Biomolecular Machines

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Mechanist (January 04, 2009, 16:54:10 PM):
Welcome to the Molecular Machines

A thread to lump together all the interesting discoveries regarding the intracellular biomolecular machinery that are crucial for life to exist. Feel free to post interesting discoveries and perhaps describe the functionality of the intracellular biomolecular machines.

Intracellular biomolecular machinery include the following:
1) DNA replication and repair machinery (replisome)
2) DNA transcription machinery and RNA processing and translation machinery (Spliceosomes and ribosomes)
3) Cell cycle signaling network (pRB, e2F, CDKs)
4) Programmed cell death machinery (Apoptosis, autophagy, mitotic catastrophe)
5) Protein processing machinery (Chaperones, ubiquitin-proteasome system)
6) Intracellular signaling networks (protein kinases and phosphatases)
7) Mechanical machines for intracellular shuttling of biomolecules and cellular movement (Microtubule network, kinesin, dynein)
8 ) Energy production machines (Electron transport chain, F0F1 ATP synthase)

Sliding clamps and the clamp-loading machine.
Sliding clamps are ring-shaped proteins that some refer to as the “guardians” of the genome or others name them as the “ringmasters” of the genome.
Interestingly these clamps are structurally and functionally conserved in all branches of life and crystallographic studies have shown that they have almost superimposable three-dimensional structures, yet these components have very little sequence similarity (Figure 1) [1].

Figure 1: Sliding clamps from the various domains of life.

The picture below is taken from the Molecular Biology Visualization of DNA video (2:14) from the freesciencelectures.com site.
Great video!

Figure 2: Replication machinery.

The following components can be seen:
Sliding clamps (PCNA in eukaryotes): Green circular shaped
Clamp loader (RFC in eukaryotes): Blue-white component in the middle
(Figure 3: Structures of PCNA connected to RFC (front))


Figure 4: Structures of PCNA connected to RFC (side)

(Figure 5: Structures of PCNA connected to RFC (back))
Helicase: Blue (Figure 6: Helicase (front))
DNA polymerase: Dark-blue components attached to the sliding clamps
Primase: Green component attached to helicase
Leading strand: Spinning off to the right
Lagging strand: Spinning off to the top

They are not ringmasters for nothing.
Sliding clamps participate and control events that orchestrate DNA replication events in the following ways:

* Enhancement of DNA polymerase activity.
* Coordinate Okazaki fragment processing.
* Prevention of rereplication
* Translesion synthesis
* Prevents sister-chromatid recombination and also coordinates sister-chromatid cohesion
* Crucial role in mismatch repair, base excision repair, nucleotide excision repair
* Participates in chromatin assembly


Other functions include:

* Epigenetic inheritance
* Chromatin remodeling
* Controls cell cycle and cell death signaling


The true ringmasters.

Clamp loaders are another group of interesting proteins (see video and figures 3-5 above). Interestingly again, their functional and structural architecture are conserved across the three domains of life with low-level sequence similarity [2]. At the replication fork during replication, they load the sliding clamps many times onto the lagging strand (after DNA priming) and only once onto the leading strand. They also act as a bridge to connect the leading and lagging strand polymerases and the helicase. Which brings us to another interesting group of proteins; the helicases.

Helicases are also known to be ring-shaped motor proteins, typically hexamers (see figure 6) and separate double-stranded DNA into single-stranded templates for the replication machinery. Replication occurs at about 1000 base pairs per second due to the highly efficient combination of sliding clamps and the polymerases. Thus, helicases need to unwind DNA at at least that speed. Unwinding DNA too slowly and the replication machinery might break down . Unwind the DNA too fast or untimely and harmful mutations might occur as single-stranded DNA is prone to degradation and cytosine deamination.

The speed at which helicase unwinds DNA is no accident though, as it is intrinsically controlled. As helicase is bound to the lagging strand, it unwinds the leading strand in a separate direction. Applying a pulling force on the leading strand leads to a 7-fold increase in the speed of DNA unwinding by helicase [3, 4]. The highly efficient DNA polymerase/sliding clamp combination provides this controlling force on the leading strand. This forms a robust unwinding/polymerization interaction whereby polymerization controls and prevents unwanted DNA unwinding.

Altogether, the replisome machinery provide a robust way for DNA replication to prevent unnecessary DNA damage and mutation.

References.
1. Vivona JB, Kelman Z. The diverse spectrum of sliding clamp interacting proteins. FEBS Lett. 2003 Jul 10;546(2-3):167-72.
2. Jeruzalmi D, O'Donnell M, Kuriyan J. Clamp loaders and sliding clamps. Curr Opin Struct Biol. 2002 Apr;12(2):217-24.
3. Ha T. Need for speed: mechanical regulation of a replicative helicase. Cell. 2007 Jun 29;129(7):1249-50.
4. Johnson DS, Bai L, Smith BY, Patel SS, Wang MD. Single-molecule studies reveal dynamics of DNA unwinding by the ring-shaped T7 helicase. Cell. 2007 Jun 29;129(7):1299-309.
Teleological (July 02, 2009, 08:43:32 AM):
The bc1-complex for electron transfer from dihydroubiquinone to cytochrome c through the Q-cycle.

The bc1-like complexes (Complex III in mitochondria) play a central role in the electron transport chains of respiratory and photosynthetic machinery.

Their function is to carry out a sequence of electron and proton transfer reactions to generate a trans-membrane proton motive force that supplies the energy for ATP synthesizing utilizing the ATP synthase (excellent video, funny clip:p) machinery. Protons and electrons are supplied by dihydroubiquinone which in turn is generated by complexes I and II of the electron transport chain.

How do the bc1-like complexes carry out their function?
First the structure:
The cyt bc1-complex contains two separate redox chains; High potential and low potential.
The high-potential chain connects the Qo-binding site with the cyt c1 through the Rieske Iron-sulphur-protein (RISP). The RISP is situated on a rotateable arm that is able to connect the cyt c1 component with the Qo-binging site.
The low potential chain connects the Qo-site with the Q1-site through the cyt BL and Cyt BH complexes.

Now the mechanism. A bifurcated electron transfer mechanism:
1) The lipid-soluble dihydroubiquinone molecule binds at the Qo-site and liberates one proton into the intermembrane space and in the process forms a semiubiquinone radical.
2) The RISP swings around to receives an electron from the semiubiquinone and donates it to cyt c1 which in turn donates it to cytochrome c. Cytochrome c plays its part in energy transfer to complex IV of the electron transfer chain.
3) A second proton is liberates into the intermembrane space and an electron is donated to the low potential chain, resulting in the formation of ubiquinone
4) At the Q1-site the electron is donated to ubiquinone to form semiubiquinone, while a proton is donated from the mitochondrial matrix.
5) In order for the formation of dihydroubiquinone at the Q1-site, two dihydroubiquinones must bins at the Qo-site.
6) Thus the end result is the formation of 1 dihydroubiquinone, 2 quinones, 4 intermembrane protons and 2 ferrocyrochrome c proteins and loss of 2 mitochondrial matrix molecules after the binding of 2 dihydroubiquinones at the Qo-site.


That is the basic general mechanism, however research is ongoing into how bypass reactions are avoided.
For example:
Why do the electrons flow in only one direction in the low electron transport chains?
Why aren't both electrons donated to the high-potential chain in the first place?
Radical hypotheses have been proposed including [SIZE="2"](From Cape et al. 2006 Trends Plant Sci. 2006 Jan;11(1):46-55.)[/SIZE]:
Quote
(i) A complex that can either stabilize the intermediate semiubiquinone, rendering it inert and invisible through some unknown mechanism, or that can use the unprecedented tactic of destabilizing its reactive intermediates.
(ii) A kinetic ‘water-park’ that tunes reaction activation enthalpies or entropies to route ‘water’ (electron) flow into productive channels.
(iii) A nano-machine that gates the electron and proton transfer reactions of semiubiquinone according to its recognition of the different redox and/or conformational states of the complex.
(iv) An extraordinary, and unprecedented, double concerted oxidation of dihydroubiquinone that simultaneously distributes two electrons and at least one proton between at least three different acceptors.

Options II and III do not exclude the possibility of quantum mechanics and coulombic interactions playing a role.

All-in-all a brilliant solution for a bifurcated electron transfer mechanism in order to generate a proton motive force from dihydroubiquinone.


Interestingly, the intermediate (semiubiquinone) generated at the Qo-site is believed to be a major contributor to the formation of reactive oxygen species by donating it's free electron to oxygen and thereby resulting in the formation of superoxide. Superoxide formation causes damage to various molecules including DNA, RNA, proteins and lipids.

Semiubiquinone

Paradoxically though, reactive oxygen specie generation at the Qo-site as a result of semiubiquinone formation is increased during periods of hypoxia (low oxygen). Hypoxia is a major initiator of cancerous growth because it activates various pro-growth signaling pathways. Hypoxia in cells usually occur as a result of poor circulation and delivery of oxygen. Obesity, lack of exercise and poor diet all contribute to these circumstances.

Thus, the bc1-complexes connects bad health choices with higher incidences of cancers and other mitochondrially related diseases through reactive oxygen species formation as a result of hypoxic conditions within various systems of the body.

Exercising and eating right are thus good for oiling your biomolecular machines...
rwenzori (July 12, 2009, 15:15:32 PM):

A thread to lump together all the interesting discoveries regarding the intracellular biomolecular machinery ...


Do your threads breed?

http://www.sciforums.com/showthread.php?t=86313
http://mybroadband.co.za/vb/showthread.php?t=126821
https://richarddawkins.net/forum/viewtopic.php?f=4&t=60319
http://teleomechanist.blogspot.com/2008/07/biomolecular-machines.html


ID evangelism?
Teleological (July 13, 2009, 09:15:37 AM):
A little more information about nuclear pores:
[SIZE="3"]Video[/SIZE] and a few images:





And a few recent discoveries:
First Detailed Map Of Nuclear Pore Complex Made
New Model Of A Nuclear Pore Complex Is Based On Crystal Structure Of Its Key Component
3-D Structure Of Key Nuclear Pore Building Block Identified

Quote
In new research, scientists have for the first time glimpsed in three dimensions an entire subcomplex of the NPC; it's the key building block of this little understood and evolutionarily ancient structure, an innovation fundamental to the development of nearly all multicellular life on earth.

The findings, by Martin Kampmann, a graduate student in John D. Rockefeller Jr. Professor Günter Blobel's Laboratory of Cell Biology, add details to an unfolding picture of cellular evolution that shows a common architecture for the NPC and the vehicles that transport material between different parts of the cell, called coated vesicles. As early as 1980, Blobel proposed that internal membranes of cells – such as those encompassing the nucleus and vesicles – evolved from folds or invaginations of the outer cell membrane.

Rockefeller scientists Brian Chait and Michael Rout suggested in a 2004 paper in PLoS Biology that both the NPC and vesicle coats, which contain similar protein folds, evolved from ancient membrane-coating proteins that stabilized these primordial internal membranes. "So far, it's been unclear how these ancient folds work in the nuclear pore complex", Kampmann says. "Now we can see that the α-solenoid folds form long, flexible arms and hinges that end in the more compact, globular β-propellers. The same architectural principle is found in clathrin, a common component of vesicle coats."

In research to be published online June 7 in Nature Structural & Molecular Biology, Kampmann isolated and purified samples of the most fundamental building block of the NPC known as the Nup84 complex, which is composed of seven proteins. The entire NPC – enormous by molecular standards – consists of 30 different kinds of proteins. Focusing on the Nup84 complex, Kampmann used an electron microscope (EM) to take thousands of images of the complex in different states or conformations, which could reflect a role in the expansion and contraction thought to facilitate the passage of various sized molecules through the NPC. By computationally averaging these many different views, he reconstructed the first three-dimensional models of the Nup84 complex. Finally, based on prior work in the Blobel lab using X-ray crystallography to determine the exact atomic structure of individual proteins in the Nup84 complex, he plugged these proteins snugly into the EM structure.

"Because the nuclear pore complex is probably too big and flexible to determine its entire atomic structure by X-ray crystallography, I think this three-dimensional EM approach could be a big help in solving the whole thing," Kampmann says. "It allows us to put the crystal structures that we do have in context." Kampmann is applying the EM approach to other subunits in hopes of fleshing out the overall picture of one of the most mysterious machines in molecular biology. "Martin's data represent an important advance toward piecing together the structure of the NPC," Blobel says.

Given the central role of the nuclear pore complex in the most basic cell processes, defects in its assembly, structure and function can have lethal consequences. Its proteins have been associated with viral infection, primary biliary cirrhosis and cancer. An understanding of how the complex works could lead to treatments for these diseases, and also reveal the evolutionary coup that led to the gene-protecting structure found in every cell more complicated than the simplest single-celled microorganisms: the nucleus.
Teleological (July 17, 2009, 09:16:42 AM):
More efficient biomolecular machines!

DNA 'Sloppier Copier' Surprisingly Efficient: Three Major Puzzles About Famous Enzyme Solved
Quote
ScienceDaily (July 15, 2009) — The "sloppier copier" discovered by USC biologists is also the best sixth man in the DNA repair game, an article in the journal Nature shows.


Quote
The enzyme known as DNA polymerase V (pol V) comes in when a cell's DNA is reeling from radiation damage or other serious blows. Pol V copies the damaged DNA as best it can – saving the life of the bacterial cell at the cost of adding hundreds of random mutations.

The July 16 Nature study reveals pol V's key attributes: economy of motion and quickness to engage.

The study also solves two other stubborn mysteries about the mechanics of DNA repair: the exact composition of the active form of pol V and the crucial role of a protein filament, known as RecA*, that is always present around DNA repair sites, but was never shown to be directly involved.

The three findings together describe an exquisitely efficient process.

"It's a beautiful mechanism for how cells conserve energy," said first author Qingfei Jiang, a graduate student of senior author Myron Goodman, professor of biological sciences and chemistry at USC College.

Cells multiply by division, which starts with the copying of DNA. Pol V kicks in when a section of damaged DNA baffles the enzymes normally involved in copying.

In experiments with E. coli, Jiang and Goodman showed that the activation signal for pol V is the transfer to the enzyme of two key molecules from RecA*.

RecA* is a nucleoprotein filament: a long line of proteins bound to single-stranded DNA. The molecules that RecA* transfers to pol V are ATP, the energy factory of the cell, and a single RecA* protein among the many that make up the filament.

The copying of damaged DNA is formally called "translesion synthesis," or TLS.

"What is RecA* doing?" had been a vexing question in the field for two decades, since the discovery that the filament was necessary for DNA repair. No one, however, could figure out why.

Goodman's group showed that the role of RecA* is limited but direct: It is needed to donate molecules to activate pol V, but it does not participate in damage-induced DNA copying and does not even need to be next to the repair site.

Instead, RecA* acts as a fuel station to put pol V to action.

With the two extra molecules attached, pol V copies the damaged DNA. As soon as it reaches the end of the damaged section, it falls off and immediately deactivates.

Pol V then waits to be called again.

In addition to saving energy, the process prevents the mistake-prone copier from trying to "repair" normal DNA.

"All the other DNA polymerases [enzymes], when they copy DNA, they go first from one and then to another DNA and copy it. Not this baby. It has to be reactivated," Goodman said.

"It's a utility player. It's the guy who does the tough jobs."

He added that the discovery "explains one of the key ways that you get mutations when you damage DNA."

Human cells use similar enzymes, Goodman said.

The study of mutations holds fundamental relevance for medicine, evolutionary biology, aging research and other fields.

Goodman's research group discovered pol V in 1999. The "sloppier copier" nickname, coined by USC science writer Eric Mankin, has since been adopted widely.

At the time, Goodman described pol V as a "last-ditch cell defense" that averts death at the cost of frequent copying mistakes, which show up as mutations in the cell's DNA.

Ironically, the sloppier copier may do more for the long-term success of the species than its accurate cousins. Some of the accidental mutations are likely to be helpful. Cells with those mutations will adapt better to their environment, and the mutations will spread through the species by natural selection.

Goodman and Jiang's co-authors were Kiyonobu Karata and Roger Woodgate of the National Institute of Child Health and Human Development, and Michael Cox of the University of Wisconsin-Madison.

The National Institutes of Health funded the research.

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