Chromatin Structure
- Negatively charged DNA wrapped around positively charged histones
- Histone octomer with DNA= nucleosome. Rich in lysine and arginine. H2A, H2B, H3, H4
H1- linker - DNA and histone synthesis in S Phase
Heterochromatin
Highly condensed and transcriptionally inactive
Euchromatin
Less condensed and DNA is accessible. Transcriptionally active
DNA methylation
Differentiates between new and old strand. Methylation at cytosines and adenine . Allows for mismatch repair.
CpG lslands repress transcription= muted.
Histone methylation
Most of the time makes DNA mute
Histone acetylation
Relaxes DNA coiling and makes DNA active
Nucleotides
PURines (AG)- Pur As Gold
PYrimidies (CUT)- CUT Py
Thymies are methylated
What does deamination of cytosine create?
Uracil
Which bond is stronger G-C or A-T?
G-C because it has three hydrogen bonds as opposed to only two in A-T. Increased G-C = increased melting temperature
What three amino acids are necessary for purine synthesis?
Glycine, Aspartate, and Glutamate
What is a nucleoside?
base+(deoxy)ribose Sugar
What is a nucleotide?
Base+ (deoxy)ribose sugar+ Phosphate
Describe purine synthesis.
Start with a sugar (ribose 5-phosphate) + Phophoribsoyl Pyrophosphate (PRPP) and then add base.
Describe pyrimidine synthesis.
Make a temporary base (orotic acid). Then add sugar + phosphate (PRPP). Then modify the base
What is the function of ribonucleotide reductase?
Converts ribonucleotides to deoxyribonucleotides
What is the role of Leflunomide?
Inhibits the dihydroorotate dehydrogenase (important in the orotic acid production in pyrimidine synthesis)
What is the role of mycophenolate and ribavirin?
They inhibit IMP dehydrogenase (important in purine synthesis pathway)
What is the function of hydroxyurea?
Inhibits ribonucleotide reductase(enzyme important in pyrimidine synthesis)
What is the function of 6-mercaptopurine and prodrug azathioprine?
Inhibition of de nove purine synthesis
What is the function of 5-fluorouracil (5-FU)?
Inhibits thymidylate synthase which decreases deoxythymidine momophosphate which results in a decrease in dTMP
What is the function of methotrexate, trimethoprim and pyrimethamine?
All inhibit the function of dihydrofolate reductase (decrease dTMP) in humans, bacteria, and protozoa, repectively.
Describe Adenosine deaminase deficiency.
Creates an excess of ATP and dAT imbalances nucleotide pool via feedback inhibition of ribonucleotide reducatase and prevents DNA synthesis and thus low lymphocyte count.
What is one major cause of autosomal recessive SCID?
Adenosine deaminase deficiency
What is hypoxanthine-guanine phosphoribosyltransferase?
An enzyme that converts hypoxanthine to IMP and Guanine to GMP.
What enzyme is absent in Lesch-Nyhan syndrome?
Hypoxanthine-guanine phosphoribosytransferse which results in an excess of uric acid production and de novo synthesis of purine production
What is the inheritance pattern of Lesch-Nyhan syndrome?
X-linked recessive
What are the symptoms of Lesch-Nyhan syndrome?
Results from HGPRT deficiency: H-Hyperuricemia G- Gout P-Pissed off (aggression and self-mutilation) R- Retardation T-dysTonia
What enzyme does fluoroquinolones inhibit?
DNA gyrase or prokaryotic topoisomerase II
Please define a transition mutation.
A transition mutation is a mutation that changes a purine to another purine (e.g. A to G) or pyrimidine to antoher pyrimidine (C to T)
Please define a transversion mutation.
A mutation that results in a change of a purine to a pyrimidine or vica versa.
Please give an example of a missense mutation
Sickle cell anemia
Please give an example of a frameshift mutation
Duchenne muscular dystrophy
Please describe nucleotide excision repair mechanism and what type of mutations they fix.
Specific endonucleases release the oligonucleotide-containing damage bases; DNA polymerase and ligase fil and reseal the gap respectively. Repairs bulky helix-distorting lesions
Describe xeroderma pigmentosum and what mutation causes it.
Mutation: Nucleotide excision repair mechanism so cell cannot repair pyrimidine dimers. Cuases early melanoma, basal cell carcinoma, and squamous cell carcinoma
Please describe base excision repair and what type of mutations they fix.
Base-specific glycosylase recognizes altered base and creates AP site. ONe or more nucleotides are removed by AP-endomucleases, which cleaves the 5’ end. Lyase cleaves the 3’ end. DNA polymerase-beta fills the gap and DNA ligase seals it. It fixes spontaneous deamination
Please describe mismatch repair.
Newly synthesized strand is recognized, mismatched nucleotides are removed, and the gap is filled and resealed.
Please name a disorder caused by a defect in mismatch repair mechanism.
Hereditary nonpolyposis colorectal cancer (HNPCC)
Please describe nonhomologous end joining
Brings together 2 ends of DNA fragments to repair double-stranded breaks. Require no homology
What causes ataxia telangiectasia?
mutation in nonhomologous end joining
What do the start codons code for in eukaryotes and prokaryotes?
Eukaryotes- mthionine
Prokaryotes- formylmethionine
Name the three stop codons
UGA- U Go Away
UAA- U Are Away
UAG- U Are Gone
Describe a promoter.
Site where RNA polymerase and multiple other transcription factors bind to DNA upstream from gene locus (AT-rich upstream sequence with TATA box)
Enhancers/ Silencer
Stretch of DNA that alter gene expression. Can be thousands of bp up or downstream or in introns.
Name and describe the functions of eukaryotic RNA polymerases.
RNA Pol I- makes rRNA
RNA Pol II- makes mRNA
RNA Pol III- makes tRNA
Name the compound found in the death cap mushroom that inhibits RNA polymerase II.
Alpha-amanitin. Causes severe hepatotoxicity. Symptoms appear between 1-2 weeks
How many RNA polymerases do prokaryotes have?
One
Name three modifications that occur in the maturation process of mRNA.
5’ methylguanosine cap
3’ poly A tail
Splicing of introns
What makes up the sliceosome?
Primary transcript + small nuclear ribonucleoproteins(snRNP)
What causes SLE?
Antibodies to spiceosomal snRNPs
Anti-U1 RNP antibodies cause…
mixed connective tissue disease
Name the parts their function in tRNA
T Loop- recognition of ribosome
D Loop- recognition of correct aminoacyl tRNA synthetase
Anti-Codon- recognizes mRNA codon
3’ CCA- binds amino acid
Describes aminoacyl-tRNA synthetase
Enzyme that loads amino acid onto the 3’ end of tRNA. If it adds the wrong amino acid it will hydrolyze and take it off
Translation-Initiation
Initiated by GTP hydrolysis; initiation factors help assemble the 40S ribosomal subunit with the initiator tRNA and are released when the mRNA and the ribosomal 60S subunit assemble with the complex
Translation- Elongation
- Aminoacyl-tRNA binds to A site
- rRNA catalyzes peptide bond formation, transfers growing polypeptide to amino acid in A site
- Ribosome advances 3 nucleotides toward 3’ end of mRNA, moving peptidyl tRNA to P site
Translation- Termination
Stop codon is recognized by release factor. and completed polypeptide is released from ribosome
Name three posttranslational modification
- Trimming
- Covalent alterations
- Chaperone protein
Name 7 covalent alterations that can be made on a protein.
- Phosphorylation
- Dephosphorylation
- Carboxylation
- Hydroxylation
- Methylation
- Acetylation
- Ubiquitination