BSR Ch.8 Regulated Proteolysis

What is the complex called that breaks down proteins?

Proteosome

Where were proteosomes first discovered?

Lysosomes in eukaryotes

In eukaryotes, proteins that are marked for degradation are marked by what?

Ubiquitination

What are the 2 major protein families involved in protein degradation?

AAA+ proteases and Hsp/Clp proteins

What does “AAA+” stand for?

ATPases associated with diverse cellular activities

What are the 2 examples of AAA+ one component proteases?

Lon and FtsH

What is the example of 2 component proteases?

Clp

What are the 2 components of a two-component protease?

Protease and unfoldase(chaperone)

(T/F) AAA+ proteases are specific to their substrates.

True

What is the name of the stretch of amino acids that marks a protein for degradation?

Degron

(T/F) A degron can be at the C-terminus, N-terminus, or in the middle of the protein.

True

What was the example of a C-degron added by tmRNA during ribosome stalling?

SsrA-tag

N-degrons can be generated by the adding of what amino acids by amino acid transferases?

Leu or Phe

In the process of removal of fMet from mature proteins, what enzyme first removes the formyl group?

Peptide deformylase (PDF)

In the process of removal of fMet from mature proteins, what enzyme removes the methionine after deformylation?

Methionine aminopeptidase (MAP)

What is the name of the rule that states that the N-terminus amino acid dictates the half-life of a protein?

N-end rule

What N-terminal residues make proteins only have a half life of ~2 mins?

R, K, L, F, W, and Y

What are the primary N-terminus destabilizing residues?

L, F, W, and Y

What are the secondary N-terminus destabilizing residues?

R and K

If the N-terminus residue is a secondary destabilizing one, amino acid transferases (Aat) add what residues?

L or F (usually L)

What protein recognizes primary destabilizing residues and delivers them to the AAA+ protease ClpAP?

Recognin / ClpS

What is the name of the protein class that identifies substrates marked for degradation and delivers them to the protoeosome?

Adaptor proteins

What adaptor recognizes SsrA-tags delivered from tmRNA?

SspB adaptor

Where does the SspB adaptor deliver the SsrA-tagged proteins?

ClpXP

What adaptor recognizes substrates following the N-end rule?

ClpS

In B. subtilis, what adaptor protein helps assemble the full protease ClpCP?

MecA

What type of antibiotic activates ClpP, leading to uncontrolled proteolysis?

Antibiotic acyldepsipeptides (ADEPs)

What are the 3 main ways DNA can be transferred between bacteria?

Conjugation, transformation, and transduction

Which DNA transfer method involves the uptake of extracellular DNA from the environment?

Transformation

Bacteria that can undergo transformation without any help are called…?

Naturally transformable

When bacteria reach a stage where they can uptake extracellular DNA are called…?

Competent

Bacteria that can naturally reach competence without any help are called…?

Naturally competent

Who performed the original experiment that showed the capability of transformation?

Frederick Griffith

What phenomenon in the Griffith experiment showed transformation?

Heat killed S cells with living R cells yielded both living S and R cells

What bacteria was used in the Griffith experiment?

Streptococcus pneumoniae

What scientists later confirmed that DNA was the transforming agent?

Avery, MacLeod, and McCarty

Induction of competence is usually a stress response due to what?

Nutrient deficiency and overcrowding

What is it called when only a certain % of a bacterial group become competent?

Bistable state

In the competence mechanism, what is the two component system?

ComAP

In the competence TCS, which is the sensor kinase and which is the response regulator?

ComP = Sensor Kinase | ComA = Response Regulator

ComP binds to what ligand?

ComX

Phosphorylation of ComA to ComA~P by ComP allows for ComA~P to upgregulate what gene?

comS

What is the protein that is the transcription activator of competence genes?

ComK

ComK induces what types of genes?

DNA uptake, DNA recombination, and comK itself (positive autoregulation)

Under normal conditions, what protein binds to ComK and brings it to ClpP to be degraded by the ClpCP proteosome?

MecA

During competence induction, what protein binds to MecA, preventing it from degrading ComK and allowing competence genes to be transcribed?

ComS

ComS is known as the “proteolytic switch” or as the…?

Anti-adaptor

What was the main toxin example provided in lecture?

Doc

What does “Doc” stand for?

Death on curing of prophage

What does Doc inhibit?

Translation elongation

Where does Doc bind to?

30S subunit of ribosome

What is the antitoxin for Doc and what does it stand for?

Phd → Prevents Host Death

Where is the phd-doc module located?

E. coli P1 bacteriophage

(T/F) The Phd antitoxin is unstable and is readily degraded by ClpXP.

True

What inhibits transcription of the phd-doc module?

Phd and the Phd-Doc complex

How many Phd molecules bind to Doc to neutralize it?

2

Which Phd terminus is the DNA binding domain?

N-terminus

Which Phd terminus is the toxin neutralizing domain?

C-terminus

What is the phenomenon called when a bacterial cell divides, and one daughter cell gets the plasmid and the other doesn’t, causing the daughter cell lacking the plasmid to die?

Plasmid addiction

What was the other toxin example that was an endoribonuclease?

MqsR

What was the other toxin example that is a gyrase inhibitor?

CcdB