the enzymatic function of gp16 during DNA packaging.</p> <p>For nanotechnological application of gp16, the gene of gp16 was genetically engineered to carry the fluorescent fusion protein (eGFP). The resulting eGFP-gp16 was shown to be biologically active in both ATP hydrolysis and DNA packaging. The eGFP-gp16 was successfully used as a marker for direct observation of DNA packaging and for direct counting of gp16 number in single molecule microscopy studies. Such engineering of gp16 will facilitate future studies on structure, function, distance, speed and motion mechanism of the phi29 DNA packaging motor by single molecule imaging.</p> 2009-12-04 English text University of Cincinnati / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=ucin1250542532 http://rave.ohiolink.edu/etdc/view?acc_num=ucin1250542532 unrestricted This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws.
collection NDLTD
language English
sources NDLTD
topic Biochemistry
Biology
Molecular Biology
DNA packaging
spellingShingle Biochemistry
Biology
Molecular Biology
DNA packaging
Lee, Tae Jin
Biochemical characterizations of DNA packaging enzyme gp16 of bacterial virus phi29
author Lee, Tae Jin
author_facet Lee, Tae Jin
author_sort Lee, Tae Jin
title Biochemical characterizations of DNA packaging enzyme gp16 of bacterial virus phi29
title_short Biochemical characterizations of DNA packaging enzyme gp16 of bacterial virus phi29
title_full Biochemical characterizations of DNA packaging enzyme gp16 of bacterial virus phi29
title_fullStr Biochemical characterizations of DNA packaging enzyme gp16 of bacterial virus phi29
title_full_unstemmed Biochemical characterizations of DNA packaging enzyme gp16 of bacterial virus phi29
title_sort biochemical characterizations of dna packaging enzyme gp16 of bacterial virus phi29
publisher University of Cincinnati / OhioLINK
publishDate 2009
url http://rave.ohiolink.edu/etdc/view?acc_num=ucin1250542532
work_keys_str_mv AT leetaejin biochemicalcharacterizationsofdnapackagingenzymegp16ofbacterialvirusphi29
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