Chromatin structure
beads on a string"
Neg charged DNA wraps twice around histone octamer (positively charged histones - Arg, Lys)
The only histone not in nucleosome core
H1
Heterochromatin
HeteroChromatin - Highly Condensed
transcriptionally inactive, sterically inaccessible
Euchromatin
Eu = true "truly transcribed"
Less condensed, transcriptionally active, sterically accessible
Purines - names & # rings
A, G
2 rings
Guanine has a ketone
Pyrimidines - names & # rings
CUT
1 ring
THYmine has a meTHYl
Uracil vs Thiamine
Uracil in RNA
Thymine in DNA
Deamination of cytosine makes
Uracil
CG bonds vs AT bonds in DNA
CG bonds - 3 H bonds -> stronger, higher melting temp
vs. AT bonds - 2 H bonds
Pyrimidine Synthesis -
rdE, ingredients
Carbamoyl phosphate synthetase II
CO2
Glutamine
Aspartate
Purine Synthesis -
rdE, ingredients
Glutamine-PRPP amidotransferase
Glycine
Aspartate
Glutamine
CO2
THF
Amino acids necessary for purine synthesis
GAG
Glycine
Aspartate
Glutamine
Nucleosides vs. Nucleotides
base + ribose
base + ribose + phosphate; linked by 3'-5' phosphodiester bond
Precursor for purines
IMP
Pyrimidines precursor
Orotate (aka, orotic acid)
with PRPP added later
Function of ribonucleotide reductase
Convert ribonucleotides to deoxyribonucleotides
(ribonucleotides are synthesized first)
Failure to thrive, megaloblastic anemia (that doesn't improve with B12 or folate), increased orotic acid in urine, no hyperammonemia
Orotic Aciduria
AR defect/deficiency in UMP synthase
(Orotate phosphoribosyltransferase + OMP decarboxylase)
--> build up of orotic acid
Tx - uridine rich diet (provides what reaction can't synthesize)
Hyperuricemia, gout, aggression, self-mutilation (lip-biting), choreoathetosis
Lesch-Nyhan Syndrome
MECH - Deficiency in HGPRT ->-> excess uric acid production
Tx - allopurinol
Severe recurrent infections, chronic diarrhea, failure to thrive.
Xray - absence of 'thymic shadow'
Severe Combined Immunodeficiency (SCID)
MECH - deficiency of Adenosine Deaminase (ADA) -> excess ATP & dATP -> inc neg feedback -> inhibits ribonucleotide reductase -> prevents DNA synthesis -> decreased lymphocyte count.
Transition v. Transversion
TransItion - (Identical)
Substituting purine for purine, pyrimidine for pyrimidine
TransVersion - conVersion
Substituting purine for pyrimidine and vice versa
Exceptions to universal genetic code
Genetic code is conserved throughout evolution.
Exceptions:
mitochondria, arhaebacteria, Mycoplasma, some yeasts
What does UV do to DNA?
UV light pairs thymine to thymine -> thymine dimers on SAME strand of DNA
Mutations in DNA, from most harmful to least
Nonsense (stop codon) >
Missense (dif aa) >
Silent (same aa)
Frameshift
Helicase
unwinds DNA template at replication fork
single-stranded binding proteins
prevent strands from reannealing
DNA topisomerases -
Function
Drugs that inhibit
Fxn: create a nick in the helix to relieve supercoils created during replication
Fluoroquinolones - inhibit prokaryotic topoisomerase (DNA gyrase)
Etoposide - inhibit eukaryotic topoisomerase
DNA polymerase III
Function
Prokaryotic only.
5'-->3' synthesis
3'-->5' exonuclease proofreading
DNA pol I
Prokaryotic only.
Degrades RNA primer (5'->3' exonuclease)
Fills in gap with DNA
DNA ligase
Seals strands of DNA together
DNA Repair -
Single strand
Double strand
Single Strand:
Nucleotide excision repair (removes damaged oligonucleotide)
Base excision repair (removes damaged base)
Mismatch repair (corrects unmethylated mismatched bases)
Double strand:
Nonhomologous end joining (brings together 2 ends of DNA, no ho
What type of repair mechanism is mutated in:
Xeroderma Pigmentosum?
HNPCC?
XP - nucleotide excision repair
HNPCC - mismatch repair (MSH-1, -2)
Direction of replication, transcription, translation
Replication: 5' --> 3'
Transcription: 5' --> 3'
Translation: N --> C
What bears the triphosphate (energy source) for bond in DNA synthesis?
5' of incoming nucleotide
What needs to be free for next nucleotide to be added in DNA synthesis?
3' OH of existing DNA chain
A final protein has one less amino acid than was coded for in the mRNA.
Which amino acid was removed?
Methionine. AUG.
Can be removed before translation is finished.
Operon includes
Promoter region (upstream - TATA, CAAT boxes)
+
Structural coding genes
+
Regulatory genes (enhancers, silencers)
Promoter
aka "transcription initiator"
Site where RNA pol II and transcription factors bind
Upstream CAAT (-75) and TATA (-25) boxes
Mutation -> dramatic decrease in gene expression
Transcription factors
Motifs
Proteins that (must) bind DNA of promoter region
Motifs - leucine zipper, helix-loop-helix, helix-turn-helix, zinc finger
Regulators
Enhancer:
Stretch of DNA that binds transcription factors -> inc rate of transcription
Silencer:
Site where repressors bind -> dec rate of transcription
Both - may be located close to, far from, or in (intron) the gene whose expression it regulates
Eukaryotic RNA pol's
Rampant, Massive, Tiny"
RNA pol I -> rRNA (most abundant)
RNA pol II -> mRNA (longest)
RNA pol III -> tRNA (smallest)
Special function of RNA pol II
Opens DNA at promoter site
What inhibits RNA pol II?
alpha-amantin (found in death cap mushrooms)
Causes liver failure
Prokaryotic RNA pol:
Fxn?
Drug that inhibits?
There's only 1 - it makes all 3 kinds of RNA.
Inhibited by rifampin
What is the template for the AAUAAA polyadenylation signal that is added to mRNA before leaving the nucleus?
There is none!
Poly-A polymerase does NOT require a template
What disease provides a good example of different exons being combined to form different proteins?
Beta-thalassemia
Mutations can be due to alternative splicing.
Where and how is an aa bound to tRNA?
Covalently to 3' end
What 2 things are responsible for accuracy of amino acid selection during translation?
Aminoacyl-tRNA synthetase
(scrutinizes anticodon-aa)
+
Binding of charged tRNA to codon
(aa-tRNA bond provides energy for formation of peptide bond)
What is tRNA "wobble"?
Codons that differ in 3rd (a.k.a., "wobble") position may code for same aa (d/t degeneracy of genetic code)
Accurate base pairing is required only in the first 2 nucleotide positions of mRNA codon.
3 Steps of Protein Synthesis (Translation)
1. Initiation (activated by GTP hydrolysis)
2. Elongation (3 sites = APE)
3. Termination (simple hydrolysis)
Where are ribosomes synthesized?
Nucleus, then transported to cytosol
3 Steps of Elongation
1. Aminoacyl-tRNA binds A site
2. 23S/Ribosomal rRNA/ribozyme in 50S catalyzes peptide bond formation, transfers growing polypeptide to amino acid in A site
3. Translocation: ribosome advances 3 nucleotides toward 3' end of mRNA, moving peptidyl RNA to P
What does the translocation step in proteins synthesis require?
Prokaryotes - Elongation Factor G (EF-G)
Eukaryotes - Elongation Factor 2 (EF-2)
What is the total energy expenditure of translation?
4 high energy phosphoanhydride bonds
(2 ATP, 2 GTP)
What are the 3 mechanisms of proteolysis?
1. Proteasomal degradation (ubiquitin tag)
2. Lysosomal degradation
3. Calcium-dependent enzymes
(why hypercalcemia causes damage?)
What bugs produce a toxin that alters protein synthesis?
Corynebacterium diphtheriae
Diphtheriae toxin inactivates EF-2 --> alters/inhibits protein synthesis --> pharyingitis and pseudomembrane in the throat
Eukaryotic DNA pol that:
replicates lagging strand, synthesizes RNA primer?
DNA pol alpha
Eukaryotic DNA pol that:
repairs DNA?
DNA pol beta, epsilon
Eukaryotic DNA pol that:
replicates mitochondrial DNA?
DNA pol gamma
Eukaryotic DNA pol that:
replicates leading strand DNA
DNA pol delta
What E catalyzes peptide bond formation during protein synthesis?
peptidyltransferase
inhibited by streptogramins and chloramphenicol