Exam 4 Flashcards
1
Q
Hypothetical protein-folding pathway
A
- Primary
- Secondary
- Structural motifs
- Domains
- Tertiary Structure
2
Q
What are the two families of chaperone proteins?
A
- Chaperones
- Binds to short segments of protein substrate and stabilize unfolded or partly folded proteins
- Prevents proteins from aggregating and being degraded
- First identified by rapid appearance after stress by heat shock (Hsp - heat shock protein)
- Chaperonins
- Forms folding chambers into which all parts of unfolded protein can be sequestered
- Gives proteins time and appropriate environment to fold properly
3
Q
Roles of chaperones
A
- Catalyze and facilitate proper folding and self-assembly of proteins
- Bind to and stabilize unfolded or partially folded polypeptides
- Prevent incorrect folding or aggregation
- Refold misfolded or unfolded proteins
- Assemble and dismantle large multiprotein complexes
4
Q
Function (MOA) of chaperones
A
- Uses ATP binding, ATP –> ADP hydrolysis, and exchange of ATP for ADP to induce conformational changes
- ATP dependent conformational switch used to:
- Optimize folding after one substrate is folded
- Return chaperone to initial state so it is avaliable to help fold another polypeptide
- Set time permitted for refolding
5
Q
What is Hsp 70?
A
- Found in cytosol, endoplasmic reticulum (BiP), mitochondria
- Stabilize unfolded polypeptide chains during translation
- Majority of proteins bind to multiple chaperones as they are synthesized
- Important for transport of proteins into subcellular compartments
6
Q
Steps of folding proteins with Hsp 70
A
- Protein binds to substrate-binding site in the substrate-binding domain (SBD).
- Hydrophobic binding pocket in Hsp70 binds to exposed hydrophobic regions in target proteins
- Accessory proteins stimulate the hydrolysis of ATP
- __Hydrolysis__ of ATP causes chaperones to assume closed form that binds to target protein more tightly.
- Protein is locked into the SBD
- ADP is __exchanged__ for ATP by accessory proteins. SBD is converted to the open form.
- The folded protein is released.
- This process can be repeated until the protein is folded correctly.
7
Q
What is Hsp40?
A
- Accessory protein for Hsp70
- Stimulates binding of a substrate and rate of ATP hydrolysis by 100-1000x
8
Q
What are NEFs?
A
- Nucleotide exchange factors
- Accessory proteins for Hsp70
- BAG
- HspBP
- Hsp 110
- Promotes exhange of ATP for ADP
9
Q
What is Hsp90?
A
- Majority of proteins involved in cell signaling
- 4 distinct types
- 2 in cytosol
- 1 in ER
- 1 in mitochondria
- Recognizes partially folded proteins
- Mediates protein folding by forming stable complexes with target proteins (called “clients”)
- Clients can dissociate when appropriate signal received
- Functions as a dimer
10
Q
Steps of protein folding with Hsp90
A
- Client protein binds to the open conformation of Hsp90
- ATP binds to the open conformation and causes change in conformation
- Hsp90 is now in closed conformation with the client.
- Nucleotide-binding domains dimerize
- ATP hydrolysis results in conformational change in Hsp90 that opens it and releases the folded protein client
11
Q
What are chaperonins?
A
- Also called Hsp60s
- Group I
- Found in prokaryotes, chloroplasts, mitochondria
- Composed of 2 rings - GroEL
- Each ring has 7 subunits
- Rings interact with seven subunit “lid” - GroES
- Group II
- Found in eukaryotic cytosol
- Called TriC in mammals
- Can bind co-translationally to nascent chains
- Composed of two rings, each ring has 8-9 subunits
- No separate “lid” - incorporated into ring subunits
- ATP hydrolysis triggers closing of lid
12
Q
Chaperonin Group I Folding
A
- Partly folded or misfolded polypeptide enters one of the chambers; second chamber of blocked.
- Each ring binds 7 ATPs, hydrolyzes them, and releases them in a set order
- GroES lid released from blocked chamber (now open)
- Second GroES lid binds to protein folding chamber (originially open, now blocked)
- Target protein folds
- ATP hydrolyzed to ADP
- ATP binds
- Another GroES binds to the open chamber
- Release of GroES lid, ADP, and protein
- Cycle can repeat if needed
13
Q
Chaperonin Group II folding
A
- Apical protrusions extend over central channel to form substrate-binding surface and act as a lid
- Unfolded substrate binds
- Protrustions close
- ATP cleaves to ADP
- ADP and folded protein released
- ATP binds
14
Q
What plagues are associated with these diseases?
Alzheimer’s Disease
Parkinson’s Disease
Creutzfeldt-Jakob Disease
Transthyretin Amyloidosis
A
- Alzheimer’s - Amyloid B, Tau
- Parkinson’s - Amyloid B, Tau, a-Synuclein
- Creutzfeldt-Jakob - Tau, prions
- Transthyretin Amyloidosis - Transthyretin
15
Q
Protein misfolding diseases caused by:
Fails to fold correctly
A
Cystic fibrosis
Marfan syndrome
Amyotonic lateral sclerosis
16
Q
Protein misfolding diseases caused by:
Not stable enough to perform its normal function
A
Many forms of cancer
17
Q
Protein misfolding diseases caused by:
Fails to be correctly trafficked
A
Familial hypercholesterolemia
A1-antitrypsin deficiency
18
Q
Protein misfolding diseases caused by:
Forms insoluble aggregates that deposit toxically
A
Neurodegenerative diseases -
Alzheimer’s
Parkinson’s
Type II diabetes
Many more
19
Q
How are secretory proteins directed?
A
- Process starts at the ER
- Translation and translocation occur simultaneously
- From ER, proteins are sent to plasma membrane, lysosomes, or outside of cell
- Signal sequences
- Located at N-terminus of transported proteins
- Charged N-terminal region, usually basic
- Have core of about 10-15 hydrophobic amino acids
- short sequence at C-terminus near cleavage site that is relatively polar
- After protein reaches ER, signal sequence is cleaved off
20
Q
How do proteins get to mitochondria?
A
- Some proteins are synthesized on ribosomes within organelle
- Proteins encoded by nucleus have N-terminal signal sequence
- Mitochondrial matrix-targeting sequences
- Rich in hydrophobic, positively charged, and hydroxylated amino acids
- Lack negatively charged amino acids
- Amphipathic
- Precursor proteins synthesized and bound to cytosolic chaperone proteins
- Proteins transported to organelle
- Signal sequence cleaved off
- Other sequences internal to N-terminus direct proteins to other compartments of mitochondria
21
Q
What are the ways proteins are modified post-translationally?
A
- Amino-terminal and carboxy-terminal modifications
- Formyl group removed from methionines initiating bacterial proteins
- Amino terminal methionine, other N-terminal amino acids, and C-terminal amino acids may be removed
- Amino group of N-terminal amino acid in many proteins modified by addition of N-acetyl group
- Cleavage
- Many proteins synthesized as longer precursors
- Proteases remove sequences
- Phosphorylation
- Addition of phosphate groups to hydroxyl groups on Ser, Thr, Tyr
- Phosphorylated by ATP through kinases
- Changes charge of protein and can lead to conformational change
- Dephosphorylated by phosphatases
- Acts as “switch’ to turn on/off function
- Glycosylation
- N-linked: carbohydrate chains attacked to amide nitrogen of asparagine
- O-linked: carbohydrate chains attached to hydroxyl group of Ser/Thr, plus hydroxylysine and hydroxyproline in collagen
- Isoprenylation
- Glycosylphosphatidylinositol (GPI)
- N-myristoylation
- S-palmitoylation
- Addition of palmitate to thiol side chain of cysteine
- Acetylation
22
Q
Acetylation
A
- Addition of acetyl group (CH3CO)
- Proteins acetylated by both enzymatic and non enzymatic processes
- Major group of enzymes attach acetyl group to terminal amine on side chain of lysine
- Modification is reversible
- Enzymes are called KATs (lysine acetyl transferases) and HDACs (histone deacetylases)
- Histones were first group of proteins known to be modified by acetylation, but now non-histone proteins are known to be also acetylated
- Addition of acetyl group alters protein structure and interactions with other biomolecules
- Lysine positively charged at physiological pH
- Enzymes use lysines to bind to negatively charged substrates
- Acetylated lysines less able to bind to substrates
- Affects protein stability, binding of transcription factors to DNA, protein interactions, mRNA stability, and enzymatic activity
23
Q
N-myristoylation
A
- Addition of myristate (14-carbon fatty acid) to N-termini of proteins
- Usually added to N-terminal glycine; can also be added to lysines
- Provides hydrophobicity and guides proteins to membranes
- Usually associated with inner face of plasma membrane
- Requires second signal to get stable membrane binding
24
Q
Glycosylphosphatidylinositol (GPI)
A
- Amphipathic molecule
- Anchors cell surface proteins to plasma membrane
- Protein cleaved and transferred to preformed GPI anchor in membrane
- Proteins with GPI anchors can readily diffuse in membrane
- Localizes proteins in regions of cells
25
Isoprenylation
1. Derived from pyrophosphorylated intermediates (isoprenes) of cholesterol
biosynthetic pathway such as farnesyl
2. Attached to Cys residues in proteins
3. Helps protein anchor in membrane
4. Irreversible
26
How are proteins degraded?
1. Three enzymes
* E1 = ubiquitin-activating enzyme
* E2 = ubiquitin-conjugating enzyme
* E3 = ubiquitin ligase
2. Ubiquitin added to E1; requires ATP
3. Ubiquitin transferred to Cys residue in E2
4. Ubiquitin transferred to lysine in target protein by E3
* Covalent bond formed between carboxyl group of C-terminal glycine 76 and amino group of side chain of lysine residue in target protein
5. Called isopeptide bond because side chain amino group, instead of A-amino
group, linked to carboxyl group
6. Subsequent ligations link C-termini of additional ubiquitins to side chain of lysine 48 of previously added ubiquitins
7. Protein to be degraded has to be unfolded
8. ATPases in 19S regulatory subcomplex involved
9. Proteases in proteasome cleave after hydrophobic, acidic, and basic residues
10. Proteosomal digestion products furthered degraded by peptidases in cytosol
11. Ubiquitin reused
27
What is Ubiquitin?
1. Marks proteins for degradation
2. 76 amino acid polypeptide that ends in glycine
3. Covalently linked to E-amino group of lysine in protein to be degraded via carboxyl group of terminal glycine in ubiquitin
4. Need at least 4 ubiquitins attached to protein for it to be degrade
28
What are proteasomes?
1. Multisubunit complex – 26S
2. Two multisubunit subcomplexes – 20S and 19S
3. 20S core protease
* Four rings – 2 outer and 2 inner
* Each outer ring composed of 7 A-subunits
* Outer rings control substrate access
* Each inner ring composed of 7 B-subunits
* Three B-subunits have protease activity
4. 19S regulatory
* Contains 6 ATPases
* Promotes opening of proteasome and provides access for substrates
29
What is multiple myeloma?
* Cancer of immune system
* Abnormal proliferation of antibody-producing cells
* Cells produce high levels of toxic, aberrant immunoglobulin proteins
* These usually degraded by proteasomes
* Proteasome inhibition leads to buildup of toxic proteins and eventually
myeloma cell death
* Myeloma cells also require transcription factor NF-kB to survive
* I-kB normally inhibits NF-kB
* If I-B degraded by proteasomes, NF-kB active
* If inhibit proteasome function, I-kB not degraded and inhibits NF-kB activity
30
Deubiquitination
1. Reverse ubiquitin modifications
2. Ubiquitin-specific proteases that edit or disassemble polyubiquitin chains
3. DUBs = deubiquitinase
4. p53
* Transcription factor that regulates cell cycle
* Ubiquitinated by Mdm2
* Mdm2 deubiquitinated by DUBs
31
What are SUMOs
1. Small ubiquitin-related modifiers
2. Covalently and reversibly conjugated to specific lysine residues
3. Four different mammalian family members coded for by different genes
* SUMO-1
* SUMO-2
* SUMO-3
* SUMO-4
4. Synthesized as inactive precursors that are cleaved to expose conjugation site
5. Found predominantly in nucleus
6. Regulate protein-protein interactions
7. Can alter localization and activity of proteins
8. Roles
* Transcription activation and inhibition
* Genome stability
* DNA repair
* Protein trafficking
* Cell cycle control
32
Potential functions of SUMO chains
* Block interaction of target protein with partner protein
* Create new binding face that can recruit other binding partners
* Change conformation leading to alteration of activity or revealing masked binding sites
33
How is function of polyubiquitination determined?
34
Types of RNAs that are important to prokaryotic transcription
* mRNA
* Fully processed messenger RNA
* 5' cap, poly(A) tail
* Introns removed by RNA splicing
* snRNA
* Small nuclear RNAs (5 + 2)
* (5) Function in removal of introns from pre-mRNAs by RNA splicing
* (2) Substitute for the first two at rare introns
* snoRNA
* Small nucleolar RNA
* Bp with complementary regions of pre-RNA molecule
* Directs cleaves of RNA chain and modification of bases during maturation of the rRNAs
* siRNA
* Short interfering RNA
* ~ 22 bases, each perfectly complementary to a sequence in an mRNA
* Together w/ associated proteins, siRNAs can cleave "target" RNA, leading to its rapid degradation
* miRNA
* Micro RNA
* ~ 22 bases that bp extensively, but not completely, with mRNAs (esp. over the 6 bp at the 5' end of miRNA)
* Inhibits translation of "target" mRNA
35
# Prokaryotic Transcription Key Words
* Holoenzyme
* Structural genes
* Operator (O)
* Promoter (P)
* Transcriptional unit
* Operon
* RNA polymerase holoenzyme
* The single RNA polymerase that transcribes genes in prokaryotes
* Consists of core enzyme and 1 of several auxiliary factors (sigma, rho, etc.)
* Structural genes
* Genes encoding enzymes or structural proteins
* Operator (O)
* DNA site at which regulatory proteins (repressors) bind
* Promoter (P)
* -35, -10 site for RNA polymerase binding
* Required for accurate, high level initiation of transcription
* Transcriptional unit
* Region in DNA that is copied into RNA by an RNA polymerase
* Operon
* Section of DNA consisting of structural genes and the cis-acting (adjacent DNA sequences) regulatory elements that regulate those genes
36
What do auxiliary factors do in RNA polymerase holoenzymes?
* The presence of auxiliary factor generates alternative forms with specific properties.
* Core + sigma holoenzymes (sigma-70, sigma-54)
* Core + rho holoenzyme
* Prokaryotic transcription begins when sigma holoenzyme (core + sigma) binds to the promoter region (-35 and –10 regions).
37
How does prokaryotic transcription begin?
1. Transcription begins when sigma holoenzyme (core + sigma) binds to the promoter region (-35 and –10 regions)
* Sigma subunit confers promoter specificity
* Sigma makes contacts in the -35 to -10 region (TATAAA element)
2. Binding to region leads to closed
complex
3. Localized helix unwinding gives rise to open-promoter complex
4. After ~10 base RNA is made, sigma is released and nus homoenzyme joins the complex
* Rate of initiation of transcription + mRNA stability determines the abundance of a given mRNA
38
Polycistronic transcript
* Transcription of bacterial operons is **polycistronic**, that is, one continuous mRNA spans several structural genes.
* Polycistronic mRNAs can be translated into several different proteins because prokaryotic ribosomes can bind to internal Shine-Dalgarno (ribosome binding sites)
* Limited to prokaryote
39
What is negative transcriptoinal control in prokaryotes?
* Uses a repressor
* Trans-acting regulatory protein that acts to prevent transcription.
Negative control can be either:
* **Inducible** - operon normally turned _off_ until an inducer molecule binds to the repressor and inactivates it.
* Example: lac I repressor.
* **Repressible** - operon normally turned _on_ since the repressor is inactive;
* A corepressor (small molecule) must bind to repressor to yield a functional repressor which turns off transcription.
* Example: in the trp operon, trpR is repressor and tryptophan is the corepressor
40
What is positive transcriptional control in prokaryotes?
* Uses activator protein
Positive control can be either:
* **Inducible** - no transcription because _apoactivator_ is _inactive_
* Coactivator binds to apoactivator to yield a functional complex
* Example: Catabolite activation operon where catabolite activator protein (CAP) is activated by binding to cAMP. lacZYA, araBAD and galETK operons
* **Repressible** - activator protein is functional in the absence of inhibitor, so transcription occurs
* In presence of an inhibitor molecule, the activator is inactivated and no expression occurs
41
What are the components of the lac system?
* **lacl**: lac repressor gene
* **lacP**: -10 + -35 region (weak promoter)
* **lacO**: lac operator (where repressor binds)
* **lacZ**: encodes B-galactosidase which cleaves lactose into glucose and galactose
* **lacY**: encodes a permease which facilitates the uptake of lactose into the cell
* **lacA**: encodes transacetylase which may detoxify lactose metabolites
42
What is diauxic growth?
* If E. coli is placed in medium containing both glucose and lactose, cells will utilize glucose first.
* Metabolize lactose after glucose has been exhausted.
* This sequential use of carbon sources is called diauxic growth.
43
How does negative control of lac operon work?
* In _absence_ of lactose, repressor binds to lacO
* Bound repressor protein overlaps lacP and physically blocks forward progression of RNA polymerase.
* No transcription of lacZYA.
* In lactose medium, initially low level B-galactosidase converts lactose to allolactose
* Binds to repressor
* Causes a conformational change
* Repressor can no longer bind to the operator
* lacZYA can now be transcribed and translated at high levels, **provided cAMP level is high**
* RNA polymerase ceases transcription when it reaches a site called a transcription termination signal
44
How does positive control of lac operon work?
1. **Catabolite activation** is required in order to turn on lacZYA transcription when lactose is present in the culture medium.
2. cAMP and cAMP binding protein (CAP, CRP) are key components of the signal transduction pathway
* Level of cAMP is determined by adenylate cyclase, which converts ATP to cAMP
* Adenylate cyclase activity is determined indirectly by the level of glucose
3. cAMP then binds to CAP
4. CAP-cAMP complex binds to CAP binding site at -61 in lac promoter, near lacP
5. Binding causes helix unwinding at downstream sites and facilitates the binding of RNA polymerase
* This increases transcription of the operon
45
What is the two-component regulatory system in prokaryotic transcription?
* Many bacterial responses are driven by two components (proteins)
* Example: Glutamine synthetase (glnA)
* NtrC – nitrogen regulatory protein C
* NtbB – nitrogen regulatory protein B
1. Histidine kinase sensor
* Regulated in response to environmental changes
* When activated, transfers phosphate of ATP to histidine residue in transmitter domain
* Then transfers phosphate
to the receiver domain of the response regulator
2. Response Regulator
* Phosphorylation then activates the effector domain
* Effector domains can have different functions depending upon system
46
# Eukaryotic transcription key terms
* Polycistronic
* Monocistronic
* Methylated cap
* Poly A tail
* Half-life
* Polycistronic
* Several genes (cistrons) per mRNA
* Found in prokaryotic mRNAs
* Monocistronic
* Only one coding region per mRNA
* This is the rule for eukaryotic mRNAs, wih a few exceptions
* Methylated cap
* Set of modified G residues at the 5' end of eukaryotic mRNAs
* Poly A tail
* String of 100-200 A nucleotides on the 3' end of eukaryotic mRNAs
* Half-life
* The time required for 50% of a given mRNA to be degraded
47
How does the eukaryotic mRNA structure compare to the prokaryotic structure?
Prokaryotic
* Polycistronic (protein A, B, Y)
Eukaryotic
* Monocistronic (just protein)
* 5' Methyl CAP Poly A tail
48
Eukaryotic RNA polymerases in transcription
* **RNA pol I**
* Transcribes a single gene - the precursor of large ribosomal rRNAs (18S, 28S, 5.8S)
* rRNA gene is present in 100s of tandem copies
* **RNA pol II**
* Transcribes all mRNA genes (i.e. those destined to be translated into protein), microRNA precursors, and most snRNAs involved in splicing
* mRNA, snRNAs, siRNAs, miRNAs
* Only pol II transcripts are polyadenylated
* **RNA pol III**
* Transcribes small genes like tRNAs, U6 snRNA, 5S rRNA, and 7S RNA
* All three RNA Pols share 9 core subunits (5 closely match prokaryotic core)
* Requires GTFs
49
General Transcription Factors (GTFs)
* Required to initiate transcription in eukaryotes
* Distinct from polymerase core subunits
* TFII- A, B, D, E, F, H
50
Key points for eukaryotic transcription process
* RNA polymerase begins transcribing at promoters
* Transcriptional units are much longer than needed to encode the protein product.
* Discrepancy can be explained by mostly by RNA splicing.
* Rate of transcription initiation varies from gene to gene, and is tightly regulated
* Cap structure is added to 5' end when synthesized
* 3' end of mRNA is generated by cleavage of primary transcript
* PolyA tail added to 3' end and introns are spliced out
* Processed mRNA is transported to the cytoplasm for translation
51
# Eukaryotic transcription
Housekeeping genes vs tissue-specific genes
Housekeeping genes:
* Most cell types express a common set of proteins
* Constitutive expression: ~same in all cells
* Involved in basic cell structure and central metabolism
* GAPDH, actin, cyclophilin
Tissue-specific genes:
* Cell type specific proteins that determine cell's phenotype
* Highly regulated
* Increased gene expression - dependent on continued presence of inducing signal
* Increased gene expression transient even in presence of regulatory signal
* Increased gene expression indefinitely after signal termination (irreversible and inherited
52
Eukaryotic transcription promoters
* **TATAA box**
* -25 to -30 region
* **Inr (initiator)**
* Span transcriptional start sites
* -2 to +4
* **BRE (B Recognition Element)**
* TFIIB recognition element
* -37 to -32
* **DPE**, DCE, MTE
* Downstream elements that play a role in recruiting RNA Pol II and GTFs
* DPE: +28 to +32
53
TFIID
* 15 protein complex
Includes:
* TBP (transcription binding proteins)
* Binds TATAA box
* TAF (TBP associated factors)
* Binds INR and other downstream elements
* TFIID binds promotor and facilitates the binding of the remaining TAFs
54
TFIIB
* Recruited and binds BRE element and TBP
* Serves as bridge to RNA Pol II
55
Eukaryotic RNA Pol II Initiation: Stepwise Assembly Model
1. TFIID binds to promoter and facilitates the binding of the remaining TAFs
2. TFIIB is recruited and binds BRE element and TBP
* Serves as bridge to RNA Pol II
3. The complex binds to the minor groove of DNA and recruits TFIIF and RNA pol II, then TFIIE and TFIIH
4. The Preinitiation Complex (PIC) is now complete
5. TFIIH has helicase activity. It phosphorylates the CTD (C-terminal domain) of RNA Pol II
6. Phosphorylation of 5th serine residue in a repeated amino acid sequence by TFIIH leads to dissociation of Pol2 from the PIC and initiation of transcription
56
What is the mediator in eukaryotic RNA Pol II initiation?
* Mediator is necessay transcriptional protein complex
* 20 subunits
* Interacts with GTFs and RNA Pol II
* Interacts with gene specific factors and "mediates" regulation of transcription initiation
* Mediator loses affinity for the complex when CTD of RNA Pol II is phosphorylated
* The CTD then binds other proteins that facilitate elongation and RNA processng
57
What is the alternative model of eukaryotic RNA Pol II initiation for eukaryotic transcription?
* RNA Pol enzyme preforms a complex (holoenzyme) with GTFs
* This complex is then recruited to specific promoters by activation factors
58
General transcription steps in eukaryotic transcription
1. TBP (in TFIID) binds to TATAA box
2. TFIIB is recruited and binds to the BRE element and TBP, which serves as a bridge to RNA Pol II
3. The complex then binds to the minor groove of DNA and recruits TFIIF and RNA Pol II, then TFIIE and TFIIH.
4. This completes the PIC
5. Mediator interacts with GTFs and Pol II
6. TFIIH phosphorylates the CTD of RNA Pol II, which leads to dissociation of GTFs from Pol II and initiation of transcription
7. Mediator loses affinity for CTD
8. CTD then binds to other proteins that faciliate elongation and RNA processing.
59
Basal transcription vs activated transcription
* General Transcription Complex (GTF) can generate low levels of transcription initiation
* Activated (high level) transcription requires GTF + trans-activator protein(s)
* **Trans-activators** work by binding to enhancers and recruiting components of the basal apparatus to the promoter.
60
Where can genes be regulated?
Process:
1. DNA
* Epigenetics and copy number variants
* Transcriptional control - enhancers, silencers, insulators, response elements
2. RNA transcript
* RNA processing control
3. mRNA (nucleus)
* RNA transport and localization control
4. mRNA (cytosol)
* mRNA degradation control - inactivates mRNA
* Translational control w/ microRNAs
5. Protein
* Protein activity control - inactivates protein
61
What transcription mechanisms are type A? Type C?
* Type A - Lac operon
* Type C - Bacteriophage "genetic switch"
62
What are the mechanisms of transcriptional control?
* Cell signaling by ligand activation of an intracellular receptor
* Glucocorticoid receptor (GR)
* Ligand activation of a plasma membrane receptor
* Ligand activation of a plasma membrane receptor
* G protein receptors
63
What are regulatory sequence elements (response elements)?
* DNA sequence usually near (upstream) of gene to be controlled - generally in promoter region
* Allows gene to be responsive to cellular environment
* Often consists of small sequence repeats (or inverted repeats) separated by a variable number of DNA base pairs
Types:
* **Cis-acting**
* Regulatory sequence in DNA that control gene on same chromosome
* **Trans-acting**
* DNA sequences encoding diffusible proteins or RNA that control genes on same or different chromosomes
* **TATA**
* Assembly site for RNA polymerase
* A1, A3, A5
* Binds pancreatic duodenum homeobox-1 (PDX-1)
* GG1, GG2
* GG1 is also known as A2
* Both may also bind PDX-1
* **CRE1, CRE2**
* cAMP response elements
* C2
* Binds PAX4 (repressor) and PAX6 (stimulator)
* E1, E2
* E1 binds USF (stimulates)
* E2 binds IEF1
* NRE
* Negative Response Element
* Binds OCT1
* G1
* Binds PUR-1/MAZ
64
What are DNA binding motifs in transcriptional control?
* Zinc finger
* Have been identified as both tumor suppressor genes and oncogenes
* Helix-Turn-Helix
* Leucine Zipper
* CRE binding protein
* c-myc, c-fos, c-jun = regulatory proteins
65
What are enhancers ("DNA control" elements)?
* Short sequence that stimulates transcription
* Not dependent on location and orientation
* Often contain multiple binding sites and can exert positive influence far from a promoter
* Can work in either direction (upstream or downstream) of promoter
* Distal enhancers can activate target gene expression by looping to promoters
* Promoters initiate transcription, enhancers may increase transcription of target gene
* **Enhanceosome** - complex of proteins that bind cooperatively to gene enhancer
* SPI: has zince finger
* CEBP: has leucine finger
* INSR: insulin receptor
66
What are silencers ("DNA Control" elements)?
* Binding sites for negative transcription factors
* Represses by blocking access for proteins required for gene transcription
* Some promote condensation of chromatin structure
67
What are insulators ("DNA Control" elements)?
* Sequences of DNA that restrict the action of enhancers and silencers in one chromatin domain affecting neighboring domains
* Blocks enhancer without hindering ability of the enhancer or promoter to function elsewhere
68
Epigenetics
* Heritable changes in gene expression that occur without a change in DNA sequence
* Regulates gene expression throughout individual's lifetime
* Reversible
* Generally paternal genome is "reset" (DNA methylation and histone modifications) following fertilization
* Maternal genome "reset" gradually
69
Post translational control: miRNA and siRNA
* miRNAs
* Regulators of endogenous genes
* Involved in early development, cell proliferation/death, fat metabolism, cell differentiation
* Aberrant miRNA expression may be correlated with cancers
* Final step of transcriptional control with miRNAs is incorporation of strand into RISC
* siRNAs
* Defenders of genome integrity in response to foreign or invasive nucleic acids such as viruses, transposons and transgenes
* Works in catalytic manner, post-transcriptionally, and at very low concentrations
* Activates endonuclease that digests a dsRNA target containing mRNA
70
p53 gene
* "Guardian of the Genome"
* Gene TP53, protein p53
* Several hundred genes regulated by p53
* Tumor suppression gene - prevents propagation of genetically damaged cells by regulating cell cycle
* Over 50% of human tumors have mutation of this gene
* In most cases the mutations are acquired, affecting both p53 alleles in somatic cells.
* Some individuals inherit one mutant gene which predisposes patients to cancer (because only one additional “hit” is needed for transformation)
* Such individuals have 25-fold increase risk of tumor by age 50 – sarcomas, brain and breast tumors, leukemia, at younger age and may be multiple
* Predisposition often characterized as dominant
71
rRNA processing
* Transcribed by RNA Pol I
* Eukaryotes have 4 special rRNAs - 3 from a single long precursor (28S, 18S, 5.8S)
* 4th is 5S, from separate gene
* 5S is transcribed by RNA Pol III
* Initially transcribed as 45S transcript that is processed by RNases to produce 28S, 18S, 5.8S rRNAs
* 5.8S rRNA hydrogens bonds to 28S rRNA in eukaryotes
72
Eukaryotic rRNA processing of subclass units
* snRNA - participate in cleavage
* scRNA (small cytoplasmic) - participate in synthesis and localization of proteins
* snoRNPs - guide base modifications in other RNAs (r-, t-, snRNAs)
* These are all transcribed by RNA Pol II and III
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RNA polymerase III transcribes...?
* tRNA, 5S rRNA, U6 snRNA
* 5S and tRNA promoters are downstream of transcription start site
* TFs bind there to recruit RNA Pol III
* Different combinations of TFIII's
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Histone modification
* Covalent modifications to histone proteins determine how tightly the nucleosomes (complex of chromosome + associated histone proteins) are packed together
* Heterochromatin - regions of tightly packed nucleosomes
* Euchromatin - less tightly packed, allows binding of RNA polymerase and regulatory proteins controlling gene expression
* Histone deacetylases, HDAC (-)
* Histone acetylases, HAT (+)
* Histone methyltransferases (-)
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tRNA Processing
* tRNA's are initally transcribed as pre-tRNA's
* Sometimes multiple tRNA's are located within same pre-tRNA transcript
* _RNase P_ processes the _5' end_ of pre-tRNA's
* Ribonucleoprotein complex
* RNA in RNase P, without the protein, is active, so it is a ribozyme
* A _conventional protein RNase_ cleaves the _3' end_ of tRNAs
* The 3' end of many tRNA's are processed through addition of a _CCA end_ (site of attachment for animo acid)
* Approx 10% of all bases in tRNA's are modified at certain positions in the tRNA structure
* Modifications change base pairing properties and stabilize hairpin loops
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Basic steps of processing eukaryotic mRNA
1. Capping
* Modification of 5' end with 7-methylguanosine
2. Tailing
* 3' polyadenylation (polyA tail)
3. Splicing
* Introns are removed, exons are linked together
4. Editing
* Changes single bases to alter amino acid sequence of protein product
5. RNA degradation
* Over 90% of pre-mRNA is introns, and thus, needs to be degraded
* Processing steps are coupled to transcription and are closely coordination.
* The CTD (C-terminal domain) of RNA Pol II plays a key role, providing a binding site for the enzyme complexes involved in mRNA processing
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# mRNA processing
mRNA degradation
* Over 90% of pre-mRNA is introns and thus, needs to be degraded
* Lariats are targeted by enzymes that recognize the 2'-5' phosphodiester bond
* 3' and 5' naked ends are targeted
* Premature termination codons may trigger nonsense mediated mRNA decay
* Rates of mRNA degradation vary from less than 30 minutes to greater than 20 hours
* Shorter half lives are usually assigned to regulatory proteins that rapidly respond
* Longer half lives are assigned to structural proteins
* The length of the polyA tail is related to the half life of the mRNA in a general sense
* Some factors bind to sequences at the 5' end of mRNA's to block translation
* siRNA's and miRNA's can also impact degradation
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# mRNA processing
mRNA editing
* Editing is the process of changing single bases to alter the amino acid sequence of the protein product
* Cytosine is deaminated to produce a uracil yielding a CAA-UAA change, creating a stop codon
* Example:
* ApoB mRNA, when unedited, leads a large product (Apo B100) for the liver, and, when edited, a smaller product (Apo B48) for the small intestine
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# mRNA processing
Alternative splicing
* Non-spliceosome factors recruit the spliceosome snRNP's to specific alternative sites, acting as activators
* Some factors can act as repressors, blocking ability of snRNPs to bind to certain splicing sites
* Changes the mRNA by splicing out exons
* This results in altered amino acid sequence in protein (after translation)
* Increases diversity of the number of proteins that are encoded within the DNA
* Example:
* Alternative splicing of transformer gene in Drosophila is controlled by Sex determination Switch Protein (SXL) that is only expressed in females
* SXL blocks binding of U2AF and results in the binding of U2AF to lower affinity site 3'
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# mRNA processing
Tailing: 3' polyadenylation
* Poly(A) tail is added to the 3' end of the pre-mRNA (polyadenylation)
* Upstream of site, there is a AAUAA signal
* Downstream of site, there is a GU rich element
* Some genes also have a GU rich sequence upstream of the poly A site
* An endonuclease cleaves the chain at the AAUAA sequence and a poly-A polymerase adds a poly A tail of roughly 200 bases
* These enzymes are attracted to the phosphorylated CTD of RNA Polymerase
* Nearly all eukaryotic mRNAs are polyadenylated
* Important for mRNA stability - prevents attack by 3' exoribonucleases
* Poly-A tails aid in translation
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# mRNA processing
5' capping
* A 7-methylguanosine cap is added to the 5' end of pre-mRNA after transcription
* Methyl groups are added to the guanosine and to the ribosomes of one or two of the next bases in the mRNA
* The cap stabilizes the mRNA and prevents attack by 5' exoribonucleases
* Aid in translation by helping to align it with the ribosome
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# mRNA processing
Basic splicing mechanism
* The pre-mRNA transcript includes 5' and 3' untranslated regions (UTRs) as well as the exons and introns
* Splicing process for the pre-mRNA where introns are removed from the RNA strand and exons are linked together
* Specific sequences in the RNA indicate the splice sites, essentially marking the end of the exon and the beginning of the intron
* **First step** of splicing is recognition and cleavage of the 5' splice site. This leaves an exon with a free 3' end
* The 5' end of the intron is joined to a 2' hydroxyl of an adenine base that is near the 3' end of the intron. This forms a loop, making the entire intron into a "lariat" like structure
* **Second step** involves simultaneous cleavage of the 3' site, releasing a free lariat shaped intron
* With concomitant joining of the 3' free end of the first exon with the newly free 5' end of the next exon
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# mRNA processing
Spliceosome mechanism
1. U1 snRNP binds to 5' splice site of pre-mRNA
* This involves bp between 5' splice site sequence and sequence at the 5' at the end of the U1 of the snRNP
2. U2 snRNP binds to internal A site within intron
3. U1 and U2 bind together, bending the intron and bringing the first exon closer the second
4. U4/U6 snRNP and U5 snRNP preformed complex enters the spliceosome
5. U5 snRNP binds to sequence upstream of the 5' splice site, meaning that U5 snRNP is holding the first exon
6. U4 leaves the complex, displacing the U1 snRNP as it leaves
7. U6 snRNP forms a complex with U2 snRNP and two things occur:
* The first exon is cleaved from the intron at the 5' sliced site
* The 5' end of the intron is joined to an internal adenine, catalyzing formation of the 2' to 5' phosphodiester bond, breating a lariat
8. The U5 snRNP is left holding a free exon positioned above the 3' splice site
9. U5 snRNP binds the 3' splice site leading to the cleavage and removal of the lariat intron and the linkage of the first and second exons
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# mRNA processing
Splicing Regulation Proteins (SR)
* Other protein factors (Splicing Regulation proteins) are involved in targeting the splicing reaction, which are not part of the snRNP's
* SR proteins recruit snRNP's to initiate spliceosome assembly
* U2AF binds to the 3' sites and recruits U2 to the branch point within the intron
* These splicing factors also may play a role through binding the CTD of RNA polymerase in maintaining the correct order of splicing through coupling it to transcription
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Codons
* Initiation codon - AUG
* Termination codons - UAA, UAG, UGA
* U Are Away, U Are Gone, U Go Away
* Degeneracy - More than one codon can code for single amino acid
* First two nucleotides in codon primarily determine specificity
* Variations occur in mitochondrial genetic code
* Anticodon
* Nucleotide ionsinate found in some tRNAs - recognizes A, U, C
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Wobble (codon)
* tRNAs can recognize more than 1 codon for a specific amino acid
* First base at 5' end of anticodon is complementary to third base of codon
* First base of anticodon can bind loosely to third base of codon
* If first base C or A, base pairing specific and only 1 codon recognized
* If first base U or G, base pairing less specific and two codons may be recognized
* If first base I, then three codons may be recognized
* Minimum of 32 tRNAs required to translate all 61 codons
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Frameshifting and RNA editing - translation
Frameshift:
* Ribosomes change reading frame during translation
* Allows two or more related but distinct proteins to be produced from one mRNA
RNA editing:
* mRNAs edited after transcription and before translation
* Nucleotides in RNA added, deleted, or altered
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Ribsomes involved in translation
Prokaryotic:
* 70S total
* 30S and 50S subunits
* 5S, 16S, 23S rRNAs
Eukaryotic:
* 80S total
* 40S and 60S subunits
* 5S, 5.8S, 18S, 28S rRNAs
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tRNAs - basic description
* Transfer RNAs
* Single stranded
* Folded into 3D structure
* Forms twisted L
* In 2D, folded into cloverleaf pattern
* Have modified bases and sugars
* All have CCA at 3' end (amino acid arm)
* Extra arm - variable in size, not present in all tRNAs
* TwC arm and D arm (horizontal on cloverleaf)
* Amino acid arm and anticodon arm (vertical on cloverleaf)
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Basic stages of protein synthesis
1. Activation of amino acids
* tRNA is aminoacylated
2. Initiation
* mRNA and aminoacylated tRNA bind to the small ribosomal subunit. The large subunit then binds
3. Enlongation
* Successive cycles of aminoacyl-tRNA binding and peptide bond formation occur until the ribosome reaches a stop codon
4. Termination
* Translation stops when a stop codon is encountered
* mRNA and protein dissociate, and the ribosomal subunits are recycled
5. Protein folding and posttranslational processing
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# Translation
Activation of amino acids
* Aminoacyl-tRNA synthetases esterify amino acids to corresponding tRNA
* Amino acid + tRNA + ATP ---> aminoacyl-tRNA + AMP + PPi
1. Form aminoacyl adenylate
* a-carboxyl of amino acid attacks a-phosphate of ATP, forming 5' aminoacyl adenylate
2. Transfer aminoacyl group to 3' end of tRNA
* Proofreading
* Critical to attach correct amino acid to tRNA, as amino acid on tRNA not checked by ribosome
* Three successive filters -
* 1) Attachment of amino acid to synthetase and activation
* 2) Binding of incorrect aminoacyl-AMP product to second site on synthetase, which is then hydrolyzed
* 3) Hydrolysis of linkage between incorrect amino acid and tRNA by synthetase
* Synthetases have to put correct amino acid on correct tRNA
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# Translation
Prokaryotic initiation
* Begins at amino-terminal end and new amino acids added to carboxyl-terminal end
* AUG initiation codon - codes for methionine
* 2 tRNAs for methionine - one initiation, other internal
* In bacteria, N-formylmethionine used for initiation
* In eukaryotes, specialized initiating tRNA used
* In bacteria, initiation is three step process:
1. IF-1 and IF-3 and mRNA bind to 30S subunit
* IF-1 binds to A (aminoacyl) site on subunit and prevents tRNA binding
* IF-3 prevents ribosomal subunits from combining prematurely
* Initiating AUG aligned by Shine-Delgarno sequence in mRNA
* AUG positioned at P (peptidyl) site
2. IF-2 + GTP and initiating fMet-tRNA binds to initiation complex
3. 50S subunit binds to complex and GTP hydrolyzed
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# Translation
Eukaryotic Initiation
1. eIF2-GTP binds to initiating tRNA-Met
2. 43S complex forms
* 40S ribosomal subunit
* eIF1, eIF1A, eIF3
* eIF2-tRNA-Met complex and eIF5
3. 5' and 3' ends of mRNA linked
* 3' end bound by poly(A) binding protein (PAB)
* eIF4E, eIF4G, and eIF4A bind to 5' cap
* eIF4G binds to both eIF4E and PAB
4. 43S complex binds eIF4F-mRNA complex
5. eIF4A RNA helicase unwinds RNA secondary structure as 40S complex scans mRNA in 5' to 3' direction until IF recognizes initiation codon
6. Ribosome scans for first AUG
7. When initiation codon recognized:
* eIF5 stimulates hydrolysis of GTP bound to eIF2
* Anticodon of initiation tRNA-Met base pairs to AUG in 40S P site
* Forms 48S initiation complex
8. 60S subunit binds to 40S subunit
9. 80S complex forms
* eIF5B-bound GTP hydrolyzed
* eIF5B-GDP and eIF1A released
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# Translation
Elongation
* Requires initiation complex, aminoacyl-tRNAs, elongation factors (EF-Tu, EF-Ts, EF-G) and GTP
* Proofreading - incorrect aminoacyl tRNAs dissociate from A site
1. Incoming aminoacyl-tRNA binds
* Aminoacyl-tRNA binds to GTP-bound EF-Tu
* This binds to A site on 70S initiation complex
* GTP hydrolyzed
* EF-Tu-GDP complex released
* EF-Tu-GTP complex regenerated using EF-Ts and GTP
2. Peptide bond forms
* fMet transferred from its tRNA to amino group of second amino acid in A site
* Now have dipeptidyl-tRNA in A site, with deacylated tRNA in P site
* Peptidyl transferase activity in 23S rRNA
3. Ribosome moves one codon toward 3' end of mRNA - translocation
* Dipeptidyl-tRNA moves from A site to P site
* Deacylated tRNA moves from P site to E (exit) site
* Third codon of mRNA now in A site and second codon in P site
* Requires EF-G and GTP
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# Translation
Termination
1. Signaled by presence of termination codon
2. Three termination or release factors (RF-1, RF-2, RF-3)
3. RF-1 recognizes termination codons UAG and UAA. RF-2 recognizes UGA and UAA
4. These bind at termination codon
5. Hydrolyze polypeptide link to tRNA
6. Ribosome subunits dissociate (may involve RF-3)
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# Translation
Rescue of stalled ribosomes
* May encounter end of mRNA before stop codon
* A site has no mRNA that can interact with charged tRNA
* Cannot recycle
* Rescused by transfer messenger RNA (tmRNA) and small protein B (SmpB)
* Binds to empty A site
* Translation continues until stop codon in tmRNA reached
* mRNA and protein degraded
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What are polysomes?
* Many ribosomes can translate single mRNA
* Transcription and translation tightly coupled in bacteria
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# Regulation of translation
Cytoplasmic polyadenylation
* Used by early embryos
* Some have mRNAs not translated until after fertilization
* Have short (20-40) polyA tail
* B/c of this, not translated efficiently
* mRNAs have AAUAAA signal and cytoplasmic polyadenylation element (CPE)
* CPE bound by CPE-binding protein (CPEB)
* In absence of signal:
* CPEB binds to Maskin, which binds to eIF4E
* This blocks translation initiation
* In presence of signal:
* CPEB phosphorylated
* Cleavage and polydenylation specificity factor (CPSF) and poly(A) polymerase (PAP) bind
* Longer poly(A) tail formed
* Translation initiation can occur
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# Regulation of Translation
RNA-binding proteins
* Ferritin - iron transport protein
* Iron response element (IRE) in 5' untranslated region (UTR)
* Can be bound by IRE-binding protein (IRE-BP)
* High iron:
* IRE-BP in inactive conformation
* Doesn't bind to IREs
* Ferritin translated
* Low iron:
* IRE-BP in active conformation
* Binds to IREs
* Small ribosomal subunit cannot bind
* Ferritin not translated
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# Regulation of Translation
Surveillance Mechanisms
* Prevents translation of improperly processed mRNAs
* Nonsense-mediated decay (NMD)
* Incorrect splicing can insert stop codon before last exon
* Mutations can create stop codon or frame-shift
* These mRNAs degraded
* Example: B0-thalassemia (missense mutation)
* Single base pair deletion
* ORF now has stop codon before last exon
* mRNA degrades and not translated
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# Translation
Antibiotics and Toxins
**Puromycin**:
* Structure similar to 3' end of aminoacyl-tRNA
* Binds to A site in ribosome
* Participates in peptide bond formation
* Cannot translocate and dissociates from ribosome
Other antibiotics:
* **Tetracycline** - blocks A site on ribosome in bacteria
* **Chloramphenicol** - blocks peptidyl transfer in bacteria
* **Steptomycin** - causes misreading of genetic code in bacteria
* **Cycloheximide** - blocks peptidyl transferase in eukaryotic ribosomes
Toxins:
* **Diphteria toxin** - catalyzes ADP ribosylation of eEF2
* **Ricin** - inactivates 60S subunit of eukaryotic ribosomes
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What is mTOR?
* Mammalian Target of Rapamycin
* Serine/threonine kinase
* Controls cell growth and proliferation = sensor
* Integrates multiple inputs from outside of cell
* Positive - growth factors, nutrients
* Negative - hypoxia, stress, low energy levels
* When activated, helps to trigger translation initiation
* 2 mTOR complexes - mTORC 1, mTORC 2
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How do amino acids affect mTORC activity?
* Increased amino acids
* ATP, oxygen, growth factor signaling increases protein kinase activity of mTORC1
* Decreased amino acids
* ATP, oxygen, growth factor signaling decreases protein kinase activity of mTORC1
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# mTOR pathway
Rheb (G protein)
* mTORC1 _active_ when bound to Rheb-GTP
* mTOR1 _inactive_ when bound to Rheb-GDP
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# mTOR pathway
TSC complex (TSC1/TSC2)
* Upstream inhibitor of mTOR
* _Negative signals (hypoxia, stress)_
* Act on TSC complex to activate it
* GTPase activity (GAP) in TSC complex activated
* Causes hydrolysis of GTP bound to Rheb
* mTORC1 bound to Rheb-GDP inactive
* _Positive signals (activation of growth factor receptors)_
* TSC complex phosphorylated to inactivate GAP
* Rheb can now bind GTP
* Rheb-GTP activates mTOR kinase activity
* Nutrients bypass TSC1/TSC2
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# mTOR pathway
S6K and 4E-BP
* mTORC1 phosphorylates ribosomal S6 kinase (S6K) and eIF4E-binding protein (4E-BP)
S6K
* Phosphorylates ribosomal subunit protein S6 - increases rate of translation elongation
* Phosphorylates eIF4B
* Cofactor of RNA helicase eIF4A
* Increases activity of eIF4A, which helps in translation of mRNAs with complex 5’ untranslated region secondary structure
4E-BP
* Binds tightly to cap-binding protein eIF4E
* Represses cap-binding activity
* Regulates translation by inhibiting initiation
* When phosphorylated, dissociates from eIF4E and translation can proceed
* Activates transcription factors for RNA polymerases I, II, III
* Increased synthesis and assembly of ribosomes, tRNAs, and translation factors
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Disregulation of mTOR pathway
* Dysregulation leads to aberrant protein translation
* Dysregulation of mTOR associated with type 2 diabetes, cancer, arthritis, obesity, and
neurodegenerative diseases
