Lec 12 - Two Component Systems

Question 1

Describe, in simple terms, what a TCS consists of and what they do.

Answer 1

They allow bacteria to sense and respond to their environment Consists of Histidine Kinase (normally membrane bound) - sensory domain, Dhp w/ conserved His residue, Catalytic ATPase domain AND Response Regulator - Receiver Domain w/ conserved Asp residue and an output

Question 2

Give 2 examples of sensory domains that are found on HK and RRs.

Answer 2

HK SD: PAS - senses O2 levels (TM PAS DHp CA) RR SD: AAA+ (sfN) and DBD - most consist of DBDs

Question 3

What class does a phosphorelay system belong in and describe it.

Answer 3

Class I Rather than P being transferred to a RD then not being transferred again, after RD has got the P it is then passed on to another conserved His residue of Hpt From there it is passed onto a RD This allows a response to multiple pathways

Question 4

Describe the roles of the 4 types of accessory protein in TCS.

Answer 4

• no SD on HK therefore a cosensor acts to transmit the signal to the HK - scaffold protein - adds an extra element of control. for the signal to be transduced from HK > RR it needs to pass through this additional protein - connector protein - eg acid response - allows the linking of 2 TCSs in response to only one signal (sometimes) - allosteric effector protein - signal from one TCS P an allosteric effector protein. This can then work on another/the same TCS to stimulate/inhibit the P of the RR

Question 5

Describe the activation of Response Regulators using 2 examples.

Answer 5

PhoB - in its unP version, exists in eqm as a dimer and monomer. in P form forms a 2 fold symmetrical homodimer w/ tandem symmetry of the DNA binding domains and recognition helices. Can now bind adjacent major grooves on DNA. in unP dimer, the DBDs are not aligned RstA - P of the RD causes dimerisation. The DBD structure is altered and one BD binds DNA. Afterwards, the other BD colocalises (as they are not originally aligned) and binds DNA so that the BDs exist next to each other. From here, EITHER… 1) RR binds a subunit of RNAP OR 2) RR binds to sigma factors of RNAP by binding at a -41 wish site both of these ways then allow RNAP to be brought to the -35 and -10 regions

Question 6

Give an example of a regulatory circuit that uses a sf and a TCS.

Answer 6

Acid Regulatory Mechanism in E. coli - a TCS recognises low pH in environment - stimulates production of SafA connector protein - SafA stimulates P of another TCS and IraM protein produced - IraM then binds to RssB to sequester it and stop it binding to RpoS/sfS - RssB + RpoS = RpoS degradation - RpoS can then activate the transcription of the acid response genes

Question 7

Describe how the 2 pathways of 2 TCS are derived and how they can become separated.

Answer 7

A gene duplication event results in an identical TCS being made Phosphotransfer & Cross talk between 2 systems because identical HK recognition sites Mutations in specificity determining residues may insulate the pathways and cross talk can no longer occur

Question 8

Even though most HKs in E. coli do not cross-talk, one system still exists that does. With the aid of a diagram, describe this system.

Answer 8

Nitrate and Nitrite system Involves the enzymes used in NO3- / NO2- metabolism NarX and NarQ still P either Nar L / Nar P (see diagram in notes) all about phosphotransfer and phosphatase activity balance