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MCB 1 > Proteins > Flashcards

Proteins Flashcards

1
Q

Genetic Code

A

Instructions contained in DNA/RNA that is translated to a specific protein

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2
Q

What makes up genetic code

A

Triplet code

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3
Q

How many codons are there for A.A

A

64
- 3 are Stop codons
- 61 code for proteogenic a.a.

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4
Q

What are the stop codons?

A
  • UAG
  • UGA
  • UAA
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5
Q

Genetic code is Redundant

A

Multiple codons can code the same amino acid
BUT each codon only codes for 1 amino acid

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6
Q

Start Codon

A

Always Methionine
AUG

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7
Q

What is special about Methionine?

A
  • Only A.A with 1 codon
  • Start Codon
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8
Q

Anticodon

A

Sequence of 3 nucleotides on tRNA that is complementary to the mRNA codon

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9
Q

Wobbling

A

3rd position of codon (3’) & 1st position of anticodon (5’) allows flexibility
- Unlike the strict Watson-Crick pairing it tolerates alternative pairings like G-U

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10
Q

Sense strand

A
  • Strand NOT used for transcription
  • Identical to the RNA transcript
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11
Q

Antisense Strand

A
  • Template strand for transcription
  • Complementary to sense strand
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12
Q

Non-overlapping meaning

A

No repeated bases in codon
XXG GXX is Overlapping

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13
Q

What is consequence of using both sense and antisense as template at the same time?

A

Formation of dsRNA

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14
Q

dsRNA

A
  • “Double Stranded”
  • Cannot be translated
  • Often prod. in Viral Infection so triggers Immune response
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15
Q

Genome reading

A
  • Ribosome reads bases in pairs of 3 called codons so it could technically read the frame 3 different ways, where only 1 is the true code needed.
  • Thats why we have START codon which tells the ribosome exactly where to start translation
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16
Q

Open Reading Frame (ORF)

A

Sequence of codons running from specific start codon to specific stop codon

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17
Q

tRNA structure

A
  • Folded RNA molecule
  • Small size (70-100 nucleotides)
  • Has modified nucleotides
  • Cloverleaf
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18
Q

Modified nucleotides on tRNA

A
  • dihydrouridine (UH2)
  • ribothymidine (T)
  • pseudouridine (Ψ)
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19
Q

tRNA arms

A
  • Anticodon Arm with anticodon loop region
  • Amino acid acceptor arm with 3’ end of tRNA (single stranded region)
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20
Q

Nucleotide sequence of Amino acid acceptor arm
+ when is it added

A

CCA on 3’ end
Post-transcriptional modification

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21
Q

5’ end of tRNA

A

Phosphorylated

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22
Q

What bond forms bw CCA on tRNA and Amino acid

A

Ester bond
OH of CCA and Carboxyl group of Amino A.

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23
Q

Aminoacyl-tRNA synthetase

A

Enzyme which ensures that the correct a.a is matched with the corresponding tRNA anticodon

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24
Q

Aminoacyl-tRNA synthetase
Double function - 1

A

Activation of amino acids
- Adenylates AA by adding AMP from ATP
- Aminoacyl-AMP forms ester bond with CCA on 3’ end

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25
Q

Aminoacyl-tRNA synthetase
Double function - 2

A

Translation of the Genetic code
- Recognizes both appropriate A.A and the Anticodon

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26
Q

Fidelity

A

Accuracy with which the correct A.A is attached to the tRNA molecule

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27
Q

What do some Aminoacyl-tRNA Synthetases have that increases Fidelity?

A

Proofreading or Editing activity
- Editing domain that can cleave ester bond if wrong A.A is bound
- Analogous to DNA polym. proofreading

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28
Q

tRNA Charging

A

Process where specific A.A is covalently attached to its corresponding tRNA
- Aminoacyl-tRNA is a Charged tRNA since it is successfully bound

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29
Q

rER Ribosomes

A
  • Bound Ribosomes on ER
  • Make proteins destined for transport outside the cell
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30
Q

Free Ribosomes

A

Generate proteins for the cell’s own needs

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31
Q

Makeup of a ribosome

A
  • 2/3 rRNA
  • 1/3 Ribosomal Proteins
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32
Q

How do we get rRNA

A

Human genome has around 200 copies of genes that code for rRNA to keep up with the demand because protein synthesis is a Fundamental Process

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33
Q

3 main types of rRNA in Eukaryotes

A
  • 18s
  • 5.8s
  • 28s
    All processed from a common Precursor RNA
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34
Q

Which rRNA is special and why

A

5S rRNA
Separate molecule transcribed differently

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35
Q

What is S in rRNA

A

Svedberg units
- Parameter proportional to the molecular size

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36
Q

Where does transcription to form rRNA happen?

A

Nucleolus of the Nucleus

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37
Q

RNA polymerase I

A

Makes Primary Transcript for 18S, 5.8S, 28S rRNAs
(mature rRNA taken to cytoplasm)

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38
Q

RNA Polymerase III

A

Transcribes 5S rRNA

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39
Q

Where are ribosomal proteins synthesized?

A

Cytoplasm

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40
Q

Ribosomal Proteins

A
  • RPL (for Large subunit)
  • RPS (for Small subunit)
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41
Q

Where do ribosomal Proteins assemble with rRNA?

A

Nucleus

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42
Q

Are Eukaryotic and Prokaryotic Ribosomes at all similar?

A

Yes, high level of similarity

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43
Q

Eukaryotic ribosomal large and small subunits

A
  • L: 60S
  • S: 40S
    Overall: 80S
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44
Q

How many rRNA molecules make up Eukaryotic Ribosomes

A

4 different rRNAs

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45
Q

Prokaryotic ribosomal large and small subunits

A
  • L: 50S
  • S: 30S
    Overall: 70S
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46
Q

How many rRNA molecules make up Prokaryotic Ribosomes

A

3 different rRNAs

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47
Q

Role of Ribosome Large subunit

A

Peptide bond formation

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48
Q

Role of ribosome small unit

A

mRNA binding to decode it

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49
Q

When do the small and large ribosome subunits form a complex?

A

ONLY during Translation

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50
Q

Are ribosomes identical?

A

No, they differ in Protein composition

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51
Q

3 tRNA binding sites on Ribosome

A
  • A site (aminoacyl)
  • P site (peptidyl)
  • E site (exit)
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52
Q

A site (aminoacyl)

A

Where the incoming aminoacyl-tRNA binds to the Ribosome during elongation phase

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53
Q

P site (peptidyl)

A

Where peptidyl-tRNA is bound to the Ribosome which has a polypeptide chain attached to it

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54
Q

E site (exit)

A

Where the tRNA that no longer carries an amino acid and released its polypeptide chain exits the Ribosome

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55
Q

Do the tRNA binding sites span 1 subunit?

A

It spans both the Small and Large subunits

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56
Q

What happens when Ribosome reaches Stop codon?

A
  • Polypeptide chain is released
  • Ribosome leaves mRNA and becomes Termination Ribosome
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57
Q

Termination Ribosome

A
  • Unstable
  • Readily dissociates to free ribosomal subunits
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58
Q

IF3 after ribosome dissociation

A

Initiation Factor 3
Recycles small ribosomal subunits (30s / 40s)

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59
Q

What happens to Free ribosomal Subunits in Sufficient IF3?

A

Small ribosomal subunits bind IF3 and become stable Native subunits (ready for translation)

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60
Q

What happens to Free ribosomal Subunits in Insufficient IF3?

A

Small & Large ribosomal bind each other without an mRNA and form a Single ribosome (non-functional) which can only function when they dissemble and form a Functional ribosome

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61
Q

Polysome

A

Clusters of ribosomes simultaneously translating the same mRNA sequence

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62
Q

Pro/Eukaryotes protein folding

A
  • Pro: Post-translational
  • Euk: Co-translational
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63
Q

Shine-Delgarno sequence

A

Located upstream of the Start Codon of Prokaryotic mRNA
(part of ribosome binding site)

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64
Q

Prokaryotic Initiation of Translation Steps

A

1) IF1 & IF3 bind small subunit (30S)
2) mRNA RBS binds 16S rRNA of small subunit
3) IF2 brings fMet-tRNA to the small subunit using GTP
4) 30S initiation complex formed
5) IF1 & IF3 released and large 50S binds
6) IF2 dissociates and GTP to GDP
7) 70S is now ready for Elongation

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65
Q

What makes up the 30S initiation complex?

A
  • IF 1/2/3
  • mRNA
  • fMet
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66
Q

Why is it fMet in Prokaryotes for 1st A.A?

A

Modified methionine Formylmethionine by Enzyme Transformylase (only Prokaryotes)

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67
Q

IF1 role

A

Prevents Large subunit 50S from binding until the 30S Initiation complex is ready

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68
Q

IF3 role

A

Orients fMet-tRNA to the correct site (P-site) not A cause we are initiating

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69
Q

Regulation of Prokaryotic Translation

A
  • Translation repressor binds Shine-Delgarno sequence (stops)
  • Environmental factors like temperature melt structures exposing SD-sequence (starts)
  • Small molecule binds riboswitch, folds and hides SD-seq (stops)
  • Antisense RNA binds SD-seq blocks ribosome binding (stops)
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70
Q

Significance of 5’ end of Eukaryotic mRNA

A

Methylguanosine cap has eIFs:
- 4E
- 4G
- 4A

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71
Q

Ternary complex Eukaryotes

A
  • Initiator tRNA (Met-tRNA)
  • eIF2 & GTP
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72
Q

Eukaryotic Initiation of Translation Steps

A

1) Ternary complex formation
2) Small subunit 40S associates with eIFs forming 43S pre-initiation compl.
3) PABP binds 3’ tail & eIF4G forming loop
4) 43S PEC binds eIFs on 5’ cap
5) 43S PEC scans mRNA 5’ to 3’ scanning UTR till Start-codon is found
6) eIFs dissociate and Large 60s subunit joins forming 80S initiation complex
7) eIF2 does GTP to GDP and dissociates

73
Q

Polyadenosine Binding Protein (PABP)

A

Binds to 3’ Poly A tail and eIF4G making the mRNA a loop/circular structure

74
Q

mRNA quality control
(Eukaryotes)

A

1) During abnormal splicing Intron might be kept
2) Intron can be a PTC (termination)
3) If ribosome reaches this it interacts with EJC and UPF proteins
4) This tells the ribosome that the mRNA is faulty and has a stop codon where Exons should be (EJC)
5) Nonsense-mediated Decay (NMD) initiated resulting in degradation of mRNA

75
Q

Regulation by microRNA
(Eukaryotes)

A

1) Silencing compl. made from Ago protein and guide strand of miRNA
2) Guide strand searches for complementary bases/binding sites called Seed regions
3) Once a site is found double helix forms bw miRNA and mRNA
4) Prevention of Translation

76
Q

Regulation by Localization
(Eukaryotes)

A

Used in regions where more translation is needed e.g. Apical side of enterocytes when food is present to make transporter proteins for more uptake of nutrients
Microtubules move mRNA transcripts to apical regions for more translation

77
Q

Regulation by Proteins
(Eukaryotes)

A

Phosphorylation of ribosomal S6 protein in 40S subunit leads to increased translation
e.g. mTOR-Kinase

78
Q

Regulation by Hairpin Structure
(Eukaryotes)

A

Hairpin is a loop formed by the folding of the RNA strand back on itself
Can block or expose binding sites

79
Q

Regulation by modification of initiation factors (eIFs)
(Eukaryotes)

A
  • Phosphorylation of eIF4E
  • 4E-BP
  • Phosphorylation of eIF2a
80
Q

Phosphorylation of eIF4E

A

Decreases eIF4E affinity to 5’ cap so Inhibits Translation

81
Q

4E Binding protein (4E-BP)

A

It binds eIF4E preventing 4E-4G interaction and prevents recruitment of 43S pre-initiation compl. due to no loop
Inhibits Translation

82
Q

Phosphorylation of eIF2a

A
  • Inhibits GTP to GDP
  • 2B (GEF) but inactive when 2a is phosph. and sequestered
    Inhibits Translation
83
Q

What increases phosphorylation

A
  • A.A deprivation: GCN2 protein Kinase activated by uncharged tRNA and phosph. eIF2a
  • Viral RNA: PKR recognizes it, 2 bound PKRs with Viral RNA form homodimer and phosph. eIF2a
84
Q

Regulation by IRES (internal rib. entry site)
(Eukaryotes)

A

5’ cap needed for translation, if its not present translation can be initiated at an IRES sequence
(IRES can be found in 5’ UTR which mimics 5’ methylguanosine cap)

85
Q

Steps if 5’ cap not present and using IRES sequence

A

1) eIF4G binds IRES sequence
2) eIF4G binds PABP to 3’ tail
3) mRNA is looped
4) Ribosome scans and starts at codon
NO eIF4E or 5’ cap needed

86
Q

Elongation Phases (3)

A
  • Decoding
  • Transpeptidation
  • Translocation
87
Q

Decoding in Elongation

A

1) Elongation factor EF-Tu with GTP facilitates binding of aminoacyl-tRNA anticodon with mRNA codon in A-site
2) GTP to GDP and EF-Tu dissociates after binding
3) EF-Tu recharged with GTP with EF-Ts (acts as GEF)

88
Q

Transpeptidation in Elongation

A

1) A.A on aminoacyl-tRNA in A-site attacks ester bond of tRNA in P-site
2) Peptidyl transferase activity forms peptide bond
3) Hybrid state where tRNA in P-site is deacylated and A-site tRNA has growing chain

89
Q

Translocation in Elongation

A
  • EF-G facilitates translocation of ribosome to next codon (GTP)
  • N to C terminal direction
  • Deacylated tRNA moved to E-site this way to form Default state
90
Q

Prokaryote vs Eukaryote Elongation factors

A
  • EF-Tu = EF1 a
  • EF-Ts = EF1 By
  • EF-G = EF2
91
Q

Termination of Translation

A
  • No tRNA for stop codon
  • Release factors are used
  • RF very similar to tRNA struct.
92
Q

Termination of Translation Steps

A

1) Ribosome reaches stop codon
2) RF recognize stop codon and bind A-site similar to how tRNA would
3) RF hydrolyzes ester bond bw tRNA and last A.A and releases polypeptide
4) RF3 GTPase removes RF1/2 from ribosome
5) RPF ribosome recycling factor dissociates ribosomal subunits

93
Q

Antibiotics that interact with Small ribosomal Subunit

A
  • Tetracycline
  • Spectinomycin
  • Hygromycin B
  • Streptomycin
94
Q

Tetracycline

A

Blocks binding of aminoacyl-tRNA to A-site

95
Q

Why does Tetracycline only affect Prokaryotes

A

Only prokaryotes have transporters that bring tetracycline into the cells

96
Q

Spectinomycin

A

Interferes with Translocation step of Elongation by binding 30S subunit

97
Q

Hygromycin B

A

Premature chain termination by inserting into A-site

98
Q

Streptomycin

A

Prevents transition from Translation initiation to Elongation & causes Miscoding

99
Q

Antibiotics that interact with Large ribosomal Subunit

A
  • Chloramphenicol
  • Erythromycin
  • Streptogramin B
100
Q

Chloramphenicol

A

Blocks peptidyl transferase activity on ribosomes so prevents peptide bond formation

101
Q

Erythromycin

A

Binds to E-site and inhibits elongation

102
Q

Streptogramin B

A

Binds P-site of 50S subunit

103
Q

Inhibitors of Translation (non-antibiotic)

A
  • Puromycin
  • Cylcohexamide
104
Q

Puromycin

A

Inhibits Pro/Eukaryotic translation by mimicking aminoacyl-tRNA and blocking A-site

105
Q

Cyclohexamide

A

Blocks the translocation phase of elongation in EUKARYOTES

106
Q

Corynebacterium diphtreriae

A
  • Produces diphteria toxin
  • Inhibits EF2 in eukaryotes
  • No translation
107
Q

Ricinus Communis

A
  • Seeds have Ricin toxin
  • Cleaves adenosine off 28S rRNA
  • No formation of functional ribosomes
108
Q

N-terminal Modifications

A
  • Cleavage of (f)-Methionine
  • Acetylation of Second amino acid after (f)-Methionine
  • Myristoylation: covalent attachment of myristoil group (14c FA) (crucial for membrane association of proteins)
109
Q

C-terminal Modifications

A
  • Amidylation: Conversion of COO- group to Amide group (2x Amide)
  • Glycosylphosphatidylinositol coupling: C-term. cleaved and attached to GPI for membrane anchoring
110
Q

Modifications of Internal A.A

A

All amino acids can go through Post-translational Modifications
- Was believed that Ile, Leu, Val, Ala, Phe did not because of hydrophobicity but proven wrong

111
Q

Hydroxylation

A
  • Proline & Lysine
  • Adds -OH on side chain allowing more H-bond formation
  • Collagen binds tightly to each other due to hydroxyproline (3 a helices)
112
Q

Hydroxylation Enzyme and consequences

A
  • Prolyl Hydroxylase
  • Succinate & Fe3+ cofactors
  • Fe3+ red. is Vit-C dependent
  • Low Vit-C = Scurvy
113
Q

Lysine Deamination

A

1) y-amino group removed by Lysyl Oxidase (forms Norleucin)
2) Aldehyde group forms reacting with amino group forming Schiff base
3) Cross-link bw Lysine and Norleucin so Intra/intermolecular cross-links

114
Q

Tethering membrane proteins

A

Adding a lipid tail to anchor protein to the membrane
- Palmitoylation (C or S)
- Myristoylation (Gly)
- Prenylation (RAS)
- Phosphatidylethanolamidation
- Cholesterylation

115
Q

Ca2+ Binding by Carboxylation

A
  • Carboxyglutamic acid (Gla-domain)
  • Vitamin K dependent carboxylase
  • VKOR system
116
Q

Protein Phosphorylation
(most general)

A
  • Mainly Serine, Threonine, Tyrosine(-OH groups)
  • Uses ATP/GTP
  • A.A from Tensed to Relaxed (can do its job)
117
Q

Methylation

A
  • Represses Transcription
  • More methylated = more hydrophobic = Heterochromatin
  • SAM
  • Lysine (x3) & Arginine (x2)
118
Q

Acetylation

A
  • Promotes Transcription
  • Loosens chromatin = Euchromatin
    • charge of NH3+ lost and histones (usually +) dont bind DNA (-) as well as before
119
Q

Bromo Domain Motifs

A
  • Special parts of certain proteins that recognize and bind to acetylated lysine residues on histones
  • Help activate gene expression by recruitment of Transcription machinery
120
Q

Glycosylation

A
  • Attaching sugar/carb to active side chain of A.A
  • Asparagine, Serine, Threonine
  • N-glycosylation (90%) & O-glycosylation (10%)
121
Q

O-Glycosylation

A
  • In Golgi
  • Random which Ser/Thr is glycosylated
  • More carbs than protein (60-95% more)
  • Proteoglycans, GAGs
  • Chondroitin/Keratan sulfate, Hyaluronate (all - charge)
  • Attract Na+ which attracts water = hydration & flexibility
122
Q

N-Glycosylation

A
  • Begins in ER
  • Strictly determined which Asn is glycosylated
  • Oligosaccharyl Transferase
  • Maturation in Golgi by trimming
  • Pyrophosphate for energy to attach sugar to Asn
123
Q

Functions of Glycosylation

A
  • Protecting proteins from degradation
  • Selective labelling of proteins
  • Determines cell-to-cell connections
  • Allows protein folding quality control
124
Q

Protein Domains

A

Independently folded structural and functional units within a protein

125
Q

Examples of PTM recognizing domains

A
  • Bromodomain: acetylated lysine residues (histones)
  • Plant Homeodomain (PHD): recognizes methylated lysine residues (chromatin)
126
Q

p53

A

“Guardian of the Genome”
Protects cells from DNA damage by either stopping the cell cycle or triggering cell death

127
Q

Anfinsen’s Dogma

A

States that secondary and tertiary structures are determined by the primary structure
Proven by denaturation then reassembly back to a functional Enzyme

128
Q

Levinthal’s Paradox

A
  • If a protein tried to fold into every confirmation it would take ages.
  • Instead it follows a funnel-like system where its structure leads it to the most energetically favorable structure.
129
Q

Hydrophobic collapse

A

Apolar/Hydrophobic side chains of A.A cluster together in protein’s interior away from the surrounding aqueous.
Establishes Molten Globule state

130
Q

Why do proteins need help folding

A
  • Macromolecular crowding inside cells
  • Protein aggregation
  • Energy Barriers
  • Chaperone proteins guide them
131
Q

Chaperones

A
  • Heat shock proteins, in High temp because proteins are most likely to make mistakes then
  • Help proteins overcome energy barrier to right conformation using ATP
132
Q

3 Levels of Chaperones

A

1) Trigger Factor = Rib. Assoc. Compl.
2) DnaK & DnaJ = Hsp70/40
3) GroEL & ES = TRic
(Prokaryotes / Eukaryotes)

133
Q

Hsp70/40

A

1) Hsp40 acts as co-chaperone for Hsp70 and binds misfolded proteins
2) Hsp70-ATP is in open state ready to accept protein
3) Hsp40 triggers Hsp70 to hydrolyze ATP to ADP, now Hsp70-ADP is closed
4) Now it is locked on misfolded protein and can help it fold

134
Q

GroEL & ES

A
  • GroEL is barrel shaped (2 ring, 7sub) central cav. where misfolded proteins are enclosed
  • GroES is the cap that closes it
  • ATP needed to close and fold
135
Q

26S Proteasome

A

19S regulatory particle recognizes polyubiquitinated proteins to unfold and translocate them

136
Q

Protein Disulfide Isomerase (PDI)

A
  • Helps proteins form disulfide bonds bw Cysteine residues
  • Oxidized: Helps create new disulfide bonds in the target protein.
  • Reduced: Helps fix incorrect DS bonds in the target protein by “shuffling” them.
    (in the ER)
137
Q

Carb Binding Chaperones (Lectins)

A
  • Calnexin: specifically recognizes glycoproteins that have undergone N-glycosylation
  • Recognizes monoglycosylated glycoproteins and binds to fold them
  • When done glucosidase removes glucose and its ready to go
  • If there is still misfolding/hydrophobicity glucosyltransferase labels it again for another round of Calnexin
138
Q

ERAD
(ER associated degradation)

A
  • Used to eliminate misfolded secretory proteins from the ER
  • Misfolded protein taken from ER back to cytosol in retro-translocation
  • Ubiq-Proteasome system deg.
139
Q

In ERAD why do we have Retro-translocation

A

The ER does not have proteasomes so the misfolded protein must be taken to cytosol

140
Q

What is the only Organelle that gets its Proteome through Gated transport?

A

Nucleus

141
Q

Nuclear Pore Complex (NPC)

A

Freely permeable to small molecules <60kDa (euk prot.: 50-60 kDa)
Ribosomal subunits can pass through freely

142
Q

Layers of NPC

A
  • Membrane layer
  • Scaffold Layer
  • FG Nups layer (30 nucleoporins)
  • 2 Rings: Cytoplasmic & Nuclear (basket)
143
Q

How does the nucleus know which proteins should enter/exit the NPC?

A

Nuclear import receptors Importins/Exportins which recognize NLS characterized by positive a.a Arginine/Lysine

144
Q

Mechanism of Intracellular Gated nuclear IMPORT

A

1) NLS (cargo) recognized by importin
2) Importin recognized by cytosolic fibers
3) Ran-GTP binds once inside to release cargo protein
4) Ran-GTP takes importin back to cytosol and turns to GDP to leave it
5) Importin ready for new Cargo

145
Q

Mechanism of Intracellular Gated nuclear EXPORT

A

1) NES Exportin binds Ran-GTP
2) Then Exportin binds Cargo
3) Passage through pore
4) GTP to GDP in cytosol to release Cargo
5) Exportin back to Nucleus

146
Q

What converts Ran-GTP to Ran-GDP and vice versa?

A

Ran-GAP and Ran-GEF

147
Q

Transport through FG-Nup

A
  • FG: Phenylalanine-Glycine both Hydrophobic
  • Importin also Hydrophobic
  • Passes through with hydrophobic-hydrophobic interactions
148
Q

Calcineurin in high Ca2+

A

1) Ca2+ activates Calcineurin
2) Calcineurin is a protein phosphatase
3) Exposes NLS and block NES

149
Q

Peroxisomal Targeting signals & what is the receptor

A
  • PTS1: Pex5
  • PTS2: Pex7/18
150
Q

What do pex do in Peroxisome

A

Once PTS binds, they form pores to allow ONLY fully folded proteins to enter

151
Q

Which organelles use Transmembrane Transport?

A
  • ER
  • Mitochondria
152
Q

TOM (mitochondria)

A
  • Allow transfer of cytosol translated proteins
  • Uses amphipathic α-helix signal
  • Recognized by recognition molecules on mitochondrial memb.
    (OMM)
153
Q

SAM (mitochondria)

A

Helps folding of outer membrane proteins
(OMM)

154
Q

TIM23 (mitochondria)

A
  • Transports proteins into the matrix and helps insertion of transmembrane proteins
  • Spans 2 membranes due to elongated hydrophobic part
    (IMM)
155
Q

TIM22 & OXA (mitochondria)

A

Aid in the insertion of transmembrane proteins
(IMM)

156
Q

Are proteins to be transported into Mitochondria folded?

A

They are Unfolded because the channels are Tiny, so chaperones keep them unfolded till they reach TOM

157
Q

Steps of Mitochondrial Import

A

1) Amphipathic a-helix signal recognized by receptor on TOM
2) Protein now in IM space
3) TIM23 allows into IMM
4) Signal cleaved by peptidase
Only import, they cant export due to size

158
Q

How does mitochondria import get its energy?

A
  • The chaperones that hold protein in unfolded state are Hsp70 which are found in an ATP-bound state
  • When they pass to IM space there are mtHsp70 that do the same
  • They dissociate the protein in matrix
159
Q

Co-Translational Translocation (ER)

A

1) Translation starts in Cytosol
2) Stops at a point N-term. sig binds SRP
3) Ribosome associates with ER using SRP and continues translation
4) The polypeptide is pushed into the ER as it is synthesized through Translocator channel (from Sec61)
No way to come out after folding

160
Q

SRP parts

A
  • Signal sequence binding pocket
  • Translational pause domain
  • Hinge domain
161
Q

DNA recombinant technology

A

Process of combining DNA from different sources to create a new DNA sequence (recombinant DNA)
e.g. Insulin

162
Q

Plasmid

A
  • Circular double-stranded DNA
  • Replicate independently
  • 1-2 protein coding genes usually for protection against toxin/antibiotic
163
Q

Vectors

A

DNA molecules which will carry foreign DNA insert into host cells
(Plasmids/Bacteriophages)

164
Q

Inserts

A
  • Target DNA which will be inserted into the vector
  • Contains a specific gene which codes for the protein we wanna produce
165
Q

Restriction Endonucleases

A
  • Bacterial enzymes (like scissors)
  • Cut DNA at specific recognition sites
  • Each Restriction Endonuclease is specific to a particular recog. site
166
Q

Sticky end

A
  • Restriction Endonucleases cuts DNA leaving single-stranded overhangs
  • If 2 sticky ends are complementary they can form H-bonds in a NaCl solution
167
Q

Blunt end

A

Restriction Endonucleases cut straight through DNA with no overhangs

168
Q

How to prepare for PCR

A
  • 2 Primers designed
  • 5’ ends have recognition sites which are not complementary but for specific Restriction Endonucleases
  • PCR product = insert
169
Q

Transferring the Insert into the Vector

A

1) Plasmid vector also modified to have 2 recognition sites for specific Restriction Endonucleases
2) Insert and Vector have sticky ends complementary to each-other
3) H-bonds form and any breaks joined by DNA ligase

170
Q

2 Types of Vectors

A
  • Expression Vector (plasmid)
  • Reporter Vector
171
Q

What makes up an Expression Vector?

A
  • Promoter
  • Selective Marker Gene
  • Gene of other protein/peptide
172
Q

Expression Vector Promoter

A
  • Strong active promoter
  • Constitutive: can be activated
173
Q

Expression Vector Selective Marker Gene

A

e.g. Antibiotic resistance gene
Important to figure out which ones would survive in presence of that specific antibiotic

174
Q

Expression Vector Gene of other protein/peptide

A
  • Tag (glutathione transferase) sequence or fusion protein
  • e.g. 6-histidine tag (in lab)
  • For purification of the produced protein
175
Q

Transformation of Vector

A
  • Introduction of recombinant DNA into host cells (we use bacteria for insulin)
  • Bacteria uses machinery to make protein the gene codes for (insulin)
176
Q

How do we make sure the bacteria have taken up the Plasmid vector?

A

By having an antibiotic resistance gene and having the bacteria in presence of that AB, so only ones that survive took it in

177
Q

How to make bacteria take up the Plasmid Vector?

A
  • Chemical via heat shock
  • Electric
    Pores form allowing DNA to enter
178
Q

Purification of the Protein

A

1) Tag sequence fused to target protein
2) Mixture poured into column and only tagged proteins stick to ligands
3) Everything else is washed away
4) To get protein out we change pH or salt conc. to let go of the Ligand
5) Protein collected in fractions and purity/conc. is assessed
(Affinity Chromatography)

179
Q

Reporter Vector

A
  • Designed to monitor/measure the activity or expression of a regulatory gene (promoter) which will be our insert
  • Reporter gene (luciferase) used which creates a signal (light) when expressed so more light means stronger promoter

MCB 1 (3 decks)

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