Exam 3 Flashcards
What is a secondary messenger?
Intracellular signaling molecule dependent on a perceived signal on the outside of the cell which must be communicated to the cell machinery the manner in which this signal transduction occurs is through the use of secondary messengers.
What is (p)ppGpp?
It is a guanosine diphosphate. 3’ is always a diphosphate and the 5’end can be either a di or triphosphate.
What is the stringent response?
Upon nutrient starvation, the cell downregulates RNA and protein production, simultaneously the cell begins synthesizing (p)ppGpp which promotes A.A. biosynthesis after this, the cell resumes growth but at a lower rate of division.
What were the magic spots?
The scientists starved cells and then saw spots which were later identified as the (p)ppGpp which is involved in the response to nutrient starvation.
What are the two proteins involved in the stringent response, how do they work and what are their roles in the transduction of the stringent response?
There is SpoT and RelA.
SpoT is a bifunctional enzyme, meaning it can do two things. It can synthesize ppGpp or it can degrade ppGpp via a synthase and hydrolase domain respectively. The signal is first given to RelA. Normally charged tRNAs bind the A site of the ribosome during protein synthesis, in the absence of a A.A. pool the proportion of uncharged tRNAs increases and these get loaded into the A site on the ribosome instead of the charged tRNAs. This results in the binding of RelA to the ribosome and is the signal to SpoT to begin ppGpp synthesis. This signals the biosynthesis of amino acids. When the A.A. pool becomes available ppGpp is then degraded by the hydrolase domain of the SpoT and presumably RelA, dissociates from the A site and charged amino acids are once again LOADED.
What is the RHS superfamily?
They are a superfamily of proteins which are homologs of RelA and SpoT which are found in all sequenced plants and bacteria. Ie, the stringent response is found in a variety of orgnaisms. The N terminal domain has the catalytic, hydrolase and synthase activity. The C-terminal domain has the TGS which binds uncharged ACP which shifts balance of hydrolase or synthase activity. It also has the ACT which is involved in ribosomal interactions.
What level of regulation is achieved with the RelA and SpoT stringent response?
The metabolism is regulated at the global level. In general, synthesis of the cell wall, DNA replication and protein synthesis will be down regulated and alternative sigma factors along with a.a. biosynthesis will be upregulated.
What is a cyclic nucleotide and what is an example of this category of secondary messenger?
This is a single phosphate nucleotide with a cyclic bond arrangement between the sugar and phosphate, that is the phosphate and sugar together make a cyclic portion. An example is cAMP.
What is catabolite repression?
This is a regulatory mechanism which determines which carbon source will be broken down for energy. The classical example is lactose vs glucose the cell will use glucose until its depleted and then switch to lactose, the mechanism governing this is cAMP.
Roles of c-di-AMP
It was discovered in 2010 so not completely characterized. Can play roles in sporulation, cell cycle checkpoints and in tetracycline resistance. It is also important in human pathogens and may be a good target. Disrupting these genes killed the cells and they could only be rescued by ectopic expression (very low levels of transcription)
Roles of c-di-GMP
Motility via flagella production/control, biofilm production and pathogenicity ie the production of cytotoxins/invasion.
How is c-di-GMP levels controlled?
Elevated c-di-GMP promotes biofilm lifestyle, made by diguanylate cyclase (DGC) and is degraded by a phosphodiesterase when the levels are depleted, the cells become motile.
what conserved amino acid domains are found on diguanylate cylcases (DGCs) and phosphodiesterases (PDEs)?
DGC will have the GGDEF conserved amino acid domain and the PDEs can have one of two conserved a.a. sequences, either EAL which degrades it to pGpG or HD-GYP which degrades it to 2 GMPS
Types of c-di-GMP domains
You can have a EZ domain with just one of of the functions so either diguanylate cyclase or the phosphodiesterase domain with either the HD-GYP/EAL (PDE) or the GGDEF (DGC)amino acid conserved sequence. OR you can have a hybrid EZ domain with both c-diGMP synthesis and degradation so both DGC and PDE.
How do the c-di-GMP enzymes both the diguanylate cyclase and the phosphodiesterase sense their respective signal?
The DGC or PDE domains of protein activity can be added to different sensing domains much in the same way that polyketide synthesis has modules that can be interchanged to make different structures. In this case the DGC and PDE can be attached to transmembrane domains, PAS domains (oxygen sensing) response regulators (REC) or many others. This is a very cool way for the cell to use these two enzymes and the levels of c-di-GMP to sense different signals and respond to them accordingly.
Describe the PAS domain.
It is found in many signal proteins and functions as a signal sensor, they are found in all organisms from animals to bacteria. They detect their signal via a associated cofactor.
Where is the c-di-GMP found?
It is nearly universal in bacteria but is not present in higher eukaryotes.
How are DGC regulated at the enzymatic level?
they have both an active site and an inhibitory site. To make the c-di-GMP they have to dimerize and undergo a conformational change and then allow the cyclization rxn t occur. At excess levels of c-di-GMP the I site is bound via negative feedback. This can prevent the dimerization or the conformational change from happening meaning that c-di-GMP is not made. The negative allosteric modulator in this case is c-di-GMP. The i site is characterized by a RxxD motif.
What are the conserved amino acid sequences for phosphodiesterases which break apart c-di-GMP?
EAL domain which is the glutamic acid, alanine, and leucine. The other is the HD-GYP domain which are not right next to each other the HD (histidine and aspartate) comes first the second is the GYP which is glycine proline tyrosine and proline.
What can c-di-GMP bind and therefore, regulate?
The best described binding target is the PilZ domain the EAL and HD-GYP domains also bing c-di-GMP (obviously because they are broken down by phosphodiesterase) it also binds riboswitches with very high affinity necessitating only picomolar ranges of the diGMP. It also binds transcription factors can be responsible for biofilm formation mRNA degradation ect.
How can the c-di-GMP made from a single DGC modulate the activity of a specific target?
There are two hypothesis:
A) The cell raises the total pool of c-diGMP in response to an environmental condition.
B) (compartmentalization) An individual DGC modifies the local levels of diGMP. To prevent the cross talk of signals ie to prevent the total pool from increasing you have cytoplasmic phosphodiesterases which can degrade the diGMP which leaks from the local site and is then broken down preventing opposing signals from conflicting with one another (prevents cross talk).
What biochemical principal allows the transduction signal system to work?
The different proteins, the DGC and PDE along with their downstream effectors and targets have different Km, Kd, and Ki, allowing for different responses at different concentrations of diGMP based on their affinities and subsequently the concentrations needed to achieve activation and subsequently, signal transduction. That is, different concentrations of c-di-GMP have different effects.
How do alternative sigma factors work?
They bind specific promoters and compete with sigma 70. Their ability to bind RNAP and subsequently, the promoter is determined by both the concentration of the sigma and its affinity for the core RNAP.
What are the four groups of sigma factors?
1: sigma 70
2: sigma 38 environmental stress response.
3: sigma 28 and sigma 32 –> flagellum biosynthesis and heat shock respectively.
4: ECF or extra cytoplasmic factors
-dont need to know group 2 apparently these aren’t important.
How are sigma factors regulated
Antisigma factors --> sequester the sigma factors and remove from functional pool Covalent mods localization rate of transcription/translation and protein turnover
What does the group two sigma 38 (RpoS) do?
Most similar to the sigma classic, is involved in nutrient depletion associated with stationary phase growth. Many genes overlap with sigma70 and again is involved with stress response during stationary phase.
Example of protein turn over regulation
Sigma 38/ RpoS when bound by RssB is degraded by proteolysis via ClpXP. This is an example of protein turn over, by having more RssB you can destroy more of this sigma factor limiting the amount of expression of genes under its charge the same is true vis versa by having less of this you can have higher transcription of the stationary response genes.
This is regulated as follows: signal comes in –> stress starvation ect activates Inhibitor of RssB Activity (Ira) proteins which prevent rssB binding the alt sigma preventing mark of death.
Anti-sigma regulation example
group 3 sigma 28(sigD) ordinarily the sigma is bound by an anti sigma factor when it senses that a flagellum is being made it dissociates from the sigma factor and is pumped out of the cell the released sigma factor not binds guides the RNAP to the flagellum promoter and synthesis is completed.
1) anti sigma factor: sigma factor complex
2) senses something and dissociates from sigma factor and is pumped out of cell.
3) alt sigma free to bind promoter –> gene transcription and translation.
