Proteins denature under heat stress. This denaturation of proteins causes the transcription of heat shock proteins (HSPs) to deal with the problem. The production of HSPs is rapid, and facilitates repair of the damaged cell. If the temperature remains high, heat shock proteins will remain at a steady-state level in the cell, which is higher than the initial, cooler temperature state, but lower than the peak reached when cell repair was active. The cell has a need for several HSPs at all temperatures, but the need is elevated at higher temperatures and results in an increased rate of synthesis.
There are two major classes of heat shock proteins: proteases (Lon) and chaperones (DnaK, DnaJ, GrpE, and GroELS). Proteases degrade proteins that are misfolded (and some normal proteins) while chaperones recognize exposed hydrophobic regions that shouldn’t be exposed, binds to them, and places those proteins in a chamber which allows the proteins to refold properly.
HSPs are grouped into families based on their molecular weight. Proteins with a weight of about 70 kDa are grouped into the Hsp70 family. Those with a molecular weight of 60 kDa are grouped into the Hsp60 family, etc.
Following a temperature upshift (ex: from 30-42 C) there is an increase in the amount of sigma factor 32, or RpoH. This factor is responsible for the synthesis of at least 30 HSPs that work in the cytoplasm. Sigma 32 is not active at lower temperatures, and becomes stable after heat shock. Because of this, sigma 32 is considered a major heat shock regulon. Organsims that don’t make sigma 32 are unable to grow at temperatures above 20 C.
During normal temperatures and growth, sigma 32 is an unstable protein with a half life of 60 seconds. After a heat shock (e.g. 30-42 C) the protein stablizes for a few minutes, and it accumulates in the cytoplasm. At non stress temperatures, cytoplasmic proteins DnaK and DnaJ bind to sigma 32, making it subject to proteolysis by proteases (including Lon). At higher temperatures, DnaK and DnaJ preferantally bind to denatured proteins, leaving sigma 32 unmolested and able to bind to RNAP. This sigma-RNAP complex protects sigma 32, and results in a holoenzyme that transcribes the Hsp sigma 32 regulon. Therefore, it is the amount of denatured protein (as opposed to temperature directly) that results in the transcription of heat shock proteins. (This point is further supported by the transcription of HSPs after other types of damage that cause protein denaturation). During heat shock periods, there is an increase in the transcription of mRNA for sigma 32. After heat stablizes, sigma 32 activity lowers rather than the concentration.
At very high temperature (45-50 C) the sigma E regulon is activated, which protects extracytoplasmic proteins from damage. Very high temperatures can cause proteins in the membrane to misfold, which is the signiling pathway to activate sigma E (or sigma 24) in the cytoplasm. When sigma E is activated it binds to RNAP, and the sigma E regulon (which consists of at least 11 genes) is transcribed. (This mimics the way sigma 32 is controlled and transcribed) These genes code for proteins and proteases that are involved in the folding, refolding, and degredation of misfolded proteins in the cell envelope. At lower temperatures, sigma E is bound by an anti-sigma factor in the inner membrane. Envelope stress allows for the release of sigma E, when then binds to RNAP.
Sigma S is considered the master regulator for general stress response, including heat shock, nutrient stress, etc.
Tuesday, March 30, 2010
The SOS response in bacteria
When damage to DNA is sensed, the SOS signal induces over 20 unlinked genes. These genes cope with the DNA damage by remairing the damaged DNA, allowing DNA replication to proceed past the damaged site (translesion synthesis), and stalling cell division to allow time for DNA repair. Normally, the SOS response system is suppressed by down regulating the SOS genes. This process is governed by a master repressor protein called LexA.
Damage to DNA is dected when DNA polymerase encounters a lesion during DNA replication. This causes replication to stop, which leaves a tract of single stranded DNA exposed without being replicated. Since ssDNA is prone to attack, the cell covers the exposed DNA with a protein termed RecA, which starts binding at the 3’ end. The RecA-DNA complex causes LexA to bind and cleave. After enough LexA is broken, the repair genes are expressed. SOS genes have LexA boxes at their promoters. The LexA boxes have slight differences in sequence, and therefore slight differences in their affinity for LexA. Genes with weak affinity for LexA get expressed earlier than genes with strong affinity for LexA, thereby allowing a cascade of gene expression based on the amount of LexA present (which is directly tied to the amount of RecA-DNA complex, which is directly caused by the amount of single strand DNA exposed, which is a measure of the DNA damage).
Some of the earliest genes expressed in response to damage are the uvr genes, which encode for nucleotide excision repair (NER). These proteins detect damage on a single strand of DNA, and excise the damaged area, along with bases on either side of the damage. The gap is filled in by DNA polymerases, which leave the DNA intact and replication can continue. Most lesions are fixed in this manner.
If NER is insufficient to repair the damage, then RecA carries out recombination. The RecA-DNA complex formed at the first sign of damage catalyses the pairing of ssDNA with duplex DNA, one strand of which is homologous to the ssDNA. Once the homologous tract is found, RecA will facilitate strand exchange.
If even recombination doesn’t work, the umuDC operon is expressed, which restarts replication at the stalled fork by employing mutagenesis. PolV, a special DNA polymerase, is encoded by the umuDC operon, and it inaccurately replicates DNA over the lesion. It may introduce incorrect nucleotides, and with them mutations. PolV is formed when the UmuD protein is cleaved by RecA, and associates with UmuC, thus forming PolV. After PolV places a few nucleotides, it is replaced by the accurage PolIII DNA polymerase, and replication continues as normal until another lesion is encountered.
The whole process of DNA repair must happen before a cell divides, and therefore cell division is delayed by the SOS protein SulA. This inhibits the function of proteins involved in cell division, including FtsZ (the septation protein). Once DNA is repaired, Lon protease can use SulA as a substrate, breaking down SulA and allowing the cell to septate as normal.
After DNA repair is accomplished, LexA concentration must be restored to shut off the SOS network. This is done by a number of proteins, one of which is called DinI. DinI structure resembles DNA, and this allows DinI to interact with the RecA filiment. This interaction inhibits both the recombinase and protease activity of RecA. Once RecA is inhibited, it no longer breaks down LexA, which begins to build up in the cell again, and once again works as a master repressor. There are other proteins involved in the shut off of the SOS network (such as RecX) but many of these have not been well studied.
Damage to DNA is dected when DNA polymerase encounters a lesion during DNA replication. This causes replication to stop, which leaves a tract of single stranded DNA exposed without being replicated. Since ssDNA is prone to attack, the cell covers the exposed DNA with a protein termed RecA, which starts binding at the 3’ end. The RecA-DNA complex causes LexA to bind and cleave. After enough LexA is broken, the repair genes are expressed. SOS genes have LexA boxes at their promoters. The LexA boxes have slight differences in sequence, and therefore slight differences in their affinity for LexA. Genes with weak affinity for LexA get expressed earlier than genes with strong affinity for LexA, thereby allowing a cascade of gene expression based on the amount of LexA present (which is directly tied to the amount of RecA-DNA complex, which is directly caused by the amount of single strand DNA exposed, which is a measure of the DNA damage).
Some of the earliest genes expressed in response to damage are the uvr genes, which encode for nucleotide excision repair (NER). These proteins detect damage on a single strand of DNA, and excise the damaged area, along with bases on either side of the damage. The gap is filled in by DNA polymerases, which leave the DNA intact and replication can continue. Most lesions are fixed in this manner.
If NER is insufficient to repair the damage, then RecA carries out recombination. The RecA-DNA complex formed at the first sign of damage catalyses the pairing of ssDNA with duplex DNA, one strand of which is homologous to the ssDNA. Once the homologous tract is found, RecA will facilitate strand exchange.
If even recombination doesn’t work, the umuDC operon is expressed, which restarts replication at the stalled fork by employing mutagenesis. PolV, a special DNA polymerase, is encoded by the umuDC operon, and it inaccurately replicates DNA over the lesion. It may introduce incorrect nucleotides, and with them mutations. PolV is formed when the UmuD protein is cleaved by RecA, and associates with UmuC, thus forming PolV. After PolV places a few nucleotides, it is replaced by the accurage PolIII DNA polymerase, and replication continues as normal until another lesion is encountered.
The whole process of DNA repair must happen before a cell divides, and therefore cell division is delayed by the SOS protein SulA. This inhibits the function of proteins involved in cell division, including FtsZ (the septation protein). Once DNA is repaired, Lon protease can use SulA as a substrate, breaking down SulA and allowing the cell to septate as normal.
After DNA repair is accomplished, LexA concentration must be restored to shut off the SOS network. This is done by a number of proteins, one of which is called DinI. DinI structure resembles DNA, and this allows DinI to interact with the RecA filiment. This interaction inhibits both the recombinase and protease activity of RecA. Once RecA is inhibited, it no longer breaks down LexA, which begins to build up in the cell again, and once again works as a master repressor. There are other proteins involved in the shut off of the SOS network (such as RecX) but many of these have not been well studied.
Bacterial Chemotaxis
Hello again! Today I'm going to tackle a broad, essay type question that pertains to bacterial movement: Chemotaxis.
Chemotaxis is the ability of a bacterium to move along a concentration gradient, either towards an attractant or away from a repellent. The attractant or repellent is termed a chemoeffector, and is monitored by a system of transmembrane sensor proteins, called methyl-accepting chemotaxis proteins (MCP), or receptor-transducer proteins. These proteins affect a two component system: CheA, a cytoplasmic histidine kinase, and CheY, a response regulator. Action upon this system affects the flagellar motor.
Bacteria swim by rotating flagella. Counter-clockwise rotation align the flagella in a single bundle, causing the bacterium to swim in a straight line (termed a "run"). Clockwise rotation causes this flagellar bundle to break apart, and results in random tumbling in place (termed a "tumble"). As few as 25% of the flagella need to rotate clockwise to cause random tumbling, but the more flagella rotating in this manner, the greater the change of direction.
Bacteria are unable to choose the direction in which they swim, and are unable to swim in a straight line (a run) for very long due to rotational diffusion; they "forget" which direction they were going. This results in random run and tumble movement across space. Chemoeffectors influence this random movement. When a bacterium senses it is going towards an attractant or away from a repellent (the "correct" direction from the bacterium's point of view), it will swim in a straight line for longer; this results in a longer run vs tumble phase. The presence of an attractant decreases the probability of clockwise rotation of flagella, keeping the bacterium from tumbling. The presence of a repellent increases the probability of clockwise flagellar rotation, resulting in a shorter run, and more change of direction. Therefore, attractants see longer, more frequent runs mixed with shorter, less frequent tumbles, resulting in an overall movement towards the attractant (or, conversely, away from the repellent).
Bacteria sense chemoeffectors on a temporal gradient: they are able to remember past concentrations long enough to compare them to present concentrations, and then use this information to make a decision. This memory is long enough for the bacteria to compare two points more distal than its body length, yet short enough to signal the bacteria before it tumbles randomly.
Six genes are required for chemotaxis: CheA, CheB, CheR, CheW, CheY, and CheZ. In mutants that have any of of those genes knocked out, chemotaxis is impossible. As mentioned above, chemotaxis is controlled by a two-component system, which is alerted by methyl-accepting chemotaxis proteins that span the membrane and monitor chemoeffectors in the periplasmic space. CheY is ultimately responsible for the way in which a flagellar motor turns. If it attaches to proteins in the flagellar motor (FliM), then the motor will turn clockwise. If it doesn't, the motor turns counterclockwise. Therefore, CheY must attach to the flagellar motor to cause tumbling. CheY is activated by accepting a phosphate group from CheA. CheA is signaled by transmembrane proteins, of which there are 5: Tar (taxis to aspartate and away from repellents), Trg (taxis to ribose, glucose and galactose), Tap (taxis to dipeptides), Tsr (taxis to serine and away from repellents) and Aer (taxis to oxygen as it oxidizes FADH to FAD). The presence of these substances in the extra cellular space causes a conformational change in the transmembrane protein. This initiates a CheW mediated response in CheA phosphorilation:
CheA + ATP=CheA-P + ADP + Pi
CheA then phosphorilates CheY: CheA-P + CheY=CheA + CheY-P
The binding of CheY-P to the flagellar motor causes clockwise rotation. If CheY-P is dephosphorilated, then it will not bind and the flagellar motor will turn counter-clockwise. CheZ is responsible for the dephosphorilation of CheY-P in the cytoplasm. Under normal circumstance, CheY is phosphorilated and dephosphorilated at a constant rate, allowing for the random run/tumble action observed in bacteria not experiencing chemotaxis. When an attractant is sensed, autophosphorilation of CheA is decreased, which decreases the phosphorilation of CheY, and therefore the probability of flagella turning clockwise. When a repellant is sensed, the exact opposite happens.
CheA also phosphorilated CheB, which is a regulator that governs adaptation to a particular concentration of attractant. CheB-P removes methyl groups from glutamate residues in the receptor-transducer proteins. CheR adds methyl groups to the glutamate residues. When an attractant is present, CheA does not become phosphorilated, and therefore CheB does not phosphorilate either. CheB cannot remove methyl groups from the glutamate residues. Higher methylation of glutamate residues stimulate tumbling, and therefore the chemotaxis stops for this concentration of attractant. When the concentration changes again, chemotaxis returns as normal.
Chemotaxis is the ability of a bacterium to move along a concentration gradient, either towards an attractant or away from a repellent. The attractant or repellent is termed a chemoeffector, and is monitored by a system of transmembrane sensor proteins, called methyl-accepting chemotaxis proteins (MCP), or receptor-transducer proteins. These proteins affect a two component system: CheA, a cytoplasmic histidine kinase, and CheY, a response regulator. Action upon this system affects the flagellar motor.
Bacteria swim by rotating flagella. Counter-clockwise rotation align the flagella in a single bundle, causing the bacterium to swim in a straight line (termed a "run"). Clockwise rotation causes this flagellar bundle to break apart, and results in random tumbling in place (termed a "tumble"). As few as 25% of the flagella need to rotate clockwise to cause random tumbling, but the more flagella rotating in this manner, the greater the change of direction.
Bacteria are unable to choose the direction in which they swim, and are unable to swim in a straight line (a run) for very long due to rotational diffusion; they "forget" which direction they were going. This results in random run and tumble movement across space. Chemoeffectors influence this random movement. When a bacterium senses it is going towards an attractant or away from a repellent (the "correct" direction from the bacterium's point of view), it will swim in a straight line for longer; this results in a longer run vs tumble phase. The presence of an attractant decreases the probability of clockwise rotation of flagella, keeping the bacterium from tumbling. The presence of a repellent increases the probability of clockwise flagellar rotation, resulting in a shorter run, and more change of direction. Therefore, attractants see longer, more frequent runs mixed with shorter, less frequent tumbles, resulting in an overall movement towards the attractant (or, conversely, away from the repellent).
Bacteria sense chemoeffectors on a temporal gradient: they are able to remember past concentrations long enough to compare them to present concentrations, and then use this information to make a decision. This memory is long enough for the bacteria to compare two points more distal than its body length, yet short enough to signal the bacteria before it tumbles randomly.
Six genes are required for chemotaxis: CheA, CheB, CheR, CheW, CheY, and CheZ. In mutants that have any of of those genes knocked out, chemotaxis is impossible. As mentioned above, chemotaxis is controlled by a two-component system, which is alerted by methyl-accepting chemotaxis proteins that span the membrane and monitor chemoeffectors in the periplasmic space. CheY is ultimately responsible for the way in which a flagellar motor turns. If it attaches to proteins in the flagellar motor (FliM), then the motor will turn clockwise. If it doesn't, the motor turns counterclockwise. Therefore, CheY must attach to the flagellar motor to cause tumbling. CheY is activated by accepting a phosphate group from CheA. CheA is signaled by transmembrane proteins, of which there are 5: Tar (taxis to aspartate and away from repellents), Trg (taxis to ribose, glucose and galactose), Tap (taxis to dipeptides), Tsr (taxis to serine and away from repellents) and Aer (taxis to oxygen as it oxidizes FADH to FAD). The presence of these substances in the extra cellular space causes a conformational change in the transmembrane protein. This initiates a CheW mediated response in CheA phosphorilation:
CheA + ATP=CheA-P + ADP + Pi
CheA then phosphorilates CheY: CheA-P + CheY=CheA + CheY-P
The binding of CheY-P to the flagellar motor causes clockwise rotation. If CheY-P is dephosphorilated, then it will not bind and the flagellar motor will turn counter-clockwise. CheZ is responsible for the dephosphorilation of CheY-P in the cytoplasm. Under normal circumstance, CheY is phosphorilated and dephosphorilated at a constant rate, allowing for the random run/tumble action observed in bacteria not experiencing chemotaxis. When an attractant is sensed, autophosphorilation of CheA is decreased, which decreases the phosphorilation of CheY, and therefore the probability of flagella turning clockwise. When a repellant is sensed, the exact opposite happens.
CheA also phosphorilated CheB, which is a regulator that governs adaptation to a particular concentration of attractant. CheB-P removes methyl groups from glutamate residues in the receptor-transducer proteins. CheR adds methyl groups to the glutamate residues. When an attractant is present, CheA does not become phosphorilated, and therefore CheB does not phosphorilate either. CheB cannot remove methyl groups from the glutamate residues. Higher methylation of glutamate residues stimulate tumbling, and therefore the chemotaxis stops for this concentration of attractant. When the concentration changes again, chemotaxis returns as normal.
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