175 terms

Bisc401 - Exam 3

STUDY
PLAY
Domains of Nuclear Receptors
Variable Region (activation), DNA Binding Domain, Ligand (Hormone) binding domain
Common hormones (3)
cortisol, retinoic acid, thryoxine
How does glucocorticoid receptor activate transcription> What type of protein? What does it bind to in the experiment? What structure does it form on nucleus?
Fusion protein that translocates to nucleus, activates transcription by interacting with chromatin-remodeling and histone acetylase complex. Forms a dimer on DNA
Steps in processing DNA
1) Transcription and 5' capping
2) Cleavage at PolyA site (Endonuclease)
3)Polyadenylation (PolyA polymerase, PAP +ATP)
4) RNA Splicing
When does splicing occur in long pre-mRNAs
Concurrently with transcription, before 3' cleavage.
What determines alternative cleavage?
Different PolyA sites selected for cleavage. For ex. think IgM protein. Upstrein cleavage - secreted, downstream = membrane
fibroblasts
connective tissue cells
hepatocytes
liver cells
fibronectin
a multifunctional extracellular matrix protein secreted from above cells
Which has alternative splicing
Hepatocytes splyce EIIIB, A
Fibroblast fibronectin
binnds to fibroblast membrane proteins
hepatocyte fibronectin
circulates i bloodstream, forms clots through fibrin domain
What cells have Group I self splicing introns
protozoans, has G in fold
Group II self splicing introns
Some plants/fungi, Has A in fold
Reaction of splicing
2 Transesterification reactions
1) OH at point base A attacks phosphate, phosphate releasex exon1
2) Hydroxyl from exon 1 attacks 3' phosphate, releasing loop
Loop of splice
Lariat
Spliceosome Factors (how many)
5 snRNPs = U1, U2, U4, U5, U6
Proteins SF1, U2AF (Yn, AG)
Why U snRNAs?
Rich in Uridine
Steps of Spliceosome
1)U1 binds to 5' splice site
2) SF1 to branc point A
3) U2AF to Y and AG
4) U2 snRNP replaces SF1, Yn, AG
5) Recruits U4,5,6 to create spliceosome
6) U1, U4 leave, catalytic core, first transesterification,
7) second transesterification and U2, 5, 6 leave
Compensatory Mutation
Restores spplicing activity in U1
ESE
Exons splicing enhancer = bounded by SR proteins
SR proteins
rich in serine/arginine
mediate cooperative binding of U1 snRNP to 5' splice site and U2 to branch point through protein protein interactions
Wht causes spinal muscular atrophy
SR proteins absence/defects
Criss Exon Recognition complex
On the exon, includes U2 at branch point, U2AF65 at Ys, 35 at AG (3' splice site), ESE/SRs, U1 at 5' splice site
Spliceosome consists of
U1 - 35, not SRs
Example of ALternate Splicing
Sex in Drosophilia
Ultimate Result for Drosophilia
No Tra in males, thus different DSX protein, different sex
Sex determination in Drosophilia
Early Sx1 promotes Sx1 in females, none in males
Sx1 promotes Tra1, none in males
Tra1 + Rbp1+Tra2 = Different Dsx
All done by alternative splicing
DSX
Double sex protein = repression of opposite sex
How does SX1 bind
Binds to intronic splicing silencer, Prevents snRNP binding
How does Tra Bind
binds exonic, splicing enhancer, brings U2AF and U2
RNA Editing
Alters 1+ nucleotide, can occur post or concurrent
apoB gene, what occurs
Post-trans modification of mRNA, deamination at 6666, C-> U
ApoB100 versus ApoB48
500 vs. 240kDa
Both associate with lipids
ApoB delivers cholesterol to tissues because it has functional LDL, low density lipoprotein, domain
IN-vivo transfection assays
a functional assay for protein activation
Plasmid 1 of in-vivo transfection assays
encodes a DNA binding protein x
protein x has
cis-regulatory sequence
Plasmid II of in-vivo transfection assays
Has x binding site and reporter gene
I-VTAs prove
DNA binding proteins
Nucleus in-> out
Nucleolus, chromatin
Nuclear pore complex, perinuclear space
3 types Nuclear pore complex (NPC)
1) Structural
2) Membrane
3) FG
FG =
Phenylalanine and Glycine
What does the FG NPC allow
small ions, metabolites, globular proteins,
Prevent?
Large proteins, RNP complexes
Center og FG NPC?
Matrix of FG repeats, fluid, extended random-coil formations, forms molexular cloud
NXP
Nuclear transporter protein
What goes through NPC first
5' capped ends, associates with ribosome, proteins stripped
Proteins Bound to mRNA in nucleus
REF
NXF1
NXT1
CBC
PABN1
REF
RNA export factor
NXF1
Nuclear export factor
NXT1
Nuclear export transporter
CBC
Cap Binding complex
PABN1
Poly A binding protein (nucleus)
Order of 3 protein in phosphorylation
REF, NXF1, NXT1
Ran
Small G protein, when active, binds to exportin
Dbp5
RNA helicase, important in mRNP remodeling
NpI3
Normally phosphorylated protein, SR, binds to nascent mRNA, promotes snRNP binding
Sky1
Cytoplasmic protein kinase phosphorylates/stimulates release of NpI3
GLe2
Adapter protein brins mRNP to NPC
Steps of Reversible phosphorylation
NpI3 binds rna
Becomes dephosphorylated by GLC7
NxF1 and NxT1 bind
GLE2 brings to NPC, passes through
Sky1 phosphorylates NpI3, NXT1 and NXf1 go bac to ucleus
If reversible phosphorylation is incorrect,
RNA degraded in nucleus
Export out Sequence and Protein
RAN
RAN-GDP -> RAN-GTP by RAN-GEF
Binds to exportin, cargo +NES binds
Leaves by concentration gradient
RAN GAP hydrolyzes,
Exportin and RANGDP come back
NLS
Nuclear localization sequence
How does this process work?
Sequential conformational changes
NES
Nuclear Export signal
Import sequence
RAN-GDP becomes GTP
Leaves nucleus with importin
GDP
Binds cargo in cytosol
Importin and cargo come back
NLS Example
Pyruvate kinase by itself is in cytosol, but add T antigen NLS, goes to nucleus
General Structure mRNA in Eukaryotes, 5'->3'
7mG(Cap) -> 5'UTR => AUG -> Coding (ORF) -> STOP
ORF
Open reading frame, typically one per protein
USe of 5' cap
Required for translation, protection from exo-ribonuclease degradation
5' and 3' UTR
Regulate translation
3 pathways of degradation
1) Deadenylation dependent degradation
2) Deadenylation independent degradation
3) endonuclease mediated mRNA decay
Deadenylationn dependent degradation (2 ways)
1) deadenylating nuclease creates deadenylase complex
5'->3'', decapping by DCP2/DCP1, exposes mRNA, XRN1 exonuclease eats mRNA
3'->5' Exosome and scavenger decapping
Deadenylation independent
Enhacer of decapping 3 (Edc3), and RNA binding protein (Rps28B) promote decapping complex, XRN1 eats.
Sequences at 5' end make it sensitive to decapping
Endonuclease mediated
Endonuclease cuts in half
XRN1 5->3
Exosome 3'->5'
What does a shortened Poly A tail do?
Doesnt allow PABC1 to bind to stabilize 5' cap and traanslation/initiation factors
How does mRNA prevent degradation
Has stems/RNA loops
AU Rich region
forms stems
IREs
Iron response elements, form loops of mRNA
TfR mRNA
Makes transferrin receptor protein to bind/transport iron into cell
IRE-BP
IRE binding protein
Active at low Iron
Inactive at High Iron
Binds to loop
REsult of ACtive IRE-BP
Masks AU rich region, prevents dedenylating enzymes, more transferrin
Ferritin
makes proteins that bind/store iron
IRE-BPs of Ferritin
Active again at low iron, but block translation
Translation Apparatus
TA
TA : Message
mRNA
Adapters
tRNA
Chargers
aatRNA synthetases
links
amino acids
Coordination 1
40s
Coord 2
60S
Initiatiors
eIF1,2,3
Elongators
eEF1,2
Terminators
eRF1,eEF3
Juice
GTP/ATP
How much RNA is rRNA? tRNA?
80, 15%
Components of ribosome large
60S = 28S + 5.8S +5S +polIII
Small rRNA
40 S= 18S + mRNA + polII
Introns for rRNA
non-transcribed spaces
Order for transcription of rRNA
18, , 5.8, 28S
Which end is going out of ribosome
N Terminal
# codons to amino acids
61 to 20
Termination sequences
UAA, UAG, UGA
Degeneracy?
More than 1 codon for 1 amino acid
How long are tRNAs
70-80
2 features of tRNA
1) Charged by specific amino acid
2)recognizes/binds to codon
3' CCA
in all tRAs, 3' end, charged with amino acid
Special chemicals in tRNA
Dihydrouridine, Inosine, Ribothymidine, Pseudouridine, some methylated
Loops of tRNA
Acceptor stem, D loop, anticodon, variable loop, TPsiCG loop
How does tRNA react with aatRNA synthetase
anticodon loop and acceptor arms haave non-covalent interactions
aatRNA special fact
has proofreading ability
Third base degeneracy
a) Irrelevant or b) only distiguishes purines from pyrimidines
Wobble Rules
G=U
G=C
A=U
Iosine can base pair with A, C, U
What is created when aatRNA synthetase adds amino acid
High energy ester bond
IMplications of Wobble effect
a single tRNA can recognize more than onne codon to amino acid
3rd nucleotide unimportant
Subunits prokaryotic rRNA
30S/50S =70S
Eukaryotic subunits
40S/60S = 80S
Reaction of tRNA
Aminoacyl tRNA has amino acid
Peptidyl tRNA releases amino acid
Deacetylated tRNA leaves
Sites of rRNA
A site for aatRNA
P peptidyl site
E exit site for tRNA
Challenges in translation innitiation/elongation
1) Get message to ribosome
2) Identify start codon)
3) Recruit correct translator/bridge for first word
4) recruit next translator
5) Connect peptide
6) move machine
Initiation checkpoints
1) eIF2-GTP binds to 40S, plus EIF1 at E, EIF1A at A, 3 at 40S, and 5 is small GTPase
2)43S initiation complex binds to mRNA eIF4-complex
3)Scanning to find AUG
4) eIF2 GTP on tRNA MET is hydrolyzed, pairing with P site
5) 60S subunit binds to 40S, association leads to hydrolysis of eIF5B-GTP, functions as proofreadng
How is mRNA prepared for initiation
CAP, eIF4G, A, B, E
KOZAK sequence
ACCaugG sequence surrounding AUG
Elongation
1) entry of aatRNA into A site, only correct one fits, brought by EF1alpha
2) Hydrolysis of EF1alpha inniduces better fit
3) formation of new peptide bond catalyzed by large rRNA
4) EF2-GTP hydrolysis translocates ribosome
Termination
Release factors eRF1 and eRF3 GTP localize to A site, hydrolysis, peptide is released
Checkpoints of Initition, elongtion, termination
All GTP swithces
1) eIF2-GTP hydrolyzed to bind Met to start site
2)Association of ibosomes by eIF5B-GTP
3)Hydrolysis of EIF1alpha to bring first tRNA
4)Translocation of ribosome by EF2-GTP
5)Release of polypeptide by erF1 and eRF3-GTP
eIF4E
Binds cap
eIF4G
Binds PABP
eIFA
helicase
eIF4B
stimulates eIF4A
Reaction name of peptide bond formation
peptidyl trasferase reaction
What binds to PolyA 3' UTR
CPEB, Maskin
CPEB
Cytoplasmic polyadenylation element binding protein, binds to U rich region
What does CPEB recruit
CPSF, PAP, PABC1, and eIF4 complex
How does CPEB recruit
When activated/phosphorylated by protein kinase from hormon stimulation
Shine-Dalgarno sequence
complementary, short upstream
Smooth ER produces
Lipids
Translocon
Gated channel protein in ER
BiP
chaaperone, peptide binding and atpase domains, binds unfolded peptide, reduces slicding
Sec63
Dephosphorylates BiP
SRP
Signal Recognition Particle
Combines with signal sequence to deliver to SRP receptor
SRP receptor
Alpha and beta subunits, in ER. SRP brings ribosome to dock hear
Cotranslational translocation steps
1) Signal sequence
2) Translation pauses, SRP brings to SRP receptor
3) GTP Hydrolysis opens translocon, SRP released
4) Translation resumes
5) Signal peptidase cleaves signal sequence
How to prove translocation and translation at same time?
Run two experiments. Allow proteins to form without microsomes, and with microsomes. If at same time, proteins will be inside microsomes and will have smalle molecular weight
SRP composition
6 proteins, 300 bp RNA
RNA is the scaffold
p9/p14 interact with ribosome
p68/p72 bind to SRP receptor
p19
p54 Binds to signal sequence
Domain of SRP
Ffh, signal sequence binding domain with hydrophobic binding groove
How do SRP and SRP receptor bind?
Both bound to GTP, when coming together, the fit allows hydrolysis of both GTPs
Sec61
Bacteria
Has gates by helicase, and pore by isoleucine residues
sealed to small molecules
Sec61 structure
alpha, beta, gamma subunits, cross linking agent grabs protein
Tail anchored proteins are
T-snares and v-snares
Type 1
Stop transfer anchor sequence,
NH3 in lumen
Type II
Signal anchor sequence
N terminal in cytosol because positive amino acids come before sequence
Type III
Signal anchor sequence
N terminal in lumen because + amino acids come after sequence
Type IVA
Multipass protein
Signal anchor sequence, followed by stop transfer anchor sequence
on repeat
GPI
Glycosyl Phosphotidyl inositol
GPI transamidase
Cleaves protei and transfers new C terminal to GPI in membrane
GPI Structure
Hydrophobic fatty acids
Polar: Phosphate, glucosamine, mannose, inositol
GPI Protein advantages,
Type I, cleaved with GPI transamidase, GPI Anchor diffuses in membrane faster
Structure of phospholipid
Polar head: phosphate, glycerol,
hydrophobic fatty acids
3 classes of membrames
Phosphoglycerides
Spingolipids
Sterols
Integral membrane protein
1+ alpha helices or beta sheets transmembrane motif
Lipid anchor membrane protein
covalently attached to lipid
peripheral
non-covalent binding to membrane protein
Transport of vessicle sequence
Donor -> Budding -> Fusion -> Target
3 types of vessivles
CopI, CopII
Clathrin
Clathrin
PM to trans golgi to endosomes
ARF G-protein!
CopI
Between Golgi, cis-golgi, er, ARF G protein!
CopII
Er to cis golgi
Sar1 g protein
G proteins for budding initiation
ARF or Saar1
Sequence of vessicle budding (SAR1)
Recruitment of GTP binding protein
Sec12 is GEF for Sar1
Coat
Sec23 is GAP
Decoat
How to lock vessicle
Rab and GTP protein recruited to uncoated vessicle
hydrolysis allos v-snares and t-snares to interact
v in v snare =
VAMP
VAMP =
Vessicle
Associated
Membrane
Protein
V + T Snare
4 helix coiled coil
Fusion
Membranes combine
Alpha snap and NSF bind to each cis-snare complex
Hydrolysis disassembles snare complex
YOU MIGHT ALSO LIKE...