Counting of six pRNAs of phi29 DNA-packaging motor with customized single-molecule dual-view system
Counting of six pRNAs of phi29 DNA-packaging motor with customized single-molecule dual-view system
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DOI：
10.1038/sj.emboj.7601506
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发表时间：
2007-01-24
期刊：
影响因子：
11.4
通讯作者：
Guo, Peixuan
中科院分区：
文献类型：
--
作者：
Shu, Dan;Zhang, Hui;Guo, Peixuan
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.
