proteolytic cleavage
done by proteases, example: removal of methionine after translation
Zymogens
inactive precursors that are activated by proteolytic cleavage, ex. clotting cascade (serine proteases cleave inactive zymogens)
Clotting cascade (main point)
To stimulate a thrombin burst-->convert fibrinogen to fibrin-->fibrin cross linkages create clot
Acylation
When a methionine is removed it is replaced by an acetyl group at the N-terminus (donated by acetyl CoA)
Mryristylation
Lipid anchors. Myristyl anchors embed themselves in the lipid bilayer; allows a protein that would normally not associate with the lipid bilayer to be attracted to that area
Prenylation
Prenylation attaches the cysteine residue and prenyl (15 residue farnesyl) group via a thioester. Fti inhibitors for Progeria try to block prenylation.
Example of prenylation
Prenylation is known to occur on proteins of the RAB family of RAS-related G-proteins, oncogenic GTP-binding and hydrolyzing protein RAS which is farnesylated
DNA methylation
Adding methyl group at CPG islands to DNA causes steric hinderance -->allows for transcription factors to bind-->STOP TRANSCRIPTION
Histone acetylation
When you acetylate histones (using HATS)->shields their charge->decreases their affinity for DNA->activate them-> ENHANCE TRANSCRIPTION, to deactylate use HDACs
Histone methylation
When histones are methylated, histones are deacetylated, DNA is methylated--> allows binding of transcription factors on outer DNA helix gene silencing (NO TRANSCRIPTION)!
Phosphorylation
Most common post translational modification, proteins are phosphorylated by kinases, AAs that are phosphorylatable are serine, threonine, tyrosine
Physiological Example of Phosphorylation
Phosphorylations that occur in glycogen synthase and glycogen phosphorylase in hepatocytes in response to glucagon release from the pancreas
Ubiquitination
ATP-dependent process that is major mechanisms for the destruction of cellular proteins, involves a complex structure referred to as the proteosome, ubiquitin carrier protein attaches ubiquitin to the protein with ubiquitin ligase
Lactose
glucose-galactose cleaved by lactase (non-inducible, brush border disaccharidase), rate limiting step in its digestioon is its hydrolysis not the transport of glucose and galactose
Glycogen
glucose molecules linked by alpha-1,4 glycosidic bonds (amylose), digestion promoted by amylase
Primary monosaccharides
glucose, fructose, galactose
Glucose
monosaccharide Na+ dependent glucose transported across brush border from mucosa-->enterocyte
Galactose
monosaccharide Na+ dependent transported across brush border from mucosa-->enterocyte
Fructose
monosaccharide Na+ independent facilitated diffusion (uses GLUT5) transported across brush border membrane from mucosa-->enterocyte
Glycoprotein
proteins with sugars covalently linked to their AAs, MOST CELL SURFACE MEMBRANES, usually contain amino sugars (N-Acetylglucosamine, N-acetylgalactoseamine), neutral sugars (D-galactose, D-mannose, L-fucose) or acidic sugars (sialic acid)
Functions of Glycoprotein
Hormone receptors at the cell surface, cell-cell interaction
Proteoglycan
long unbranched sugar chains, hallmark of disaccharide repeats, MOST ER and Golgi membrane proteins
Functions of Proteoglycans
Many of ER and Golgi membrane proteins, also proteins secreted from the cell like serum and mucus proteins, Glycosylation is the major enzymatic modification in the body
N-linked proteins
N-glycosidically linked oligosaccharides widespread in nature, characteristic of membrane and secretory proteins, linked by N-acetylglucosamine (GlcNAc)--connected to asparagine
O-linked proteins
O-linked found in mucous fluids, but can also be present in membrane and secretory proteins, 3 or more sugars linked by N-acetylglalactosamine (GalNAc)--connected to serine, theronine. O-linked found a lot in collagen
Collagen Synthesis
First procollagen (N-linked glycoprotein)-->N linke oligosaccharide is removed ("N")-->only O linked remain in mature collagen
Collagen glycosylation
Degree of glycosylation impacts structure--> less glycosylated collagen=ordered fibrous structure (tendons) while heavily glycosylated are more like meshwork structure in basement membrane
N-linked protein in hormones
GlcNAc is linked to serine residue that become phosphorylated by protein kinases in hormonal stimulation
High mannose N-linked protein
all N-linked glycoproteins have oligosaccharide chains coming off a common core 3 mannose residues+2 GlcNAc, high mannose is how they all start some glycoproteins are modified further after this
Tetra-antennary type N-linked protein
Complex chains which have terminal trisaccharide sequence of sialic acid0galactose-GlcNAc attached to branched core mannoses
LDL receptor glycoprotein structure
has 2 N-linked oligosaccharides near LDL-binding domain (not involved in binding) and cluster of O-linked oligosaccharides near membrane spanning region (sialic acid residues to hold it up)
Biosynthesis of N-linked glycoproteins
Synthesized in the ER. Dolihcol is the anchor in the lipid bilayer of the ER membrane-->first sugar is GlcNAc-1-P-->another GlcNAc-1-P --> 4 or 5 mannose residues--> 3 glucose residues (which are later removed) NEXT STEP modificaton in the ER-->sent to Golgi-->elongation
Biosynthesis of O-linked glycoproteins
Occurs in the Golgi occurs in stepwise fashion of addition of sugars
High mannose oligosaccharides in Lysosomes
Lysosomal enzymes are N-linked oligosaccharides are synthesized in the ER and Golgi
Lapatinib (HERCEPTIN)
Small molecule drug to treat HER2 resistant breast cancer by bind to ATP recptors-->Blocks the tyrosine kinase from binding
Imatinib
Small molecule drug to treat CML by bind to ATP recptors-->Blocks the tyrosine kinase from binding
Proteoglycans
Gel forming compounds made of protein backbone with covalently bound sugars, oligosaccharide chains have disaccharides repeats usually composed of amino sugar and uronic acid
Glycosaminoglycans (GAGs)
the carbohydrate part of proteoglycans, each has unique disaccharide repeat, usually includes hexosamine and uronic acid (EXCEPT FOR KERATAN SULFATE), amino sugars are usually glucosamine (GlcNH2) or galctosamine (GalNH2) present in their N-acetylated form
Hyaluronic acid
proteoglycan, no sulfation--located in joint and ocular fluids
Chondroitin sulfates
proteoglycan, located in cartilage, tendons, bone
Dermatan sulfate
proteoglycan, located in skin, valves, blood vessels
Heparan sulfate
proteoglycan, amino group sulfated (not acetylated) located in cell surfaces
Heparin
proteoglycan, amino group sulfated (not acetylated) located in mast cells and liver
Keratan sulfate
proteoglycan, uronic acid replaced by galactose, located in cartilage, cornea
Synthesis of proteoglycans
synthesized by a series of glycosyl transferases, epimerases, sulfo transferases. Synthesis of core oligosaccharide while still in the RER, then synthesis of the repeating oligosccahride and other modifications take place in the Golgi
Germline mosaicism
X-inactivation causes one X chromosome to be inactivated in some tissues and the other X chromosome to active in others
X-linked mental retardation
X chromosome has a high frequency of mutations, microdeletions, duplication that cause X linked mental retardation
LDL receptor
defects in this receptor are responsible for familial hypercholesterolemia (autosomeal dominant) it's a transmembrane glycoprotein!
factor VIII
Allel coding for this causes hemophilia A--X-linked recessive, usually seen in males not females
X-linked recessive (can females ever have phenotype)?
If the father is a carrier on his X and the mother is a carrier then female offspring would be homozygous affected (rare because of the low incidence of X-linked recessive disorders)
Skewed X-inactivation
X inactivation (usually random) the fraction of mutant alleles that remain active is much greater than normal, the deleterious allele finds itself located on active X, if this is present in pertinent tissues then you will have disease
Unstable repeat expansions
Genetic diseases caused by the expansion within an affected gene of DNA with repeating units of three or more nucleotides in tandem (CAG or CCG)-->primarily neurological conditions result
Unstable repeat expansion diseases
Myotonic dystrophy, fragile X syndrome, Huntington's Disease (polyglutamine disoder), spinocerebellar ataxias (polyglutamine disoder)
Anticipation
Genetic term referrring to the progressive severity and decrease in the age of onset for diseases that are passed through the pedigree
Disease associated with Anticipation
Fragile X, Myotonic Dystrophy, Huntington's
Huntington's disease
CAG repeats
Spinocerebellar ataxia
CAG repeats
Fragile X
CGG repeats
Myotonic Dystrophy
CTG repeats
Fatty acid synthesis
Step 1: turn acetyl CoA--> malonyl CoA (using Biotin and acetyl CoA carboxylase), Step 2: elongation of fatty acid chain in two
Phospholipid synthesis
occurs on the cytoplasmic face of the ER, uses CDP-->CMP and flippases who flip the phospholipid one leaflet of the bilayer to the other
Cholestrol
Phospholipid, has a polar head group and a largely non-polar hydrocarbon tail. When it enters the bilayer, it causes stiffening. It will sit in hydrophobic tail region. It exists at quite a high level at the bilayer. By stiffening the bilayer, you alter the properties, even changing membrane permeability.
Fluid mosaic model
Singer, Nicholson said that the phospholipid bilayer has assymetry that is caused by differential packing of p-serine vs. p-choline
Phospholipids in the bilyaer
phosphotidyl serin phosphotidyl cholinelipid rafsanother example of asymmetry--mobile--float around bilayer, environment inside rafts is different from outside- Caveoli
Dominant negative effect
Mutation in gene regulatory region, If you get a mutation in the regulatory region it has an enhanced effec as a result of kinase activity
Dominant negative effect (disease example)
Amylotrphic lateral sclerosis, causes proteins to aggregate and interfere with cellular function
PDZ Domain
scaffold protein to bind ion channels to membrane, 80-90 AAs 6 strand Beta sandwich flanked by alpha helices, recognizes the C-terminus of receptors, involved in anchoring the CFTR in lung epithelial cells
SALT BRIDGE in Hb
ionic bond between lysine and glutamate
Hemoglobin
Oxygen carrier, iron in heaxcoordinate (binds 4 porphyrin, 1 proximal histidine, 1 oxygen) porphyrin ring is tetra coordinate
Cooperativity
When the binding of one oxygen molecule increases the binding affinity of the other sites, tense (low affinity), relaxed (high affinity)
Allosterism:
when the binding of one molecule at a site other than the active site increases or decreases the finction there
Bonding between base pairs in DNA
Hydrogen bonding
Alpha helix (macrodipole)
N terminus is positive, C terminus is negative
Hydrophobic interactions
Ex. oil in water-->increase entropy of system by liberating water molecules, all aromatice molecules are nonpolar (hydrophobic)
Sterochemistry of enantiomers
Animals are almost exclusively L-amino acids, sugars are all D-sugars
# exons in B-globin gene
3
# exons in BRCA1
24
# exons in B-myosin heavy chain (MYH7)
40
Prokaryotic vs. Eukaryotic DNA
Prokaryotic Genes On, Eukaryotic OFF, Prokaryotic no DNA-protein complexes, Eukaryotic has DNA-protein complexes
Stop codons
UGA, UAA, UAG (you go away, you are away, you are gone)
Kozak sequence
Sequnce that the AUG start codon in included within (in the 5'UTR region)
Exon-Intron splice
GT (3' end of exon, 5'end of intron)----->AG(5' end of exon, 3'end of intron)
Transcription factors
TATA box (complimented by iniator), CAAT box, GC rich
Large genes (>100bp)
Factor VIII, CFTR, Dystrophin, BRCA1
Small genes (<10 kb)
B-globin, insulin
Medium gene (10-100bp)
collagen, LDL receptor
Avergae number of exons per gene
10 exons
Pseudogene
transcribed but not translated or not transcribed at all--can lead to unequal crossing over
Unequal crossing over
misalignment of two alpha globin genes on a chromsome causes alpha-thalassemia
tandem repeats
Repetitive DNA sequences-satellite DNA near centromeres
SINEs
short interspersed nucleotide repeats (10% human DNA), can interfere with crossing over, ex. Alu repeats SINEs cause familial hypercholesterolemia
Which exon is lost in the unequal crossing over Familial Hypsercholesterolemia
exon 5
LINEs
long interspersed nucleotide repeats (20% human DNA), can be transposable elements
Mitochondrial DNA
37 genes, amternal inheritance, no introns, highly conserved, makes more mutations, 2 strands heavy-guanine rich and light-cytosine rich
Heteroplasmy
the mixed population or normal and mutant mtDNA
Types of DNA
A,B,Z
Z DNA
left handed turn
Histones (#)
two copites of each of the four core histone (H2A, H2B, H3, H4)
Histones that can substituted
H3 and H2A
Histones that can be chemically modified
H3 and H4
Histone code
the pattern of major and specialized histone types and their modifications
Cell division
4-6 hours to reproduce 6.4 Gb
High fidelity
DNAs ability to reliably replicate
DNA error rate
1 in 10^9 is the limit, actual rate is 1 in 10^6 it fixes it by proofreading!
DNA licensing
ensure that DNA replication is limited to once/cycle
Replication origin
replication origins are rich in A-T (easier to break)
Number of replication sites (Prokaryotic vs. Eukaryotic)
E. coli have 1, humans have 100,000
Steps of DNA Replication
1. unwinding & replication forks, 2. stabilization with SSBPs, 3. Iniation (priming with DNA polymerase alpha), 4. Elongation (5'-->3'), 5. Lagging strand synthesis (3'-->5'' semgents) discontinuous, 6. Licensing: ensuring each replicates only once/cell cycle
Helicase
DNA strands are separated in ATP-dependent fashion
Topoisomerase
prevent DNA from becoming supercoiled
Single stranded binding protein
stabilize leading strand
DNA Polymerase
Can only add to the 3' end nucleotides, needs Magnesium to function
DNA Pol alpha
AKA RNA primase, synthesizes an RNA primer and then acts as a DNA pol to elongate for about 20 bp
DNA Pol delta
Lagging strand synthesis, Highly processive, proofreading 3'-->5'
DNA Pol E
Leading strand synthesis, Highly processive, proofreading 3'-->5'
Processivity
stays on strand longer
PCNA (clamps)
ATP-dependent way to increase the processivity of the Polymerase (stay on the strand)
Prokaryotic Polymerazes
DNA Pol 1, II, III
Main polymerase in bacteria
DNA Pol III
RNAase H
takes off the small RNA primer and DNA pol delta and epsilon fill it in
Semi-discontinuous synthesis
Lagging strand is built in segments (Okazaki fragments)
Okazaki fragments
segments of DNA on the lagging strand
DNA Ligase
glues together Okazaki fragments
Replisome
the whole complex of helicase, SSBPs, Polymerase, PCNAs
Replication
4-6 hours for 6.4 Gb, 100,000 replicons, during S phase
CDC 6
CDC6 recruited to form pre-replication complex, licensing to ensure 1 DNA replication/cell cycle, by the end of G2 there is no CDC6 left
Type of DNA Damage
Spontaneous, Exogenous (UV, Radiation, Chemical)
Spontaneous DNA damage
Deamination (standard bases are exchanged for nonstandard bases), base loss (Depurination>depyrimidation), ROS
Ionizing radiation
Causes double strand breaks by hydrolysis of water which breaks down into ROS
UV Radiation
Photo activate nucleotides, causes thymine (pyrimidine) dimer formations
Adduct formation
covalent attachment to DNA nucleotides, ex. benzo[a]pyrene
Alkylating agents
Carbn comound group to one of the bases-->disrupts structures, Ex. CYTOXAN
Crosslinking
Bi functional-->2 adduct forming entities can bond two 2 positions on DNA (can be inter or intra strand), Ex. CISPLATIN
Type II topoisomerase inhibitor
etoposide
Microsatelitte Instability
Occurs during replication of repetitive sequences, Forward slippage (parent) causes deletion, backwards slippage (daughter) causes insertion
Translesion synthesis
DNA backbone is still intact but you will get replication error
DNA Double strand break
caused by ionizing radiation, most deleterious form of DNA damage-->leads to aneuploidy
Cell cycle checkpoint
Eukaryotes have cell cycle checkpoints at G1 and G2, focus on checkpoint for G2--if you have mutations in the genes that code for checkpoints
Why use alkylating agents, crosslinking agents in cancer therapy?
Tumor cells grow faster than normal counterparts, should be more susceptible to checkpoint
DNA Repai pathways
Base Excision repait, Nucleotide Excision Repair, Translesion Synthesis, Mismatch Repair, Homologous Recombination, End joining
Base Excision Repair
Deaminations, depurinations-->uses glycosylases to cut out damaged base (least flexible)
Nucleotide Excision Repair
UV photoproducts, cross links-->RNA pol encounters DNA lesion, stops, uses a multiprotein complex (>10 proteins) for primary repair mechansim of UV photoproducts, cuts 5 bases 3' of the damage and 23 bases 5' of the damage, gaps filled by DNA pol
Mismatch Repair
Replication error (ex. Lynch Syndrome)
Homologous recombination
Double strand break, adducts, cross links
End joining
Double strand breaks
Translesion synthesis
Not high fideltity, can pass by damage, very error prone, non-templated manner
XPA and XPC addount for (%) of all XP cases
50%
How to test for XP?
Unscheduled DNA synthesis with skin biopsy (use radiolabeled thymine)
Where is rRNA synthesized?
nucleolus
Where does capping and polyA tail are added after transcription, where?
nucleus
Transcriptional unit
TATA, GC, CAAT box, Enhacers and Silencers (on the DNA), TFIID
Transcription Termination sequence
C-G-C-G
What causes mushroom poisoning?
alpha-Amanitin, is a mushroom poisoning RNA pol II inhibitor-->block mRNA transcription
RNA polymerases
Enzymes that synthesize the RNA strand from a DNA template during transcription
RNA pol II transcribes which RNA?
rRNA
mRNA is transcribed using what RNA Polymerase?
RNA Pol II
RNA pol II transcribes which RNA?
mRNA and microRNA
RNA pol III transcribes which RNA?
tRNA and ribozymes
Stages of transcription
1. initiation (construction of RNApol complex on the promoter, recruitment of transcription factors), 2. Elongation, 3. Termination (cessation of RNA transcription with CG repeats)
Does RNA Polymerase has its own helicase activity?
Yes
TATA Box
10-20% of human promoters, 25-30 bases upstream of trranscription start site, allows correct positionaing of RNA pol
TBP
TATA binding protein, first to bind the DNA, causes the DNA to bend-->recruits TFIID and other transcriiption factors
General transcription factors (GTFs)
required for PolII in a test tue are TFIIA, B,D, E, F, and H (but basal level is achieved with purified B and F)
Elongation
RNA pol requires energy to add ribonucleotise to the 3' end of the growing strand, 17 bp transcription complex with 8 bp DNA-RNA hybrid
Abortive transcription
RNApol shows strong binding to promoter and generates short 9bp RNA fragments-->eventually it clears the promoter
Rho factor (prokaryotic)
Termination factor dependent for termination, used in bacteria
How is transcription different in prokaryotes
RNA pol directly recognizes sequences in DNS for binding and transcription, no nuclear envelope, no introns, transcription/translation occur simultaneously, use of polycystronic messages (like the LAC operon)
Antibiotic that target prokaryotic transcription
Rifampin binds to beta subunit of prokaryotic RNApoly, Dactinomycin (actinomysin D) binds to DNA template and interferes with RNApol progression
Reverse Transcription
Viral RNA used reverse transcriptase. Take mRNA strand and reverses transcription to turn it into DNA. Then it inserts itself into our genome
Gene Regulatory Proteins
Helix turn helix, zinc finger, leucine zipper, winged helix, winged helix turn helix, helix loop helix
Helixa turn helix
Repressor protein
Zinc Finger
Zn ion to stabilize structure/finger/specific triplet of base pairs, Zn ion causes secondary structure, bind major groove
Leucine zipper
Leucine every 7th residue, "zips" up to dimerize the protein, needs a dimer to inhibit DNA
Winged Helix
4 helices and two strand beta sheet
Winged helix turn helix:
3 helical bundle and 4-strand beta sheet
Methylate histone
Block charge-->activate transcription
Acetylates histone
Block charge-->activate transcription
Methylate DNA
Methylate DNA at the CpG island-->inhibit transcription
Nucleosome
Histone complex (8 total) with the DNA wrapped around it
Histone acetyltransferases (HATS)
Acetylate histones
DNA Binding Proteins (enhancer/silencers)
Regions that can contain multiple elements for assemply of large protein complexes--can be 1000s of bps away
Multi-domain proteins
activators and repressors can have multiple functions besides serving as transcriptional regulators
Polycystronic message
message with a length of RNA with whole process associated through several consecutive genes--all related to same process
Lac Operon
B-galactosidase cleaves lactose into allolactose-->bind repressor subunits to prevent assembly-->cAMP starvation signal forms CAP cAMP and promotes RNA pol attachment--> RNA pol transcribes genes to produce B-Galactosidase, permease and acetylase
Halflife of RNA
10 hours
Chronic Myeloid Leukemia
15-20% of all adult leukemias, BCR-ABL-->ABL (tyrosine kinase) --> speeds up cell division and inhibits DNA repair
Regulation of RNA turnover- AUUUA sequences
Experiment deomstrating the destabilizing effect of AUUUA sequences-->shortened the halflife of B-globin mRNA from 10h-->1.5h
Nonsense Mutation
is when you get a stop codon before you should
Nonsense mediated decay
Inserting stop codon where they shouldn't be
Regulation of RNA turnover-- IRE-bps
In high iron, the mRNA that codes for transferrin is off. When you have low iron, you want to pump the iron in so the IREbps stabilize the stem loops and turn the transferrin on-->you get lots of transferrin and protect your little iron
What exon is removed through alternative splicing in CF?
exon 9
RNA splicing-Lariat
Just 5' of acceptor site is pyrimidine rich acceptorregion that forms lariat site, branch site a single A binding is sitting just upstream of the pyrimidine rich region and is where the lariat lands
What is the reason for alternate splicing?
The human genome is limited
Alternate RNA Splicing include or exclude certain exons?
Ex. alpha-tropomyosin, alpha-TM Exon Gene organization uses alternate splicing to produced different types of muscles fibers
Exonic splicing enhancers (ESEs)
Enhance recognition of splice site- can bind directly or indirectly
Intronic splicing enhancers (ISEs)
Enhance recognition of splice site- can bind directly or indirectly
Exonic splicing silencers (ESSs)
Silence recognition of splice site- can bind directly or indirectly
Intronic splicing silencers (ISSs)
Silence recognition of splice site- can bind directly or indirectly
Cryptic splice site
splice site that is not supposed to be there, causes competition between splice sites
Gain of function mutation
Creation of cryptic splice site
Loss of function mutation
A splice site is weakened or destroyed
In CF, regulation of splice site
A splice site in intron 8 regulates inclusion of exon 9, in CF you don't get exon 9 included
CF compound heterozygote, R117H and 7T
mild presentation of disease, congentical bilateral absence of vas deferans
CF compound heterozygote, R117H and 5T
mild CF, disease symptoms present
deltaF508
most severe mutation that is associated with CF
MAPT
codes for tau protein, chromosome 17, involved splicing for exon 2, 3, 10 mutations can disrupt the balance of isoforms and cause disease--> ALZHEIMERS
miRNA synthesis
Synthesized in the nucleus as double stranded RNA, forms hair pin loops in nucleus, Drosha suts the hair pin loops in the nucleus, Exportin 5 sends it out to the cytoplasm where Dicer cuts it into 20-30 bp fragments, transciptional cleavage, now the miRNA can recognize its homologous RNA on the RISC complex
Drosha
cuts the hairpin loops of Pri miRNA in the nucleus to turn it Pre-miRNA
Exportin 5
Exports Pre-miRNA into the cytoplasm
Dicer
chops up the Pre miRNA into 20-30 bp fragments
RISC
complex that the anti sense strand binds to-->blocks translation
Epigenetic
study of heritables changes in gene function that occur without a change in the sequence of DNA
CpG island
C and G rich area in the 5' regulatory region close to the promoter region of DNA that gets methylated-- YOU METHYLATE THE C (cytosine)
Location of LncRNAs
Located in the nucleus and then trafficked to the cytoplasm
Heterochromatin
Highly compacted DNase resistant DNA
When you acetylate histones which residue do you tag?
Lysine
When you methylate the histone, which residue do you tag?
Cytosine (the C's of the CpG islands)
Can drugs demethylate DNA?
yes-through drug reversal you can restore transcription
Rett Syndrome
MECP2--Methyl CpG binding protein--no males can have Rett Syndrome unless they have Klinefelters (XXY) because if you have only on mutant X you won't have any normal protein
What gets ADP ribosylated in Cholera?
G protein
Sumoylation, Small Ubiquitin like Modifier (SUMO)
ADP ribosylation
What genetic condition are likely to have epigenetic component?
Imprinted genes--prader willie and angelman (7 genes missing on chromosome 15, in normal the maternal allele is expressed, paternal allele is silenced-when the maternal allel is lost you get Angelman)
Example of secondar structure of RNA
Stem-loop and small subunit of rRNA
rRNA
structural RNA (80% of all processed DNA)
tRNA is the only RNA with non standard bases, what are they?
Inosine and pseudouridine
Are tRNA's aminoacylated?
Yes, it uses ATP to give a high energy bond to the AA which is transferred to the RNA
aminoacyl tRNA synthetase
What glues the AAs onto the tRNA. There is one for each amino acid
How many tRNAs do we need?
61
How many tRNAs do we have?
31
Wobble
Since we need 61 tRNAs and only have 31, we have to alternate position 3 with the inosine and pseudouridine
Prokaryotic Ribosome
30S and 50S subunits, total 70S
Eukaryotic Ribosome
40S and 60S subunits, total 80S
Svedburg coefficient
centrifugation coefficient used in antibiotics
Shine-Dalgardo Sequence
What prokaryotic DNA use to determine start sequence (like the eukaryotic Kozak sequence)
Ribosome scanning
Small ribosomal subunit scans (with tRNA attached) by ribosome scanning the mRNA 5' UTR to look for start AUG codon
What happens once tRNA find start sequence?
Important Initiation factors are recruited (EIF Ii) and then the 60S is recruited
What does every protein start with?
Methionine
A-site
donor tRNA-amino acid (amino acid)
P-site
tRNA growing peptide chain (peptide)
E-site
tRNA (exit)
Peptidyl transferase
anzyme that is responsible for elongating the polypeptide chain
Release factor
Once the stop codon comes into A site, RF Binds to the A site with the help of GTP
Ubiquitination
sends proteins to be destructed in the proteosome
Secondary structure of proteins
relies on hydrogen bonding interactions, defined by rotation restriction around phi and psi, alpha helix (interchain H bonding) and Beta sheets (interchain H bonding)
Ramachandran plot
used to calculate the islands stability of stability by minimizing steric hindrance
Tertiary structure
3-D structure, relies on the interactions between side chains (not the backbone)
Only covalent bonds in tertiary structure
Disulfide bonds, cysteine-cystine
Quaternary structure
Several multiple protein subunits, ex. human hemoglobin, heterotetramer (2 alpha and 2 beta)
Two sites of protein translation
ribosomes in the cytoplasm and ribosomes on the RER
Direction of protein translation, ribosomes move
from 5'-->3' (synthesis of protein from N terminus-->C terminus)
Signal recognition protein (SRP)
Signal sequence at the beginning of the protein signals SRP used
Chemical environment of the mitochondria
Oxidizing
Chemical environment of the cytoplasm
Reducing
ER tanslation
Signal recogniition sequence on the protein recognized by the SRP-->direct protein into the ER membrane--> enzyme signal peptidase suts off the signal sequence at the beginning of protein
Translation termination
Once translation is complete, the ribosomal subunits dissociate-->completed protein is sent to the Golgi-->trafficked out of the cell
Protein folding
goal is to get to the local minimum energy conformation-->decrease Gibb's free energy and increase entropy (of water molecules)
Disulfide bonds
Don't want to be in the cytoplasm, weaker thand C-C bonds-->mostly found in secretory proteins, lysosomal proteins and exoplasmic domains on the membrane proteins
Where do disulfide bond form in the protein?
In the hydrophobic interior region of the protein-->they have lower energy
Connotoxin
importnat for pain management-->uses disulfide bonds to hold peptide architectures together