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Counting of six pRNAs of phi29 DNA‐packaging motor with customized single‐molecule dual‐view system
Author(s) -
Shu Dan,
Zhang Hui,
Jin Jiashun,
Guo Peixuan
Publication year - 2007
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1038/sj.emboj.7601506
Subject(s) - biology , dna , dual (grammatical number) , computational biology , genetics , literature , art
Direct imaging or counting of RNA molecules has been difficult owing to its relatively low electron density for EM and insufficient resolution in AFM. Bacteriophage phi29 DNA‐packaging motor is geared by a packaging RNA (pRNA) ring. Currently, whether the ring is a pentagon or hexagon is under fervent debate. We report here the assembly of a highly sensitive imaging system for direct counting of the copy number of pRNA within this 20‐nm motor. Single fluorophore imaging clearly identified the quantized photobleaching steps from pRNA labeled with a single fluorophore and concluded its stoichiometry within the motor. Almost all of the motors contained six copies of pRNA before and during DNA translocation, identified by dual‐color detection of the stalled intermediates of motors containing Cy3‐pRNA and Cy5‐DNA. The stalled motors were restarted to observe the motion of DNA packaging in real time. Heat‐denaturation analysis confirmed that the stoichiometry of pRNA is the common multiple of 2 and 3. EM imaging of procapsid/pRNA complexes clearly revealed six ferritin particles that were conjugated to each pRNA ring.