Download Bacteriophage DNA packaging

Molecular engines and proteins as nano-biomachines
Highways for molecular motors – kinesin and dynein
Kinesin
10 different kinesin subfamilies – same globular head, but different tails
Two broad functional groups— cytosolic and mitotic kinesins.
Cytosolic kinesins: vesicle and organelle transport. Responsible for shuttling
mitochondria, Golgi bodies, and vesicles within eukaryotic cells
Mitotic kinesins: spindle assembly and chromosome segregation in cell
division
https://www.youtube.com/watch?v=y-uuk4Pr2i8
Kinesin
Kinesin head is structurally similar to myosin motor domain
The MT-binding site in Kinesin is placed like the actin-binding site in myosin
Common ancestor?
Kinesin moves in 8 nm steps corresponding to the distance between successive
alpha- and beta-tubulin monomers in a protofilament. Moves from –ve to +ve
direction of microtubules.
Molecular Cell Biology. 5th Edition. 2004.
Dynein
A microtubule-based molecular motor involved in axonal transport, mitosis, and
cilia/flagella movement
Two types: Cytoplasmic and Axonemal
Forms large molecular complexes
Cargoes: membranous organelles, including lysosomes, endosomes, phagosomes,
and the golgi complex
Cytoplasmic dynein heavy chain can adapt to diverse cargos and functions by
changing its composition
Dynein mediates transport to the (-) end of MTs
Structure of Dynein
Tail located at the N-terminal ~1,400 amino acid residues and involved in cargo binding (gray ).
Next ~550 residues comprise the “linker” (pink ), which changes its conformation depending
on the nucleotide state.
Head (motor) domain is composed of six AAA+ (ATPase associated with diverse cellular
activities) modules.
Among the six AAA+ domains, hydrolysis at the first AAA domain mainly provides the energy
for dynein motility.
Stalk domain of dynein was identified as the microtubule binding domain.Tip of the
buttress/strut is in contact with the middle of the stalk and probably works as a mechanical
reinforcement of the stalk..
Electron microscopy of
dynein.
J Cell Biol. 2013. 202:15-23.
Dynein present in cilia and flagella are termed axonemal dyneins.
Cilia and flagella are found on a variety of cell types, ranging from single cell
protozoa and sperm to the ciliated epithelia of the respiratory and reproductive
tracts.
Ciliary motility is generated by the dynein-driven sliding of doublet microtubules.
Defects in the dynein motors or components that regulate their activity can have
profound consequences; in vertebrates, these include infertility, respiratory
disease, and defects in the determination of the left–right axis during embryonic
development. These defects are called “Ciliopathies”.
Cilia. 2012. 1:1-16
Cilia of Paramecium
Cell Motil Cytoskel. 2009. 66:220-336
Flagella of sperm
Rotary motors
ATP synthase
Located in the inner mitochondrial membrane
and the chloroplast thylakoid membranes of
eukaryotes, as well as in prokaryotic
plasma membranes.
Ancient family of proteins, highly conserved
ATP synthase
Function similar to turbines of
hydroelectric water plant
Proton Gradient like water of the dam
Protons flow from higher to lower
gradient
Synthase components rotate in response
to proton flow
Generated rotational energy coupled
to ATP synthesis
F1 has α3β3γδε conformation
Fo proton pump, blocked by
oligomycin
ATP synthase
“loose”, “tight” and “open” conformations – John Walker and Paul Boyer
Modular evolution model
A proton pump and a helicase got together…....
ATP synthase
Medium contains 2
mM ATP.
Noji et al., 1997, Nature
Cilia and Flagella
Propels cells forward or sweeps materials across
Cilia and Flagella
Same axonemal structure
Microtubule arranged in 9+2 pattern
Associated dynein, other proteins
Cilia and Flagella
Sliding force in the axoneme converted to bend by regions that resist sliding
Nucleic acid motors
Bacteriophage
RNA polymerases- transcribe RNA from DNA
DNA polymerase- Synthesize double stranded DNA
Helicase-reduce double strands of DNA
Topoisomerase- reduce supercoiling of DNA
Viral DNA packaging motors
Bacteriophage
Bacteriophage
Bacteriophage
When stretched out to its full extent, the DNA is around 10µm long, 200 times
the size of the capsid
The DNA packaging motor “terminase” can work against loads of up to 57pN
on average
Contains ATPase function
1 ATP = 2 bp packaging
Bacteriophage DNA packaging
Simpson et al., Nature, 2000
Bacteriophage DNA packaging
Simpson et al., Nature, 2000
Bacteriophage DNA packaging
Simpson et al., Nature, 2000
Bacteriophage DNA packaging
Lander et al., Science, 2006
Lander et al., Science, 2006
DNA polymerase
DNA polymerase
References
Lehninger Principles of Biochemistry. 5th Edition. 2008. David L. Nelson and
Michael M. Cox.
Molecular Cell Biology. 5th Edition. 2004. Lodish, Berk, Matsudaira, Kaiser,
Krieger, Scott,Zipursky and Darnell.
Alberts B and Lye RM (1992) Unscrambling the puzzle of biological machines:
The importance of the details. Cell 68:415-420.
Howard J (1997) Molecular motors: Structural adaptations to cellular functions.
Nature 389:561-567.
Kikkawa M (2013) Big steps toward understanding dynein. J Cell Biol 202:1523.
Porter ME and Sale WS (2000) The 9+2 axoneme anchor multiple inner arm
dyneins and a network of kinases and phosphatases that control motility. J Cell
Biol 151: F37-42.
Sowa Y and Berry RM (2008) Bacterial flagellar motor. Quarterly Rev Biophy
41:103-132.
Vallee RB, Williams JC, Varma D, Barnhart LE (2004) Dynein: An ancient motor
protein involved in multiple modes of transport. J Neurobiol 58:189–200.
Videos
https://www.youtube.com/watch?v=Ct8AbZn_A8A
https://www.youtube.com/watch?v=y-uuk4Pr2i8
https://www.youtube.com/watch?v=tMKlPDBRJ1E
https://www.youtube.com/watch?v=BbI47l2nbDQ
https://www.youtube.com/watch?v=mu72Qoy1xq0