Lecture 9.doc

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Department
Microbiology (Biological Sciences)
Course
MICRB316
Professor
Nicolas Vozza
Semester
Winter

Description
Lecture 9 1 2 Component Systems Generally, bacteria live in environments that undergo DRASTIC CHANGE (resources and conditions) To survive, they must respond rapidly, through: 1. adaption 2. Movement (fight or flight) Therefore, bacteria have evolved mechanisms to sense external conditions & transfer this information to cellular sites where an appropriate response is exerted. > altered patterns of gene expression This SIGNAL TRANSDUCTION generally involves the transfer of phosphoryl groups between proteins. Many of these signals are sensed and transmitted to the Tn apparatus by PAIRS of proteins, called Called a 2 component system. Sensor Kinases (SK) & Response Regulators (RR) > 1000 pairs of 2 component systems known. These transfer environmental stimuli to bacteria via cognate pairs of Lecture 9 2 SK + RR. Seen NtrB/C already. Also, FixL/FixJ. NtrB phophorylates NtrC. NtrB is SK. NtrC is RR. Autophsorylation of sensor kinase > phosphorylation of RR > altered confromation > DNA binding by (1) RR itself or (2) other proteins > altered Tn. NtrB is an example of a bi-functional kinase/phosphates. (needs to by able to chut off signal when not needed. Either SK is bi-functional or there is another phophotase involved. Lecture 9 3 Various regulatory mechanisms (some of which act through HPK / SK) regulate intracellular concentration of RR in response to stimuli. ~P When the phosphorylated form of RR (RR ) reaches a critical level, the DNA is bound, leading to RNAP recruitment (activation) or interference (repression). The physiological adaption is regulated via control of the concentration of the phophorylated for RR-P of the RR. Lecture 9 4 Autophosphorylation Phosphorylation of RR Response Regulators N-terminal conserved domain ~125 AA with 30% identity between proteins and several invariant AA’s. This is the phosphate Receiver Domain – 3 conserved Asp / 1 Lys The aspartates form an acidic pocket; lysine protrudes into the pocket to stabilize the phosphoryl group. C-terminal conserved domain = OUTPUT Domain (or effector domain). These interact with the transcription complex (RNAP). They mediate Tn Regulation by either: 1. Altered DNA binding (by themselves) 2. Altered RNAP binding. Lecture 9 5 The change in the activity of the OUTPUT domain is brought about through reversible transfer of phosphoryl groups to invariant aspartates at receiver domains. 3 distinct sub-families of RRs Based on structural and functional organization of the output domain. OmpR C-term DNA-binding HTH = 150 AA – linked to receiver When receiver domain is phosphorylated, a conformational change causes STRONG DNA binding by output domain. Dephosphorylation causes DNA-binding interference. Lecture 9 6 Dephosphorylation causes DNA-binding interference. OmpR-like 2 component systems act at σ70 promoters. Tn can be either activated or repressed by OmpR-like 2 component systems, depending upon where RR binds. For some of these 2C systems, they bind DNA and make direct contact with αCTD of RNAP. FixJ Conserved C-terminal DNA-binding HTH = 100 AA; different sequence from OmpR type Act in combination with σ70 RNAP / promoters Variations 1. many homologs of FixJ RR’s have an output domain, but no reciever domain. 2. Other FixJ RR’s have receiver domains, but no output domain (no gene regulation). e.g. – CheY: phosphorylated CheY interacts with flagellar switch proteins to cause reversals in flagellar rotation. This is protein-protein interaction. Not DNA interaction. Lecture 9 7 NtrC - 3 separate domains • N term – Reciever domain • C term – Dna-binding HTH • Central domain = ATPase – similar to ATP binding proteins • Unlike OmpR & FixJ which act by improving RNAP-DNA interaction (RNAP recruitment), NtrC-like RR’s facilitate PROMOTER melting This RXN involves the ATPase function. ATPase activity is dependant on prior phosphorylation of the reciever domain leading to formation of tetramers and aggregates of Ntr-C like proteins Lecture 9 8 In the de-phosphorylated state these RR’s exist as dimers and have no activity. Sensor Kinases Transfer of the phosphoryl group is maintained by HPK. However, for each RR, a COGNATE HPK can be assigned. They are named HPK because the source of the phosphoryl group is a phospho histadine AA. Modular structure; conserved 250 AA’s; 20% ID; 5 sub- domains: H box histidine is the active site which catalyzes transfer of γP from ATP to histidine AA. ATP binding site is in the G box. ATP binding site in G box Lecture 9 9 Usually HPKs exist as dimers; Phosphorylation occurs
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