Structure and Function Study of Phi29 DNA packaging motor
type: CreativeWork
description: A powerful nanomotor is employed by the tailed dsDNA virus to package the genome into a preformed protein shell during the process of replication. The bacteriophage phi29 is an excellent model for investigating the viral DNA packaging mechanism. The phi29 DNA packaging motor is composed of three ring structures: the dodecameric connector ring, the hexameric pRNA ring and the hexameric ATPase gp16 ring. The connector is the central hub for the DNA to enter and to exit. There are four positively charged lysine rings scattered inside the highly negatively charged connector channel. It is speculated that these positive charged lysine rings may play active roles during DNA packaging in many models. To test this prevalent view, the basic lysine residues were mutated to neutral alanines and the pH environment was altered. Amazingly, the results were beyond expectation. Neither the DNA translocation nor the one-way traffic property of the channel were measurably influenced by the alteration of the charge of lysine residues when the basic lysine residues mutated to neutral alanines or the pH environment changed to acid or basic. The ATPase or the terminase is the central part of the viral DNA packaging motor. The phi29 ATPase is highly hydrophobic and tends to aggregate in solution. A green fluorescent protein tag (eGFP) fused to the N-terminus of gp16 enhanced its solubility and stability. The eGFP-gp16 showed similar activity to wild type gp16 and was easily detected by fluorescent instruments. The interaction between eGFP-gp16 and DNA in the various conditions were investigated by electrophoretic mobility shift assay, FRET and sucrose gradient. γ-S-ATP dramatically increased gp16 binding affinity to DNA and ATP, ADP, phosphate could release gp16 from gp16-DNA-γ-S-ATP complex. The sliding of gp16 out of the gp16-DNA-γ-S-ATP complex could be blocked by addition of Steptavidin to ends of dsDNA which is conjugated with biotins. Also, we found that six eGFP-gp16 molecules were…
abstract: A powerful nanomotor is employed by the tailed dsDNA virus to package the genome into a preformed protein shell during the process of replication. The bacteriophage phi29 is an excellent model for investigating the viral DNA packaging mechanism. The phi29 DNA packaging motor is composed of three ring structures: the dodecameric connector ring, the hexameric pRNA ring and the hexameric ATPase gp16 ring. The connector is the central hub for the DNA to enter and to exit. There are four positively charged lysine rings scattered inside the highly negatively charged connector channel. It is speculated that these positive charged lysine rings may play active roles during DNA packaging in many models. To test this prevalent view, the basic lysine residues were mutated to neutral alanines and the pH environment was altered. Amazingly, the results were beyond expectation. Neither the DNA translocation nor the one-way traffic property of the channel were measurably influenced by the alteration of the charge of lysine residues when the basic lysine residues mutated to neutral alanines or the pH environment changed to acid or basic. The ATPase or the terminase is the central part of the viral DNA packaging motor. The phi29 ATPase is highly hydrophobic and tends to aggregate in solution. A green fluorescent protein tag (eGFP) fused to the N-terminus of gp16 enhanced its solubility and stability. The eGFP-gp16 showed similar activity to wild type gp16 and was easily detected by fluorescent instruments. The interaction between eGFP-gp16 and DNA in the various conditions were investigated by electrophoretic mobility shift assay, FRET and sucrose gradient. γ-S-ATP dramatically increased gp16 binding affinity to DNA and ATP, ADP, phosphate could release gp16 from gp16-DNA-γ-S-ATP complex. The sliding of gp16 out of the gp16-DNA-γ-S-ATP complex could be blocked by addition of Steptavidin to ends of dsDNA which is conjugated with biotins. Also, we found that six eGFP-gp16 molecules were…
author: Fang, Huaming
language: English
keywords: Biomedical Research, nanomotor, viral DNA packaging motor, bacteriophage, ATPase, phi29, nanotechnology
date published: 2012
date modified: 2026-07-24T03:36:23Z
provider: OhioLINK
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of Cincinnati