2-Post-transcription Flashcards
(177 cards)
1
Q
Regulation of Euk. Gene expression by UTR binding
A
- RNA Binding proteins (RBP)
- Bind UTR regions (5’/3’)
2
Q
UTR binding example
A
IRE & IREBP in iron starvation
3
Q
IRE & IREBP in Iron Starvation
A
- Cytosolic Aconitase is an IREBP
- Binds IRE of 5’ UTR of mRNA coding Ferritin
- Represses translation of mRNA strand of Ferritin, No Ferritin so less storage
- Stabilizes 3’ end of another mRNA for transferrin-R, More Transferrin-R
4
Q
IRE & IREBP in Iron Excess
A
- Iron binds Cytosolic Aconitase (IREBP) bound to IRE on mRNA
- Aconitase removed and no more repression
- Ferritin Made
- Aconitase off of 3’ exposes poly-A and causes instability
- No Transferrin-R made
5
Q
Alternative maturation of Eukaryotic mRNA
A
- Alternative splicing
- Alternative promoter
- Alternative excision (e.g. repressor on 5’/3’ sites)
- PolyA site (B-cells, weak/strong sites)
6
Q
Alternative Promoter
A
- More than one promoter on a strand
- If first promoter is used whole thing is transcribed
- If second promoter is used the previous part is not incorporated in the pre-mRNA
7
Q
Alternative Poly-A site (B-cell example)
A
- Resting B Cell: Low Cstf so weak poly-A site not recognized (membrane bound IgM)
- Active B Cell: High Cstf recognizes weak poly-A site and early cleavage
(free IgM)
8
Q
Alternative splicing
A
- Constitutive (normal)
- Cassette exon: can be eliminated with introns
- Alternative 5’/3’ splice sites
- Intron retention
- Mutually exclusive exons
9
Q
What enzyme transcribes miRNA and how many are there
A
RNA Polymerase II
(500-2000 genes across different species)
10
Q
miRNA main functions summary
A
Inhibitory
- Cleavage of mRNA into 2 pieces
- Destabilization of mRNA by shortening poly-A tail
- Less efficient translation of mRNA to proteins by ribosomes
11
Q
Biogenesis of Pri-miRNA (primary)
A
- Synthesized in Nucleus from microRNA genes
- Introns of protein coding genes or non coding RNA genes
- Almost all synthesized by RNA polymerase II
12
Q
Structure of Pri-miRNA
A
- Self-complementarity (fold back on themselves)
- 35bp (ds) stem and single stranded loop/hairpin (not strict pairing)
- 5’ cap structure and 3’ poly-A tail
13
Q
Pri-miRNA Motifs
A
- Basal UG motif
- Flanking CNNC motif (3’)
- Mismatched GHG motif
- Apical UGU motif
14
Q
Biogenesis of Pre-miRNA
A
From Pri-mRNA
- By Microprocessor (heterotrimeric complex)
- Pre-miRNA Exported to cytoplasm by Exportin5 & GTPase
15
Q
Microprocessor
A
Primary to Pre-mRNA
- Enzyme drosha (ribonuclease III) cuts pri-mRNA at 22nd nucleotide of ssloop
- 2 Proteins: DGCR8
16
Q
Biogenesis of miRNA
A
- By heterotrimeric complex from pre-miRNA
- Dicer enzyme (ribonuclease III) + TARBP proteins
- Loop is lost and dsRNA=miRNA formed
17
Q
miRNA activation
A
- Binding to Ago (argonaut) protein using guide strand
- Forms RNA induced silencing complex (RISC)
- Other strand (passenger) is degraded
18
Q
How does Ago protein decide which strand to chose as guide from mi-RNA
A
Prefers one with A or U at 5’end or less stable 5’end
19
Q
RNA editing - miRNA formation modification
A
- ADAR enzymes catalyze deamination of Adenine to Inosine within the pri-mRNA transcript
- Inosine is structurally similar to guanine and is read as guanine by machinery
- So A to I, binds C instead of U
- Causes either Destabilization and degradation of pri-mRNA or an edited mature miRNA with altered specificity
20
Q
What does RNA editing + Alternative cleavage make?
A
IsomiRs
21
Q
What is edited more frequently RNA transcripts of miRNA genes or protein coding genes?
A
miRNA since they have a regulatory role
22
Q
Mitrons
A
- Form from introns of protein coding genes
- When intron is spliced it escapes degradation and remains in nucleus
- Debranched (DBR1) and forms pre-miRNA then steps continue = miRNA
23
Q
Regulation of mRNA via RISC
A
1) Argonaute proteins bind 3’/5’ ends of miRNA guide.s
2) Scans cytoplasm for target mRNA
3) Seed region of miRNA binds weakly to mRNA
4) If good match stays bound, if not it dissociates
5) If 100% complementary (rare), Ago cuts mRNA into 2 & RISC keeps scanning (typical for siRNA)
24
Q
Seed region (& first base)
A
- Small part of miRNA sequence that is important for finding its target mRNA
- First base very important for its specificity: usually Adenine
25
Where does miRNA bind mRNA (& what is recruited)
At 3' UTR of target mRNA
(recruits TNRC6)
26
TNRC6 outcomes
- TNRC6 brings proteins to break down mRNA through PolyA (main way)
- TNRC6 binds 5' to stop its translation to protein without degradation (less efficient)
27
miRNA vs siRNA
- miRNA: reduce expression of target gene
- siRNA: completely eliminate the expression of a target gene
28
Methods of Epigenetic Control
- DNA methylation
- Histone modification
29
DNA methylation
- By SAM
- Changes activity of DNA segment without changing the sequence
- Almost exclusive in CpG dinucleotides (5th carbon of c then g on backbone)
- Silences genes
30
Importance of Cytosine deamination in methylation
- Usually C deamination forms Uracil
- But, methylated C deamination forms Thymine
- This is one of the most frequent mutations involving Arginine
31
DNA demethylation mech
1) After methylation by DNMT
2) 5-mc cant be broken easily due to C-C bonds
3) TET oxidises 5-mc to formylC then 5-caC (carboxylic)
4) caC removed by TDG making AP site then then Base excision repair to return C
32
Pioneer TFs
Weirdly prefer methylated sites on DNA rather than the usual TFs that prefer unmethylated
33
How are some introns spliced and some not in other cells?
We have Negative & Positive control with Splicing repressors and Splicing activators which binds intron splice sites
34
mRNA degradation methods
- Poly-A tail removal/shortening
- Decapping
(allows exonucleases to act)
35
RNA editing (e.g. apo)
Example that mRNA coding for ApoB-100 has base CAA
- In liver: No RNA editing so full mRNA translated
- In intestines: RNA editing by deamination and C forms U. (UAA)
Early stop codon and truncated protein formed ApoB-48
36
Non-Sequence Specific Approach
Measuring overall gene expression in a sample
1) mRNA reverse transcribed to cDNA (dsDNA)
2) SYBR Green dye used to bind cDNA
3) Fluorescence only when bound to cDNA
4) PCR amplifies cDNA making more and hence more fluorescence detected
37
Advantages of Non-Sequence Specific Approach
- The SYBR Green dye can be used for any genes being studied
- It is a non-specific DNA binding dye
38
Disadvantages of Non-Sequence Specific Approach
- Lack of specificity may lead to detection of non-specific amplification products
- Primers can form complex with dye giving signal (false signal)
39
Probe
Short ssDNA oligonucleotide complementary to target gene
(e.g. TaqMan)
40
Probe parts
- Reporter molecule (5'): Fluorophore
- Quencher molecule (3'): Absorbs emitted fluorescence
41
Sequence specific approach when Probe is intact
1) Reporter excited by λ1 emitting λ2
2) λ2 absorbed by Quencher
3) No signal is detected
42
Sequence specific approach when Probe is Degraded
1) During PCR, DNA polymerase with 5' exonuclease activity cuts probe
2) Reporter, Nucleotides & Quencher released
3) Fluorescence can be detected
43
Advantages of Sequence specific approach
- Always reliable
- If probe not digested then no signal
44
Disadvantages of Sequence specific approach
A difference Probe is needed for each gene we want to analyze
45
Threshold intensity
Amount of PCR product that can be reliably detected during efficient phase that surpasses baseline background noise
46
Threshold cycle
Cycle number at which the amount of PCR product reaches the threshold intensity
47
Types of Quantification
- Relative Quantification
- Absolute Quantification (uses standard calibration curve)
48
DNA Microarray/Chip
Solid surface (glass/silicone) where thousands of known DNA sequences (probes) representing genes are immobilized in an organized grid/array
49
DNA Microarray method
1) RNA extracted from sample
2) Reverse Transcription to cDNA
3) cDNA labelled with fluorescent dye
4) Placed on microarray
5) If cDNA binds probes it means the gene was expressed & fluorescence detected
(analysis of genome wide expression)
50
What makes up genetic code
Triplet code
51
How many codons are there for A.A
64
- 3 are Stop codons
- 61 code for the 20 proteogenic a.a.
52
What are the stop codons?
- UAG
- UGA
- UAA
53
Genetic code is Redundant
Multiple codons can code the same amino acid
BUT each codon only codes for 1 amino acid
54
Start Codon
Always Methionine
AUG
(the only a.a with only 1 codon)
55
Anticodon
Sequence of 3 nucleotides on tRNA that is complementary to the mRNA codon
56
Wobbling
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
57
Sense vs Antisense strand
- Sense: Strand NOT used for transcription, Identical to the RNA transcript
- Antisense: Template strand for transcription, complementary to sense strand
58
Non-overlapping meaning
Every base is read in sets of 3, once read it moves to next 3, they do not overlap
59
What is consequence of using both sense and antisense as template at the same time?
Formation of dsRNA which can not be translated
It is a signal of a viral attack against cell
60
Genome reading & importance of START
- 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
61
Open Reading Frame (ORF)
Sequence of codons running from specific start codon to specific stop codon
62
tRNA structure
- Folded RNA molecule
- Small size (70-100 nucleotides)
- Has modified nucleotides (7-15)
- Cloverleaf, but 3D L-shape
- Phosphorylated 5'
- 3' CAA +a.a
63
Modified nucleotides on tRNA
- dihydrouridine (UH2)
- ribothymidine (T)
- pseudouridine (Ψ)
64
tRNA arms
- Anticodon Arm with anticodon loop region
- Amino acid acceptor arm with 3' end of tRNA (single stranded region)
65
Nucleotide sequence of Amino acid acceptor arm
+ when is it added
CCA on 3' end
Post-transcriptional modification
66
What bond forms bw CCA on tRNA and Amino acid
Ester bond
OH of CCA and Carboxyl group of Amino A.
67
Aminoacyl-tRNA synthetase
Enzyme which ensures that the correct a.a is matched with the corresponding tRNA anticodon
- Different one for each of the 20 standard A.As
68
Aminoacyl-tRNA synthetase
Double function - 1
Activation of amino acids
- Linking appropriate a.a to tRNA
1) Adenylates AA by adding AMP from ATP (PPi hydrolyzed so irreversible)
2) Aminoacyl-AMP forms ester bond with CCA on 3' end of tRNA
= aminoacyl-tRNA
69
Aminoacyl-tRNA synthetase
Double function - 2
Translation of the Genetic code
- Recognizes both appropriate A.A and the Anticodon
70
Fidelity
Accuracy with which the correct A.A is attached to the tRNA molecule
- Some aminoacyl-tRNA synth. have proofreading/editing activity using Editing domain
71
tRNA Charging
Process where specific A.A is covalently attached to its corresponding tRNA
- Aminoacyl-tRNA is a Charged tRNA since it is successfully bound
72
Bound vs Free ribosomes
- Bound: on RER make proteins destined for transport outside the cell
- Free: in cytoplasm make proteins for cell's own needs
73
Makeup of a ribosome
- 2/3 rRNA
- 1/3 Ribosomal Proteins
74
How do we get rRNA
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
75
3 main types of rRNA in Eukaryotes
- 18s
- 5.8s
- 28s
All processed from a common Precursor RNA
76
Which rRNA is special and why
5S rRNA
Separate molecule transcribed differently
77
What is S in rRNA
Svedberg units
- Parameter proportional to the molecular size
78
Where does transcription to form rRNA happen & using what enzyme?
Nucleolus of the Nucleus by:
- RNA Polymerase I (18S, 5.8S, 28S)
- RNA Polymerase III (5S)
79
Ribosomal Proteins (& synth/assembly)
- RPL (for Large subunit)
- RPS (for Small subunit)
Synthesized in Cytoplasm then assemble with rRNAs in Nucleus
(1/3 of ribosome)
80
Are Eukaryotic and Prokaryotic Ribosomes at all similar?
Yes, high level of similarity
81
Eukaryotic ribosomal large and small subunits + rRNA makeup
- L: 60S
- S: 40S
Overall: 80S
+ 4 different rRNAs
82
Prokaryotic ribosomal large and small subunits + rRNA makeup
- L: 50S
- S: 30S
Overall: 70S
+ 3 different rRNAs
83
Role of Ribosomal Large & Small subunit
- Large: Peptide bond formation
- Small: mRNA binding to decode it
84
When do the small and large ribosome subunits form a complex?
ONLY during Translation
85
Are ribosomes identical?
No, they differ in Protein composition
86
3 tRNA binding sites on Ribosome
- A site (aminoacyl)
- P site (peptidyl)
- E site (exit)
87
A site (aminoacyl)
Where the incoming aminoacyl-tRNA carrying next a.a binds to the Ribosome during elongation phase
88
P site (peptidyl)
Where peptidyl-tRNA is bound to the Ribosome which has a polypeptide chain attached to it
89
E site (exit)
Where the tRNA that no longer carries an amino acid and released its polypeptide chain exits the Ribosome
90
Where are tRNA binding sites on Ribosome?
(Subunits)
It spans both the Small and Large subunits
91
What happens when Ribosome reaches Stop codon?
- Polypeptide chain is released
- Ribosome leaves mRNA and becomes Termination Ribosome
92
Termination Ribosome
- Unstable
- Readily dissociates to free ribosomal subunits
93
IF3 after ribosome dissociation
Initiation Factor 3
Recycles small ribosomal subunits to native subunits (30s / 40s) ready for translation
- If IF3 not sufficient, single ribosome forms until it dissociates to subunits again
94
Polysome
Clusters of ribosomes simultaneously translating the same mRNA sequence
95
Pro/Eukaryotes protein folding
- Pro: Post-translational
- Euk: Co-translational
96
Shine-Delgarno sequence
Located upstream of the Start Codon of Prokaryotic mRNA
(part of ribosome binding site)
97
Prokaryotic Initiation of Translation Steps
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 (formyl-m)
4) 30S initiation complex formed (IF1/2/3, fmet-tRNA, 30S, mRNA)
5) IF1 & IF3 released and large 50S binds
6) IF2 dissociates and GTP to GDP upon large subunit binding
7) 70S is now ready for Elongation
98
Why is it fMet in Prokaryotes for 1st A.A?
Modified methionine Formylmethionine by Enzyme Transformylase (only Prokaryotes)
99
IF1 role
Orients fMet-tRNA to the correct site (P-site) not A cause we are initiating
100
IF3 role
Prevents Large subunit 50S from binding until the 30S Initiation complex is ready
101
Regulation of Prokaryotic Translation
- 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)
102
Significance of 5' end of Eukaryotic mRNA
Methylguanosine cap has eIFs bound:
- 4E
- 4G
- 4A
103
Ternary complex Eukaryotes
- Initiator tRNA (Met-tRNA)
- eIF2 & GTP
104
Eukaryotic Initiation of Translation Steps
1) Ternary complex formation
2) Small subunit 40S associates with more eIFs (1, 1A, 3, 5) 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
105
Polyadenosine Binding Protein (PABP)
Binds to 3' Poly A tail and eIF4G making the mRNA a loop/circular structure
106
Regulation of Eukaryotic Translation (examples)
- mRNA quality control
- miRNA
- By localization
- By Proteins
- By Hairpin
- Mod. of eIF (4E)
- Phosph. of eIF2a
- IRES (internal ribosome entry site)
107
mRNA quality control
(Eukaryotes)
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
108
Regulation by microRNA
(Eukaryotes)
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
109
Regulation by Localization
(Eukaryotes)
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
110
Regulation by Proteins/kinases to increase Transcription
(Eukaryotes)
Phosphorylation of ribosomal S6 protein in 40S subunit leads to increased translation
e.g. mTOR-Kinase
111
Regulation by Hairpin Structure
(Eukaryotes)
Hairpin is a loop formed by the folding of the RNA strand back on itself
Can block or expose binding sites
112
Regulation by modification of initiation factors (eIFs)
(Eukaryotes)
- Phosphorylation of eIF4E
- 4E-BP
- Phosphorylation of eIF2a
113
Phosphorylation of eIF4E
Decreases eIF4E affinity to 5' cap so Inhibits Translation
114
4E Binding protein (4E-BP)
It binds eIF4E preventing 4E-4G interaction and prevents recruitment of 43S pre-initiation compl. due to no loop
Inhibits Translation
115
Phosphorylation of eIF2a
- Inhibits GTP to GDP so no eIF2a recycling
- 2B (GEF) but inactive when 2a is phosph. and sequestered
Inhibits Translation
116
What states increase phosphorylation of IFs
- A.A deprivation: GCN2 protein Kinase activated by uncharged tRNA and will phosph. eIF2a
- Viral RNA: PKR recognizes it viral RNA, 2 bound PKRs with Viral RNA form homodimer and phosph. eIF2a
117
Regulation by IRES (internal rib. entry site)
(Eukaryotes)
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)
= cap independent initiation of translation
118
Steps if 5' cap not present and using IRES sequence
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
119
Elongation Phases of Translation
- Decoding
- Transpeptidation
- Translocation
120
Prokaryote vs Eukaryote Elongation factors
- EF-Tu = EF1-a
- EF-Ts = EF1-By
- EF-G = EF2
121
Decoding in Elongation
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)
122
Transpeptidation in Elongation
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
123
Translocation in Elongation
- EF-G facilitates translocation of ribosome to next codon (GTP)
- N to C terminal direction
- Deacylated tRNA moved to E-site, tRNA in A-site moves to P-site with chain to form Default state
124
Termination of Translation
- No tRNA for stop codon
- Release factors are used to recognize stop codon
- RF very similar to tRNA struct.
125
Release Factors Pro vs Eukaryotes
- RF1/RF2 = eRF1
- RF3 = eRF3
126
Release factor recognition of stop codons Prokaryotes
- RF1: UAA & UAG
- RF2: UAA & UGA
(in euk. RF1 for all 3)
127
Termination of Translation Steps
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) RRF ribosome recycling factor dissociates ribosomal subunits
128
Antibiotics that interact with Small ribosomal Subunit
- Tetracycline
- Spectinomycin
- Hygromycin B
- Streptomycin
129
Tetracycline
Blocks binding of aminoacyl-tRNA to A-site
130
Why does Tetracycline only affect Prokaryotes
Only prokaryotes have transporters that bring tetracycline into the cells
131
Spectinomycin
Interferes with Translocation step of Elongation by binding 30S subunit
132
Hygromycin B
Premature chain termination by inserting into A-site
133
Streptomycin
Prevents transition from Translation initiation to Elongation & causes Miscoding
134
Antibiotics that interact with Large ribosomal Subunit
- Chloramphenicol
- Erythromycin
- Streptogramin B
135
Chloramphenicol
Blocks peptidyl transferase activity on ribosomes so prevents peptide bond formation
136
Erythromycin
Binds to E-site and inhibits elongation
137
Streptogramin B
Binds P-site of 50S subunit
138
Inhibitors of Translation (non-antibiotic)
- Puromycin
- Cylcohexamide
139
Puromycin
Inhibits Pro/Eukaryotic translation by mimicking aminoacyl-tRNA and blocking A-site
140
Cyclohexamide
Blocks the translocation phase of elongation in EUKARYOTES
141
Corynebacterium diphtreriae
- Produces diphteria toxin
- Inhibits eEF2 in eukaryotes
- No translation
142
Ricinus Communis
- Seeds have Ricin toxin
- Cleaves adenosine off 28S rRNA
- No formation of functional ribosomes
143
N-terminal Modifications
- Cleavage of (f)-Methionine (deformylase & meth. aminopeptidase)
- Acetylation of Second amino acid after (f)-Methionine
- Myristoylation: covalent attachment of myristoil group (14c FA) (crucial for membrane association of proteins)
144
C-terminal Modifications
- Amidylation: Conversion of COO- group to Amide group (2x Amide)
- Glycosylphosphatidylinositol coupling: C-term. cleaved and attached to GPI for membrane anchoring
- Cholesterylation (hydrophobic lipid groups)
145
Modifications of Internal 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, just not as much mods.
146
Hydroxylation
- On Proline & Lysine
- Adds -OH on side chain allowing more H-bond formation
- Collagen binds tightly to each other due to hydroxyproline (3 a helices)
147
Hydroxylation Enzyme and consequences
- Prolyl Hydroxylase
- Succinate & Fe3+ cofactors
- Fe3+ red. is Vit-C dependent
- Low Vit-C = Scurvy
148
Lysine Deamination
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
149
Tethering membrane proteins
Adding a lipid tail to anchor protein to the membrane
- Palmitoylation (C or S)
- Myristoylation (Gly)
- Prenylation (RAS)
- Phosphatidylethanolamidation
- Cholesterylation
150
Ca2+ Binding by Carboxylation
- Carboxyglutamic acid (Gla-domain) from Glu by adding Carboxyl
- Vitamin K dependent carboxylase
- VKOR system
- Ca2+ can bimd 2 negative Carboxyl groups of Gla & membrane so it can fix proteins to membrane (blood coag.)
151
Protein Phosphorylation
(most general)
- Mainly Serine, Threonine, Tyrosine (-OH groups)
- Uses ATP/GTP
- A.A from Tensed to Relaxed (can do its job)
152
Primary Protein structure
A.A sequence linked by peptide bonds
153
Secondary Protein Structure
Primary structure coils & forms hydrogen bonds between carbonyl & amino groups
- a-Helix (right handed, H-b every 4th a.a)
- B-pleated sheets (takes longer to form)
154
Tertiary Protein Structure
3D shape of protein held by weaker bonds like hydrophobic, ionic, hydrogen, disulfide bridges
155
Quaternary Protein structure
More than one polypeptide chain linked together to form complex proteins (may have prosthetic groups like Heme)
156
Anfinsen's Dogma
States that secondary and tertiary structures are determined by the primary structure
Proven by denaturation then reassembly back to a functional Enzyme
157
Levinthal's Paradox
- 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.
158
Hydrophobic collapse
Apolar/Hydrophobic side chains of A.A cluster together in protein's interior away from the surrounding aqueous.
Establishes Molten Globule state
159
Why do proteins need help folding
- Macromolecular crowding inside cells
- Protein aggregation
- Energy Barriers
- Chaperone proteins guide them
160
Chaperones
- 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
161
3 Levels of Chaperones (pro/eu)
1) Trigger Factor = Rib. Assoc. Compl.
2) DnaK & DnaJ = Hsp70/40
3) GroEL & ES = TRic
(Prokaryotes / Eukaryotes)
162
Hsp70/40
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
163
GroEL & ES
- GroEL is barrel shaped (2 ring, 7sub each) central cav. where misfolded proteins are enclosed
- GroES is the cap that closes it
- ATP needed to close and fold
164
26S Proteasome
19S regulatory particle recognizes polyubiquitinated proteins to unfold and translocate them
165
Protein Disulfide Isomerase (PDI)
- 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)
166
How can we reuse PDI
We re-oxidize it using:
- Ero1 (ER-related oxidase)
- GPx7/8 (GSH peroxidase)
- Prx4 (peroxiredoxin)
(all produce H2O2 as byproduct)
167
ER Chaperones
- Grp78 (BiP) & Grp94: bind hydrophobic part of protein
- Calreticulin & Calnexin: bind carbohydrates
168
Carb Binding Chaperones (Lectins)
- 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
169
Methylation (& amount)
- Represses Transcription
- More methylated = more hydrophobic = Heterochromatin
- SAM
- Lysine (x3) & Arginine (x2)
170
Acetylation
- Promotes Transcription
- Loosens chromatin = Euchromatin
- The + charge of NH3+ lost and histones (usually +) dont bind DNA (-) as well as before
171
Bromo Domain Motifs
- Special parts of certain proteins that recognize and bind to acetylated lysine residues on histones
- Help activate gene expression by recruitment of Transcription machinery
172
Glycosylation
- Attaching sugar/carb to active side chain of A.A
- Asparagine, Serine, Threonine
- N-glycosylation (90%) & O-glycosylation (10%)
173
O-Glycosylation
- 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
174
N-Glycosylation
- Begins in ER
- Strictly determined which Asn is glycosylated
- Oligosaccharyl Transferase
- Maturation in Golgi by trimming
- Pyrophosphate for energy to attach sugar to Asn
175
Functions of Glycosylation
- Protecting proteins from degradation
- Selective labelling of proteins
- Determines cell-to-cell connections
- Allows protein folding quality control
176
Protein Domains
Independently folded structural and functional units within a protein
177
Examples of PTM recognizing domains
- Bromodomain: acetylated lysine residues (histones)
- Plant Homeodomain (PHD): recognizes methylated lysine residues (chromatin)
