MIC 360 Lecture Notes - Lecture 21: Signal Peptide, Arginine, Lysine
Monday, April 9, 2018
MIC 360 Notes - Class 21 - Monday April 9, 2018
2. TAT System - Slide 1!
-The only protein secretion system that can export the folded protein!
-Why do some proteins need to be folded before secreted?!
•In many substrates, they contain co-factor!
-Co-factor either not available in target region outside of cell, or cofactor cannot
be assembled after the protein is folded!
-Thus, has to be first folded into the cytoplasm and then sent tot he target region!
-TAT system has unique function in that it recognizes one calss of specific signal
peptides!
•Signal peptides= amino acid sequences recognized by protein secretion
systems!
•Signal peptides recognized by TAT system = what’s shown on the bottom of
the slide!
•Structural feature = twin arginine !
•K = lysine!
•Normal there is a tandem arginine = located next to each other = twin arginine!
3. Slide 2!
-In the absence of the substrate, there are three type of subunits of this transporter!
-TatA protein!
-TatB and TatC!
-Three different proteins are required for assembly of one TAT system!
-When substrate absent = subunit is distributed and assembled separately!
-When substrate appears, signal peptide (RR), binds first to substrate and binds to
BC, A moves and assembles with it into a channel!
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Monday, April 9, 2018
-Once channel is formed, the substrate will be able to swim out of the membrane!
-Signal peptide remains inside and faced towards the cytoplasm!
-An enzyme comes in and cuts it = peptidase = recognizes signal peptide region
and cuts it out!
4. Test of the TAT translocation System slide 1 & 2!
-How do we know that TAT ABC are required for transport of TAT dependent
substrate?!
-Twin RR = can function as signal peptide for a substrate that is dependent on the
TAT system for secretion!
-We can construct a fusion protein = green fluorescent protein - Linked to this
GFP = reporter protein to observe the localization of this protein in a cell under
microscope!
-When this peptide is linked to GFP, what happens to the GFP?!
-It’s going to be secreted to the periplasm!
-TAT system is located in the cytoplasm and will send a substrate to the
periplasm!
-Mutation of each protein will knockout the system (the three proteins required
for TAT system)!
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Monday, April 9, 2018
Homework 2: construct a pUC19 plasmid to direct
expression of this fusion protein in E. coli. Present the whole
sequence of your plasmid.!
First convert this amino acid sequence on the slide to nuclear
acid sequence - then you can see the whole sequence of plac 19
and then figure out the fusion of the ORF which can express this
peptide as well as the GFP!
Then put the DNA fused sequence into the Plac19 = use
restriction map!
Due Tuesday!
Wants the whole DNA sequence - each nucleotide!
In order to express a gene, what do you need? In order to
transcribe? In order to translate? You need to find out
promoter, SD site, etc. !
Find one in literature or create one - find something that
has SD site!
5. The SEcY Pathway - Most commonly used export pathway!
-SecYEG = complex that mediates protein secretions of denature proteins!
-Not folded proteins!
-Only TAT system mediates export of folded proteins!
-One protein called SecB = chaperone protein!
-Chaperone protein is a class of protein factors which can bind to denature protein!
-Denatured protein when bound to chaperone will be either folded or remain
unfolded !
-When secB binds to nascent protein = this protein will not be folded!
-Will remain in unfolded form!
-Unfolded protein bound to secB will be targeted to the secretion system
SecYEG!
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Document Summary
Mic 360 notes - class 21 - monday april 9, 2018. Monday, april 9, 2018: tat system - slide 1. The only protein secretion system that can export the folded protein. Why do some proteins need to be folded before secreted: in many substrates, they contain co-factor. Co-factor either not available in target region outside of cell, or cofactor cannot be assembled after the protein is folded. Thus, has to be rst folded into the cytoplasm and then sent tot he target region. In the absence of the substrate, there are three type of subunits of this transporter. Three di erent proteins are required for assembly of one tat system. When substrate absent = subunit is distributed and assembled separately. When substrate appears, signal peptide (rr), binds rst to substrate and binds to. Bc, a moves and assembles with it into a channel. Once channel is formed, the substrate will be able to swim out of the membrane.
