Chromatin: DNA + histone proteins wrapped in a tightly compacted situation -DNA is negative; histones are positive
Mitochondrial genome has ... (3)
-circular molecules -multiple different copies -encodes 13 proteins
Euchromatin vs Heterochromatin
Euchromatin: light stain, loose DNA, allows xC
Heterochromatin: tight DNA, dark stain
Active Transport into nucleus
Importin binds molecule -taken into nucleus, importin comes off -importin binds ranGTP, taken back into cytosol -Ran-GAP cleaves complex; importin stays in cytosol
Active Transport out of nucleus
Exportin binds mRNA; binds RanGTP as well -complex exported to cytoplasm -Ran-GAP cleaves complex
on 3' end of DNA; prevents digestion by enzymes
-Telomerase add to telomeres
-different versions of the same gene
-one base change -can change AA, or may remain same
Insertion or deletion, can result in frame shift
If adenine get's depurinated ....
then you can get thiamine dimers
End-joining Mutations (2)
non-homologous: resulting from helix breakage and repair
homologous: resulting from sister chromatids acting as template for repair
Triplet Expansion: what is it, and what disease?
-CpG repeats cause incorrect adherence during meiosis
Non-disjunction: what is it, and what disease?
-separation in Anaphase 1 leads to uneven separation of chromosomes
-single base change, no AA change
-single base change, different AA creates
-single base change, stop codon (UGA, UAG, UAA) created
-result from in/dels
randomly insert selves into DNA
Exicision Nuclease (and associated disease)
Repairs DNA double strand breaks -Xeroderma Pigmentosa
One person can have different genes in different cells; -during development, certain gene mutations can be acquired at different times ... resulting in different cellular karyotypes in same person
DNA Replication (leading and lagging strand)
1) DNA Helicase opens DNA *topoisomerase 1 prevents supercoiling 2) Primase adds primer 3) DNApol-alpha starts adding dNTPs 4) DNApol-delta takes over
*leading strand; in the direction of opening; continuous addition *lagging strand; in opposite direction of opening; discontinous addition; RNAase needed to remove primer; DNApol-delta needed to fill in gaps; ligase needed to ligate fragments
DNA pol delta: special ability?
3' to 5' exonuclease ability
adds to lagging strand on 3' end of DNA (has RNA component that is uses as guide)
Cytarabine: drug mechanism
Good cancer treatment -inserts arabinose instead of ribose into cytosine nucleotides -prevents DNA replication
Cyclophosphamide: drug mechanism
-converted to phosphamide in liver -ALKYLATES DNA, blocking replication
Doxorubicin: drug mechanism
Cancer treatment -inhibits topoisomerase 2, so no religation of broken strands occurs
Imelestat: drug mechanism
binds RNA template on telomerase
-mismatch repair system messed up -leads to HNPCC
DNA helicase defect -Okizaki fragment joining impaired
RNA polymerases (differences from DNA pol)
-no exonuclease activity -no primer needed -more errors
Which RNA pols make tRNA, mRNA, and rRNA
1) TF2D binds DNA (helicase unwinds) 2) RNA pol 2 binds, get's p-lated, and rapid xC occurs
pre-mRNA Processing ... (3 things)
1) 5' methyl-guanosine cap 2) splicing by splicosome 3) 3' poly-A tial
1) Adenine at branch point cuts 5' splice site 2) Free OH at 5' splice site attacks 3' splice site intron 3) lariat shaped intron made!
-presence of cryptic splice site ... leads to incorrect splicing
Chromatin Remodeling Complexes (HATs vs HDACs)
HAT's; histone acyl transferase; transfer acyl group to lysine, making it less positive; less attraction between DNA and histone ... leads to -INCREASED xC!!!
HDAC's: histine deacetylases; remove acyl groups from histones -DECREASES xC!!!!
1) BH3 only protein triggered ... activates BAX 2) BAX creates channel into mitochondria 3) cytochrome C's excape 4) Create apoptosome 5) Increase caspase 9; this increases executioner caspaces (3,6,7)
Compare Total Size: Euk cytoplasm ribosome, mitochondrial ribosome, and bacteria
Cytoplasm: 80S Mito: 60S
Small subunit size differences
Cytoplasm: 40S (18S RNA) mito: 35S (19S RNA)
Bacteria: 30S (16S RNA)
Large subunit size differences
Cytoplasm: 60 S (25S RNA) Mito: 45S
Main function of small/large subunit
Small: binds RNAs (tRNA, mRNA)
Large: catalytic activity (peptide bond former)
Euk/ProK mRNA comparison
Euk mRNA is monocistronic (just one protein made from the strand)
ProK is polycistronic (multiple ORFs, multiple proteins from same strand)
How do tRNA's work?
-have 3 base anticodon at end -have amino acid linked at acceptor terminus
-has inosine; can pair with A, C, or U!!!
1) EIF2A (eukaryotic initiation factor) activated by binding GTP 2) EIF2A-GTP binds initiator tRNA-met to form TERNARY COMPLEX 3) Ternary complex binds small ribosomal subunit 4) mRNA binds to complex 5) Large subunit binds, EIF2A hydrolyzed to GDP; released; PREINITIATION COMPLEX COMPLETE
1) tRNA met bound to P-site of ribisome 2) 2nd AA-tRNA placed into A site; EF-1-GTP binds; gets hydrolyzed 3) Peptidyl bond formed 4) Ribosome moves one codon down 5) EF-2-GTP binds, gets hydrolized; A-site is empty 6) next AA comes in
1) One of the stop codons binds the A-site 2) eRF-GTP (release factor) pairs with stop codon 3) eRF-GTP gets hydrolized, peptide is released from P-site 4) Ribosome separates
binds small subunit; prevents initiation; wrong protein made
mistranslation of codons
blocks A-site (mimics tRNA)
prevents peptidyl-bond formation
RIP: ricin inactivating protein; removes adenine bases (acts as enzyme)
adds ribose from NAD to EF-2; inactivates EF-2
RIBOSYLATION OCCURS AT DIPTHAMIDE (A MODIFIED HISTIDINE)
1) proteins can bind 5'UTR, masking start codon
2) P-lation of eIF-2A can inactivate it
3) Use of IRES (internal ribosomal entry sites) -xL can start on uncapped mRNA
-bind ATP; take misfolded protein and loosen it up
-allow protein to refold itself correctly
Exported Proteins; Mechanism
1) emerging peptide on ribosome has signal sequence; xL stops 2) SRP (signal recognition particle) binds signal sequence 3) Complex binds a translocon via docking protein 4) Peptide continues xL into ER lumen 5) Signal peptidase (on lumen wall) cleaves SRP off of peptide
Unfolded Protein Response
1) Accumilation of unfolded proteins ... UPR 2) xL stopped; HSP's (chaperones) made 3) If inadequate, then APOPTOSIS
-specific to donor, acceptor, and linkage (O or N)
N-linked Glycosylation Mechanism
ADDED TO PROTEIN IN ER: b-1,4 linkages
1) Dolichol-P adds N-acetyl-glucosamine via UDP-transferase to ITSELF 2) 2nd NAGA added 3) Mannoses added 4) Compound flipped into ER 5) Dolichol-P donates glycoslation molecule to ASN on peptide
Two types of N-linked glycosylation
More modification occurs later in the golgi
1) High Mannose 2) complex (galactose and sialic acid added)
O-linked Glycoslation Mechanism
ADDED TO PROTEIN IN GOLGI
1) Glycosyltransferases add to fully folded proteins at serine or threonine residues in GOLGI
What are two types of O-linked peptides?
Proteoglycans of ECM and H-antigen on RBC
Congenital Defects in Glycoslation: 2 types
1) CDG-1; defective synthesis of lipid-linked oligosacharide precursor
2) CDG-2: defective trimming of disacharide chains
can create formyl-glycine
Hydrophibic entity additions
1) Myristic acid to N term 2) Palmitic Acid to cysteine 3) Prenylation of cysteine at C term
GPI anchor (hydrophobic)
inositol anchor added to C term
How does protein get localized to lysosome?
1) UDP-mannose x2 added to protein 2) Mannose falls off 3) P-lated protein goes to ribosome
How is protein moved to the mitochondria?
1) Presequence on peptide allocates peptide for mitochondria 2) HSP70 orients protein properly 3) Moved into mito through TOM and TIM 4) HSP-60 guides folding in matrix
CFTR-1 not glycated properly; gets degraded
surface mannose residues not P-lated; lysosomal proteins appear in cytoplasm and serum
-lysosomes appear dense
Protein Degradation: Lysosomal Mechanism
1) Autophagy: cytosol protein enters lysosome, taking some cytoplasm with it 2) Endocytic: clathrin pit create, clathrin vessicle fuses with lysosome; peptide degraded
Protein Degradation: Proteosome
1) Protein gets highly ubqn'ed 2) degraded by proteosome
Mechanism: C-term glycine on UBQ creates amide bond with lysine on peptide
Types of Ubiquination
1) Mono: regulate a protein (like, histones)
2) Poly: degradation
What determines how long a protein will survive in cells?
1) Misfolding and PEST shorten lifespan 2) Arg/Lys rich proteins; shorter lifespan 3) Ser/Met rich proteins; long lifespan
Differences in RNA Pol (from DNA Pol)
RNA pol has no exonuclease activity (no proofreading) RNA pol does not need primer More errors
Which RNA pols make mRNA, tRNA, and rRNA
mRNA: pol 2
rRNA: pol 1 (3)
tRNA: pol 3
What is concensus sequence?
set of promoters, enhancers upstream of start site; -most common nucleotides found at this area -includes TATA box
1) TF2D binds DNA 2) TF2H (helicase) attaches 3) DNA opened at A-T rich regions 4) RNA pol 2 binds (slow TC) 5) C-terminus of RNA-pol get's p-lated, other TF's fall off, 6) Elongation complex now rapidly does xC
*chromatin remodeling complex part of RNA pol complex
Typical Layout of Gene
_____enhancer____gene specific elem____CAAT or GC-rich _____TATABOX
-found in death-cap mushroom
Mechanism: 1) eat DC mushroom 2) increase in alpha-amanitin levels
-alpha-amanitin inhibits RNA pol II
inhibits RNA Pol 2 in Gram (+) bacteria -can induce cP450's in humans
pre-RNA processing (3 things)
OCCURS IN NUCLEUS
1) Capping 2) Splicing 3) Poly-adenylation
-methyl-guanosine added to 5' end -distinguishes mRNA; protects against degradation
RNA Splicing (3 steps)
Done by splicosomes (protein +snRNA)
1) Adenine at BRANCH POINTS cuts 5'splice site intron end 2) Free OH on exon attacks 3'splice site intron end 3) lariat intron made + mRNA
1) small poly-A tail (or GU/U rich tail) is on mRNA 3' 2) That part is cleaved, and replaced with 200 A's
RNA Nuclear Export
mRNA + exportin + CBC (cap binding complex): mRNA transported into the cytosol
Different introns can be removed; different proteins can be made from the same gene
BETA-THALESEMIA (2 forms)
-mutation of HbB gene
B-chain of Hb deformed
B(0) mutant: normal acceptor at intron 2 destroyed ... cryptic splice site : no b-chain made
B(+) mutant: new acceptor siteon intron 1 ... leads to no B-chain
What are 2 types of chromatin remodeling complexes?
HATs (upregulate xC) and HDACs (downregulate xC)
-acetylate lysine on histones; less (+) charge -less attraction between histone and DNA
-remove acetyl group from histones: more (+) charge: more attraction between histones and DNA
What can happen at N terminus of histone?
-acetylation, methylation, P-lation
What happens if histone is methylated?
-more HDAC's bind
3 classes of DNA binding proteins: types, actions?
Bind DNA, recruit HATs or HDACs
2) Zinc finger proteins
3) Leucine Zipper Motif
2 alpha helices connected by short AA
-side chain of AA recognizes DNA
alpha helix + zinc
-alpha helix binds DNA
Leucine Zipper Motifs
two alpha-helices form dimer
every 7th AA is leucine
LDL receptor gene
-codes for an integral membrane protein -Recognizes ApoB, E on LDL and VLDL
How is LDL receptor gene regulated?
In low cellular cholesterol conditions ...
1) Basal factors are always bound to gene: SP-1, CRSP 2) SREBP-1A binds to SRE ... increases HAT 3) This increases xC speed!
In normal cellular cholesterol conditions ...
1) SP-1and CRSP bound to G-C rich regions; slow xC
homologous: during division; sister chromatid can guide reassembly of nucleotides
Triplet Expansion Mutations: what disease?
-due to constant repeats, leading to strand slippage -can result in loops structure (meiosis)
If loops kept, DNA gained If loops lost, DNA lost
Found in people with Huntington's
Occurs in Anaphase 1: uneven separation of chromosomes
-found in Down's Syndrome
-Single base change -No AA change
-Single base change -Different AA created
-single base change -early stop codon created (UAA, UGA, UAG)
Deletions or Additions -can result in different AA's, stop codons, etc
Genes moved around, inserted randomly (usually into introns)
How do DNA double strand brakes arise? How do you fix?
Through ionizing radiation (X-ray)
1) Dimer recognized; helicase binds DNA 2) Excision Nuclease cleaves DNA (30bp) 3) DNA Pol and Ligase fill in the gap REMEMBER, ONLY ONE STRAND OF DNA IS REMOVED
-usually leads to loss of DNA
Extreme sensitivity to sunlight, pigmentation changes, skin cancers
-due to lack of excision nuclease
Mosaicism (and example)
One person, but they have different genes in different parts of their body;
Trisomy 21: during embyology, one cell gives rise to daughter cell with no copies of 21, and itself has 3; this is one way of getting trisomy; other ways exist
Origins of Replication: what occurs?
1) DNA helicase unwinds DNA *topoisomerase I cuts one strand, P-lates base to prevent supercoiling 2) Primase creates primer 3) DNA pol-alpha beings adding NTP 4) Sliding clamp added, DNA pol-delta comes in and continues adding NTPs
Moves in the direction of opening replication fork
Okisaki fragments made; RNAase removes primers -DnA pol delta fills in gaps -DNA ligase ligates DNA gaps DISCONTINUOUS REPLICATION
DNA Pol Delta: what's special?
has 3' to 5' exonuclease activity (proofreading)
Strand-directed mismatch repair
New DNA might have nicks and gaps due to errors; -repair enzymes can fix this using the original strand as template
-linear molecules, gets shorter every time you divide -GGGTTA repeats -replicated by TELOMERASE
1) Sees incomplete lagging strand in telomere region 2) uses RNA template to complete lagging strand
blocks DNA replication -inserts arabinose instead of ribose into cystidine
converted to phosphamide in liver -alkylates DNA, resulting in crosslinking -blocks replication
-cancer treatment -prevents religation of broken strands by blocking Topoisomerase 2
bind RNA template on telomerase
reactivate telomerase in CD4+ cells
-Hereditary non-polyposis colorectal carcinoma -patients have 80% chance of getting colorectal cancer
MISMATCH REPAIR SYSTEM MESSED UP
-dwarfism, photosensitivity, big nose
DNA helicase defect -OKIZAKI FRAGMENT JOINING IMPAIRED
What happens if methylated cytosine gets converted to thymine?
-Then DNA repair enzyme will have hard time distinguishing which is correct base: 50% chance
If thymine dimer occurs ...
then you have p53 activation, increase POMC, and increase melanin
Balanced: no genes lost
Unbalanced: genes and DNA lost
single nucleotide polymorphism: 1 base change in DNA; account for most of the differences in people
Main cause of aneuploidy?
Maternal Age: Sperm eggs remain in arrest for 45 years ... older the mother, the higher the chances of chromsomes separating badly
Which carbon atoms of the ribose are linked to phosphates?
3' O-P link and 5' C-O-P link
Where are bases attached to ribose?
What's the diff between ribose and deoxyribose?
Ribose: OH at 2' position
Deoxyribose: H at 2' position
How do you calculate percentage of AA's?
What are the B, A, and Z forms of DNA?
B-form: right handed helix, 10.4 base pairs per turn (most DNA)
A-form: right handed, 11 bp per turn; found in DNA-RNA hybrid or RNA ds
Z-form: left-handed helix stabilized by methylated cytidine bases
3 DNA stabilizing forces
1) H-bonding between bases 2) Hydrophobic/stacking interactions of base-pairs 3) Interaction of polyanionic backbone with cations
What is supercoiling?
Condition DNA is in when it is not getting xC
DNA opens up helix, unwinds (negative supercoiling) causes positive supercoiling upstream and downstream
DNA Topoisomerase I
cuts one strand of DNA double helix, allows other to rotate, and then gets religated
DNA Topoisomerase II
Manipulates two in-tact double helices; allows other helix to pass through; later, religates
How many genes, nucleotides, chromosomes in human genome?
Nucleotides: 3.2 x 10(9th)
Categories of non-coding DNA
tRNA, siRNA, miRNA, rRNA:
enhancers, promoters, etc etc
Single Copy DNA vs Multiple Copy DNA
Single Copy: make proteins, make RNA, spacers (introns)
Multiple Copy: Satelite DNA, and Dispersed Repetitive DNA (LINES and SINES)
What's special about dispersed repetitive DNA?
-LINES and SINES can cut and paste themselves in random parts of the genome
DNA sequence that is transcribed to make to eventually lead to a protein or an RNA molecule
Exons: expressed, can create protein
Introns: cut out by splicosome; can serve regulatory purpose
What has most introns? Least?
Dystrophin gene: 99% Introns
Histone H4: no introns
What are gene families? How do they arise?
Genes that have similar AA sequences, but yield different protein; Arise from unequal cross-over events in meiosis
A gene that once coded for a protein, but is now silenced by a better version of the gene; -a duplicate gene
1/3 DNA, 2/3 proteins (histones)
a) DNA + protein= histone b) histones wrap around each other = solenoids c) solenoid gets looped = 300 nm looped domains
What are the structures of nucleosome? Charge of DNA and histones?
DNA + H1 and 2(H2A, H2B, H3, H4) -DNA: - charged -histones: + charged AA's
Function of centromere
Where mitotic spindle attaches ... during cell division
1) allows for separate areas for xC and xL 2) controls access of regulatory proteins, viruses, enzymes etc
Light stain in micrograph -loose DNA; allows xC
Dark stain in micrograph -tight DNA: no xC
Big, large dark stain in micrograph -xC of rRNA
Active transport mechanism into nucleus (TFs)
1) Importin binds protein outside in cytosol: shuttled into Nucleus 2) Ran-GTP binds importin; protein released into nucleus; 3) Ran-GTP-Importin transported to cytosol 4) RanGTP (Ran-GAP) RanGDP, and importin goes free
Active transport out of nucleus (RNA, proteins)
1) exportin binds RNA or protein in nucleus 2) Ran-GTP binds exportin-RNA complex 3) Exported to cytoplasm 4) Ran-GTP complex (RanGAP) Ran GDP + RNA + exportin
inhibits topisomerase II: cancer treatment
inhibit bacterial topoisomerase II
inhibit bacterial topoisomerase II
What is an intercolating agent?
wedges itself into DNa; opens space; -EthBromide is one of these
can cause alternate splicing in same gene
B-globulin gene: E to V change at AA#6: HbS creates polymers
Treatment: hydroxy-urea (reactivates HbF)
A chromosome map: shows all chromosomes
Fluorescent in-situ hybridization: -allows individual chromosomes to be stained
3-strand DNA structure: prevent digestion by enzymes; on ends of DNA