Mutations altering a structurally conserved loop-helix-loop region of a viral packaging motor change DNA translocation velocity and processivity.
Many double-stranded DNA viruses employ ATP-driven motors to translocate their genomes into small, preformed viral capsids against large forces resisting confinement. Here, we show via direct single-molecule measurements that a mutation T194M downstream of the Walker B motif in the phage lambda gpA packaging motor causes an 8-fold reduction in translocation velocity without substantially changing processivity or force dependence, whereas the mutation G212S in the putative C (coupling) motif causes a 3-fold reduction in velocity and a 6-fold reduction in processivity. Meanwhile a T194M pseudorevertant (T194V) showed a near restoration of the wild-type dynamics. Structural comparisons and modeling show that these mutations are in a loop-helix-loop region that positions the key residues of the catalytic motifs, Walker B and C, in the ATPase center and is structurally homologous with analogous regions in chromosome transporters and SF2 RNA helicases. Together with recently published studies of SpoIIIE chromosome transporter and Ded1 RNA helicase mutants, these findings suggest the presence of a structurally conserved region that may be a part of the mechanism that determines motor velocity and processivity in several different types of nucleic acid translocases.
Department of Physics, University of California at San Diego, La Jolla, CA 92093, USA.
James M
JM
Tsay
Jean
J
Sippy
Damian
D
DelToro
Benjamin T
BT
Andrews
Bonnie
B
Draper
Venigalla
V
Rao
Carlos E
CE
Catalano
Michael
M
Feiss
Douglas E
DE
Smith
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000378:metabolism; D002384:Catalysis; D004247:DNA / Q000235:genetics*; D004265:DNA Helicases / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics*; D008863:Microspheres; D008969:Molecular Sequence Data; D009154:Mutation; D052898:Optical Tweezers; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D014764:Viral Proteins / Q000378:metabolism
D004279:DNA, Viral; D014764:Viral Proteins; C065038:gp17 protein, Enterobacteria phage T4; D004247:DNA; D004265:DNA Helicases