1
Q
Chromatin
A
DNA exsits in condenssed, chromatin form in order to fit into the nucleus
2
Q
Chromatin structure
A
- ‘beads on a string’
- (-) charged DNA wraps around (+) charged histone twice to form a nucleosome = ‘bead’
- Histones= rich in AA lysine and arginine
- H1 binds nucleosome and ‘linker DNA’ thereby stabilizing chromatin fiber
- have nucleosome core ( H2A, H2B, H3, H4 x2) and the core are connected by **H1 ( **only histone not in the core)
- mitosis = DNA condenses to form chromos
3
Q
Heterochromatin
A
Condensed, transcriptionally inactive, sterically inaccessible
HeteroChromatin= Highly Condensed
4
Q
Euchromatin
A
Eu = true –> truly transcirbed
Less condensed, transcriptionally active, sterically **accessible **
5
Q
DNA methylation
A
template strand cytosine and adenine methylated in DNA replication
allows mismatch repair enzymes to distinguish between old and new strands in prokaryotes
DNA methylation at **CpG islands **represses transcription
‘CpG Methylation makes Mute’
6
Q
Histone methylation
A
reversible repression of DNA transcription BUT can activate it sometimes
7
Q
Histone acetylation
A
relaxes DNA coiling and allows for transcription
8
Q
Nucleotides
A
base + deoxyribose+ phosphate
linked by **3’-5’ phosphodiester bone **
are Purines and Pyrimidines
9
Q
purines
A
Adenine
Guanine
2 rings
10
Q
Pyrmidines
A
Cytosine, Thymine and Uracil
1 ring
uracile ONLY in RNA and thymine in DNA
deamination of cytosine makes uracil
11
Q
Bonds between nucleotides
A
A:T = 2
C:G = 3
12
Q
Purine synthesis: de novo
A
start with sugar + phosphate ( PRPP)
Add base
13
Q
Things necessary for purine synthesis
A
GAG: **glutamine, aspartate and glycine **
and THF
14
Q
Pyrimidine synthesis: de novo
A
**requires aspartate **
make temporary base ( orotic acid)
- add sugar + phosphate ( PRPP)
- Modify base
- rubonucleotides are synthesizes first and are converted to deoxyribonucleotides by **ribonucleotide reductase **
- Carbamoyl phosphate is involved in 2 metabolic pathways: **de novo pyrimidine synthesis and urea cycle **
- various anti-neoplastic and antibiotic drugs function by interfering with nucleotide synthesis
15
Q
Drugs affecting nucleotide synthesis
A
- Leflunomide - inhibits dihydrorotate dehydrogenase
- Mycophenolate and ribavirin = inhibit **IMP dehydrogenase **
- Hydroxyurea = inhibits **ribonucletide reductase **
- 6-mercaptopurin and its’ prodrug azathioprine - inhibit **de novo purine sythesis **
- 5-fluorouracil ( %-FU) - inhibit thymidylate synthase ( decreases dTMP)
- Methotrexate (MTX), trimethoprim( TMP) and pyrimethamine - inhibits DHFR ( dihydrofolate reductase) –> decreases dTMP in humans, bacteria and protozoa
16
Q
Purine salvage deficiencies
A
adenosine deaminase deficiency
Lesch-Nyhan syndrome
17
Q
Adenosine deaminase deficiency
p.68 for drawing
A
excess ATP and dATP imbalances nucleotide pool via feedback inhibitionof ribonucleotide reductase –> prevents DNA synthesis and thus decrease lymphocyte count
is one of the major causes of **autosomal recessive SCID **
18
Q
Lesch-Nyhan syndrome
p.68 for drawing
A
defective purine salvage due to absent **HGPRT ( **converts hypoxanthine –> IMP and guanine –> GMP)
results in excess uric acid production and **de novo purine synthesis **
**X-linked recessive **
19
Q
Lesch-Nyhan syndrome findings
A
HGPRT:
Hyperuricemia
Gout
Pissed off ( aggression and self-mutilation)
Retardation ( intellectual disability)
DysTonia
20
Q
Lesch-Nyhan syndrome Tx
A
allopurinol
febuxostat ( 2nd-line)
21
Q
unambiguous genetic code
A
each codon specifies only 1 AA
22
Q
degenerate/ redundant genetic code
A
most AA are coded by multiple codons
Exceptions: methionine( AUG) and tryptophan(UGG) endcoded by only 1 codon
23
Q
Severity of mutations
A
frameshift > nonsense > missense>> silent
- For silent, missense and nonsense mutations:
- Transition: purine to purine OR pyrimidine to pyrimidine
- Transversion: purine to pyrimdine and vice versa
24
Q
Silent Mutation
A
nucleotide substitution but still codes for the same AA; often a base change in 3rd position of codon - tRNA wobble
25
Missense Mutation
nucleotide substitution resulting in changed AA
called **conservative** if AA is similar in chem structure
ex: sickle cell
26
Nonsense mutation
nucleotide substitution resulting in early stop codon
27
Frameshift mutation
deletion or insertion of a number of nucleotides not divisible by 3 resuling in misreading of all the nucleotides downstream
usually results in a truncated or non-functional protein
ex: duschenne muscular dystrophy
28
Types of single strand DNA repair
Nucleotide excision repair
Base excision repair
Mismatch repair
29
Nucleotide excision repair
type of DNA repair
specific endonucleases release the oligonucleotide containing damaged bases
DNA polymerase and ligase fill and reseal the gap
mech to repair **bulky, helix-distorting lesions **
defective in **xeroderma pigmentosum** - inability to repair **pyrimidine** dimers due to UV exposure
30
Base excision repair
type of DNA repair
base-specific **glycosylases** recognize altered base and create **AP site** ( apurinic/apyrimidinic)
one or more nucleotides are remved by **Ap-endonucleases** which cleave the **5' end**
lyase cleaves the **3' end**
**DNA-polymerase-Beta** fills the gaps
DNA ligase seals it
important in repair of spotaneous/toxic deamination
31
Mismatch repair
Newly synthesized strand recognized
mismatched nucleotides are removed
gap is refilled and resealed
defective in hereditary non-polyposis colorectal cancer (HNPCC)
32
Doble stranded DNA repair
**non-homologous end joining **
brings together 2 ends of DNA fragments to repair DB-stranded breaks
**no** requirement for homology
mutated in **ataxia taelangiectasia **
33
5' --\> 3'
direction in which DNA and RNA are **synthesized **
5' end = incoming nucleotides bear the triphosphate ( energy source for bond)
triphosphate bond is the target for the 3' hydroxyl attack
drugs blocking DNA synthesis often have a modified 3-OH grp to prevent further DNA elongation - chain termination
34
5' --\> 3'
direction in which RNA is read
35
N --\> C
direction of protein synthesis
36
mRNA start codon
**AUG or GUG**( rarely) - AUG inAUGrates DNA synthesis
Eukaryotes - AUG codes for methionine which may be removed before trnalstion is complee
Prokaryotes - AUG codes for formylmethionin ( f-met)
37
mRNA stop codons
UGA - U Go Away
UAA- U Are Away
UAG - U Are Gone
38
elements involved in regulation of gene expression on a DNA strand
Promoter
Enhance
Silencer
39
DNA strand - promoter
site where RNA polymerase and other multiple transcription factors bind to DNA **upstream** from gene locus
is an **AT-rich** sequence with **TATA** and **CAAT** boxes
mutation results in dramatic decrease in gene transcription
40
DNA strand - enhancer
stretch of DNA that alters gene expression by binding transcription factors
can be locate far, close or within ( intron) of the gene whose expression it reguates
41
DNA strand - silencer
site where negative regulators ( repressor) bind
can be locate far, close or within ( intron) of the gene whose expression it reguates
42
RNA polymerase - eukaryotes
RNA polymerase I, II, III
numbered in order of when their products are used in protein synthesis
have **no **proofreading function but **can **initiate chains
43
RNA polymerase I
makes rRNA
most numerous
r = rampant
44
RNA polymerase II
mRNA
is the largest RNA
opens DNA at promoter site
**alpha-amantin** - found in death cap muschrooms **inhibits** RNA polym II and causes **severe hypotoxicity** if ingested
45
RNA polymerase - prokaryotes
onne RNA polymerase ( multiunit) - makes all 3 kinds of RNA
46
RNA processing
initial transcript = hnRNA ( heterogeneous RNA)
hnRNA modified to become hnRNA via these processes:
Capping on 5' end ( addition of 7-mehylguanosin cap
polyadenylation of 3' end ( approx 200As)
splicing out of introns
capped tailed and spliced transcript called = mRNA and is transported out of nucleus into cytsol to be translated
47
Polyadenylation
carried out by Poly-A polyemrase which does NOT require a template
polyadenylation signal = AAUAAA
48
mRNA quality control
occurs at cytoplasmic P-bodies which contain exinucleases, decapping enzymes and microRNAs
mRNAs may be stored here for future translation
49
pre-mRNA splicing
primary transcript combines with **small nuclear ribonuclearproteins ( snRNPs)** and other proteins to form spliceosome
**lariat-shaped( looped)** intermediate is generate
lariat is released precisely to remove intron and join 2 exons
auto-Abs to **spliceosomal snRNPs ( anti-Smith Abs) are highly specific for SLE**
Anti-U1 RNP Abs are highly assoc with mixed CT disease
50
tRNA - structure
clover leaf form
**anticodon** is at the **3' aminoacyl end**
ALL tRNA, eukaryotic and prokaryotic have **CCA** at the **3'** end with a high percentage of chemically modified bases
the AA covalently binds 3' end of tRNA
**T-arm** = contains _thymnie, pseudouridine, cytosine_ sequence necessary for tRNA-ribosome binding
**D-arm** = contains _dihydrouracil_ residues necessary for tRNA recognition by the correct **aminoacetyl-synthetase **
51
acceptor stem of tRNA
3' CCA is the amino acid accpetor site
52
aminoacyl-tRNA synthetase: charging
1 per amino acid; matchmaker; uses ATP
scrutinizes AA before **and **after it binds to tRNA
if incorrect, the bond is **hydrolyzed**
the AA-tRNA bond has energy for formation of a **peptide bond **
a mischarged tRNA reads usual codon but inserts wrong AA
aminoacyl-tRNA synthetase and binding of charged tRNA to the codon are responsible for accuracy of AA selection
53
IF2
initiation factor for tRNA
54
tRNA wobble
accurate base pairing required only in the first 2 nucleotide positions of an mRNA codon so codons iddfering the 3rd, 'wobble' position may code for the same AA/tRNA as a results of the **degeneracy **of genetic code
55
three steps of protein synthesis
initiation
elongation
termination
56
initiation of protein synthesis
initiates by GTP hydrolysis
inititation factors ( eukaryotic IFs) help assemble 40S ribosomal subunit with the initiator tRNA and are rleased when mRNA and the ribosomal 60S unit assemble with the complex
57
Ribosomal subunits
**E**ukaryotic:40S + 60S ( **E**ven)
Pr**o**karyotic: 30S + 50S ( **O**dd)
58
ATP and GTP in protein synthesis
**A**TP - tRNA **A**citvation ( charging)
**GT**P - tRNA **G**ripping and **g**oing places ( translocation)
59
Elongation: Protein synthesis
1. aminoacyl-tRNA binds to A site ( **except for initiator methionine) **
2. rRNA ( 'ribozyme') catalyzes peptide bond formation, transfers growing polypeptide to AA in the A site
3. ribosome advances 3 nucleotides towards 3' end of mRNA, moving peptidyl tRNA to P-site ( translocation)
think APE
* **A** site = incoming **a**minoacyl-tRNA
* **P** site = accomodates growing **p**eptide
* **E**-site = holds **E**mpty tRNA as it Exits
60
Termination: protein synthesis
stop codon is reconized by release factor, and complete polypeptide released from ribsome
61
types of pottranslational modifications
trimming
covalent alterations
62
trimming
a kind of **post-**transaltional modification
removal of N or C-terminal propeptides from zymogen to generate mature protein
63
Covalent alterations
a kind of post-transaltional modification
phosphorlation/glycosylation/hydroxylation/methylation/acetylation/uniquitination
64
Chaperone protein
intracellular protein involved in facilitating or maintaining protein folding
in yeast, some are heat shock prteins expressed at high temps to prevent prtein denaturing/misfoldings
65
commaless, nonverlapping genetic code
read from a fixed starting point as a continuous sequence of bases
66
universal genetic code
code conserved throughout evolution
excpetions in humas: mitochondria
67
DNA replication
is both prokaryotes and eukaryotes is **semi-conservative **and involves bth **continuous and discontinuous ( Okazaki fragment)** synthesis
68
origin of replication
particular consensus sequence of base pairs in genome where DNA replication begins
have multiple in eukaryotes and single in prokaryotes
69
replication fork
Y-shaped refgion where leading and lagging strands are synthesized
70
helicase
unwinds DNA template at the replication fork
71
Single stranded binding proteins
prevents strands from reannealing
72
DNA topoisomerases
create single or DB-stranded breaks in the helix to remove **or** add supercoils
**fluoroquinolones** - inhibit DNA gyrase ( prokaryotic topoisomerase II)
73
Primase
makes RNA primer on which DNA polymerase III can intitiate replication
74
DNA polymerase III
prokaryotic ONLY
elongate leading strand by adding deoxynucletides t the 3' end
elongs lagging strand until it reaches primer of preceding fragment
3' --\> 5' exonuclease activity 'proofreadds' each added nucleus
SO: DNA polymerase III has 5' --\> 3' synthesis and proofreads 3' --\> 5' exonuclease
75
DNA polymerase I
prokaryotic only
degrades RNA primaer and replaces it with DNA
has same functions as DNA polym III but ALSO excises RNA primer with 5' --\> 3' exonuclease
76
DNA ligase
catalyzes the formation of a phosphodiester bond within a strand of DB-stranded DNA - aka joins okazaki fragments
77
Telomerase
An RNA-dependent DNA polymerase that adds DNA to the 3' ends of chromosomes to avoid loss of genetic material with every duplication
First Aid - Biochemistry
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