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DNA Structure
Structures + #ing of
nucleic acids
Proofreading
5 principal bases in
Base Excision repair
Nucleotide structure in DNA
Mismatch repair
Transcription
Complementary base pairs that exist in DNA, + #
Holoenzyme: core + sigma factor
of H bonds.
Basal promoters: euk vs prok?
DNA Synthesis
1. DNA denatured at replication origin.
RNA poll, II, III : function, amanitin
sensitivity
Terminators
How many on in eukaryotes vs.
prokaryotes?
Gene Regulaton
Operon, polycistronic message
What’s in replisome in prok,? euk?
Function of each one?
2. Primer Synthesis DNA primase -- de
novo synthesis Leading vs lagging
Introns, splicing, spliceosomes
Capping
Lac operon
Translation
strand
tRNA: intrastrand base pairing
3. Chain Synthesis
aminoacyl tRNA synthetases: function. how
many?
Order of events
Function, processivity, +
exonuclease activity of DNA poll,
DNA ~Ol III, DNA poi alpha, delta,
+ epison
A site -
P site
Peptide bond formation
Energy requirements
DNA Synthesis Fidelity
Wobble Rules
What’s the result + related disease for:
frameshift.and nonsense mutation
Antibiotics
Disease
Type 0
Enzyme Affected
Glycogen Synthase
Type I (Von Guirke’s) Glucose-6-P
Phosphatase
Type IV (Anderson’s) Lack of Branching
Enzyme
Type V (McArdle’s) Muscle
Phosphorylase
MODY
Glucokinase
(maturity-onset
diabetes)
EFT or EFT:CoQ
reductase
defieciency
Carnitine Deficiency
EFT or EFT:CoQ
reductase
Carnitine
.
CAT-I Deficiency
Carnitine
Acyl-Transferase I
MCAD Deficiency
Medium Chain
Acyl-C0A
Dehydrogenase
G-6-P
Dehydrogenase
defieciency
Beriberi
G-6-P
Dehydrogenase
Wernicke-Korsakoff
Transketolase:
nutritional shortage of
thiamine
Transketolase:
thiamine shortage.
Symptoms
Hypoglycemia
Hypoglycemia
Enlarged Liver
Gout
Cirrhosis of Liver due
to insoluble glycogen
Weakness after
exercise
Hyperglycemia;
higher glucose %
needed for insulin
release.
Death: Inability to
harness energy from
FADH2
Weakness and
muscle cramping;
elevated F.A. levels
in blood wlo elevated
ketones or
dicarboxylic acids
High F.A. in plasma,
no ketone bodies.
Hypoglycemic after
fasting.
Hypoglycemia; high
medium chain in
urine.
Low energy levels
due to inability to fully
oxidize F.A.
Anemia during drug
treatment.
Fluid retention, pain
and paralysis.
Neuronal
dysfunction, found
mostly in alcoholics
Type I Diabetes
Insulin
Hyperglycemia
Type II Diabetes
Insulin Receptor (non Hyperglycemia
functional) ~
Acanthosis nigrecans Insulin Receptor (Ab Hyperglycemia
against receptor
created)
Treatments
Small frequent
meals.
Liver Transplant
Carnitine in diet
High Medium Chain
Fatty Acids in diet.
Frequent, small
meals
Thiamine in diet.
Reduce alcohol
increase, thiamine in
diet.
Injection of insulin
Diseases
Eukaryotes
Prokaryotes
“Simple” RNA polymerase enzyme (subunit
composition o~3~’ao) transcribes all genes
Complex RNA polymerases 3 species poll
5.8S, 18S, 28S rRNA pol II protein-encoding
genes (mRNA) p01 III tRNAs, 5S RNA
-
-
-
-
a protein recognizes promoter (-35 and -10
consensus) in context of holoenzyme
a factor leaves “holoenzyme” after approx. 10
phosphodiester bonds synthesized elongating
-
polymerase is the “core” enzyme
No processing of mRNA
•
no capping
•
no splicing
•
no polyadenylation
Termination through either p-dependent or
independent pathways
polil promoter typified by TATA box at
-30; recognized by TFIID factor pol II binds
subsequently along with other TF’s
-
Most TFII factors remain at promoter as
polymerase II commences elongation, BUT
TFIIH accompanies polymerase (contains
helicase activity)
Processing of mRNA
•
capping at 5’ end
•
•
splicing of introns
termination through polyadenylation
No comparable mechanism (see polyadenylation)
Transcription (and processing to mature mRNA)
occur in nucleus transport to cytoplasm
required for translation
-
No nucleus, therefore coupled transcription
and translation
Monocistronic organization of genes variant
proteins can be produced through alternative
-
Genes frequently organized in polycistronic
arrays - single mRNA can produce multiple
proteins, often have related functions, e.g.
lac operon
Gene expression regulated primarily through
transcriptional initiation control
Transcriptional control mediated through
balance of activating and repressing
activities in “transcription factors” that bind
to DNA regulatory elements
splicing
Gene expression regulated at many points,
e.g., transcription control, mRNA stability,
alternative splicing, mRNA transport
Prokaryotic paradigm of combinatorial
control of levels of gene transcription
through transcription factors appears to
hold. DNA regulatory elements often much
more complex,
Comparin2 transcription and gene re~u1ation in prokarvotes an(1 euKaryotes
e.g., proximal promoter elements, enhancers,
locus control regions.
Comparin2 replication in prokaryotes and eukaryotes
Eukaryotes
Prokaryotes
Circular chromosomes have single origin of
replication
‘-1000
Linear chromosomes have multiple origins
of replication
nucleotide/sec
Slower rate of replication -~100
nucleotide/sec
DNA polymerase III is the main synthetic
enzyme works on both leading and
lagging strands
DNA polymerase y/~ is/are the main
synthetic enzymes work on both leading
and lagging strands
DNA primase synthesizes RNA primer
primer ‘~l0 nucleotides long
DNA primase/pol a synthesizes mixed
primer composed of short RNA chain
followed by short DNA chain primer —‘20
nucleotides in length
Replication extremely fast
-
-
-
-
-
-
-
DNA polymerase I eliminates RNA
primer through its 5’-->3’ exonuclease
activity, synthesizes DNA in its place
DNA polymerase I also important in base and
nucleotide excision repair
RNA primer eliminated by a distinct 5’-->3’
exonuclease protein DNA resynthesized by
polymerase ‘y/ö
-
DNA polymerase used for most repair
Comparin2 transcription and gene re~u1ation in prokarvotes an(1 euKaryotes
RNA synthesis
DNA synthesis
Multiprotein enzyme complex loads at origin of
replication
Multiprotein enzyme complex loads at promoter (1
species in prokaryotes, 3 species in eukaryotes
No primer required
Primer required/DNA primase
Only rNTP’s required
All dNTP’s and rNTP’s required
Mg2-dependent process
Mg2~-dependent process
Only one strand of DNA acts as template
Both strands of DNA act as template, i.e., is
bi-directional
-
selection depends on promoter sequence
Synthesis is 5’-->3
Synthesis is 5’-->3’
Template read in 3’-->5’ direction
Template read in 3’-->5’ direction
Only continuous synthesis
Continuous or discontinuous synthesis dependent on
strand
No proofreading
Proofreading/3’-->5’ exonuclease
Not relevant to process
Okazaki fragments/DNA ligase
differ between
prokaryotes and eukaryotes
Stop signals of various kinds
Stop signals not defmed
-
Always uses Watson-Crick base pairs