Biology Reference
In-Depth Information
Fig. 4.
A model depicting sequential action of six pRNAs to gear the DNA
translocation machine. Hexagon
→
connector; Surrounding pentagon
→
capsid membrane; six protrusions
six pRNAs. Arrows point to the differ-
ent energetic states of pRNA 1 with variable patterns to illustrate different
energetic conformation. A to G
→
→
six steps of 12
°
rotation, (a 5/6-fold mis-
match generates 30 equivalent orientations, 360
°÷
30 = 12
°
). Each pRNA
rotates 72
after six steps to move from one vertex to an adjacent vertex
(pRNA 1 moves from vertex a to b). If one ATP is needed for one pRNA to
move from one vertex to another via one cycle of contraction and relaxation,
then 30 ATPs (6 RNA X 5 vertex) are needed for a 360
0
rotation that cause
the translocation of one helical turn or 10.5 bases of DNA. (Adapted from
Ref. 124, with permission from the author and the publisher, American
Society for Microbiology.)
°
binding domain is located in the central region,
106,123,126,127
bases
23-97, while the 5'/3'-paired ends are required for an unknown role
involved in DNA translocation into the procapsid.
123,127-130
Mutant
pRNAs with mutations in the 5'/3'-paired ends can compete with
wild-type pRNA for procapsid binding and inhibit phage assembly
in vitro
and
in vivo
.
107,131
It is able to assemble infectious phi29 virions
in vitro
using nine
purified and two partially purified proteins overproduced from cloned
genes as well as synthetic pRNA and phi29 genomic DNA (Fig. 6).
132
Mixing purified procapsids, pRNA, packaging enzyme gp16, genomic
DNA-gp3 (gp3 is a viral protein gp3 covalently attached to each 5'
end), ATP, and Mg
++
results in the accumulation of DNA-filled heads.
35
The subsequent addition of purified phage neck and tail proteins con-
verts the DNA-filled heads into infectious virions (Fig. 7).
31,132
Omission of any one of the essential phi29 assembly components from
the reaction results in no plaque formation,
31,35,132
thus providing an
assay with a sensitivity of over eight orders of magnitude. The system is
sensitive enough that pRNAs with 10
5
- or 10
6
-fold reductions in DNA
