Exam 1 Flashcards
Failure of NER (Nucleotide Excision Repair) results in what two clinical conditions?
Xeroderma pigmentosum (failure of global NER) and Cockayne syndrome (failure of transcription-coupled NER)
Failure of the mismatch repair system results in what clinical condition?
Hereditary nonpolyposis colon cancer (HNPCC)
The hMSH is the eukaryotic homolog of ____ in E. coli
MutS, part of the Mismatch repair (MMR) system
This DNA repair system is dependent on specific DNA glycosylases which remove an incorrect base to create an AP site
Base Excision Repair (BER)
How is Uracil created from Cytosine?
Deamination
How is Thymine created from Uracil?
Methylation
How is Guanine created from Adenine?
Deamination
What do Type I Topoisomerases do?
Creates single stranded cuts in DNA, no ATP required, relaxes negative and positive supercoils in eukaryotes
What is the function of DNA helicase?
Unwind the DNA duplex during replication
What do Type II Topoisomerases do?
Creates a double stranded break, requires ATP, relieves positive and negative supercoils
What is the deal with DNA gyrase?
Gyrases are a subset of Type II Topoisomerases mostly specific to prokaryotes that introduce negative supercoils into the circular genome to facilitate replication. Antibiotic target of quinolones (novobiocin, ciprofloxacin)
What are the functions of the DNA Pol III?
5’—>3’ synthesis, 3’—>5’ exonuclease proofreading
Functions of DNA Pol I?
(1) 5’-to-3’ DNA polymerase activity
(2) 5’-to-3’ exonuclease activity for RNA primer removal
(3) 3’-to-5’ exonuclease activity for proofreading
DNA control elements? Give 3 examples.
DNA control elements act in cis.
1) TATA box (core promoter sequence.
2) Promoters (<200 bp upstream
3) enhancers (up to 50kb upstream, could be downstream, often cell-type specific)
Rifampicin mode of action
Binds bacterial RNA polymerase at the RNA exit channel and blocks it. Current moratorium on its use.
Why is eating “death cap” mushrooms (Amanita phalloides), a bad idea?
contain alpha-amanitin, a non-competitive inhibitor of RNA pol II. Blocks RNA chain elongation by preventing translocation
Syndromes associated with mutations in TFIIH (3)
Xeroderma pigmentosum, Cockayne’s Syndrome, Trichothiodystrophy. (TFIIH recruits NER apparatus)
Actinomycin D mode of action
Intercalates into double helix between successive CG base pairs, deforming the DNA
Coding strand
aka non-template strand or sense strand, this opposes the template strand and is NOT transcribed.
Template strand
aka antisense, anticoding, noncoding. Transcribed, complementary to mRNA.
Splice site at 5’ end of intron:
5’ GU. Marks the beginning of every intron.
Splice site at 3’ end of intron:
5’ AG. Last two bases of every intron.
Consensus sequence at poly-A site:
5’AAUAAA
Functions of 5’ cap (4)
1) protect from exonucleases
2) recruit cap binding complex
3) recognition by eIF4E for transport to ribosome
4) removal signals decay
The PolyA tail ____ eukaryotic mRNA, but ____ of prokaryotic mRNA
protects; signals decay
What does U1 snRNA do?
Recognizes GU site for intron splicing
What does U2 snRNA do?
binds to the branch point (A) on the pre-mRNA sequence between the 5’ and 3’ splice sites
Let’s talk about Marfan syndrome.
Caused by mutations that disrupt splicing of the fibrillin gene transcript (fibrillin is a connective tissue protein that is important for the integrity of the walls of the heart and blood vessels). They are tall and prone to aneurysms.
What does U2AF snRNA do?
Binds to the 3’ splice site of intron (AG)
Phosphodiester bonds link:
The 3’C and the 5’C on the backbone of DNA and RNA
Phosphoanhydride bonds
High-energy bonds between phosphate molecules on NTPs
Somatic mutations
i.e. UV damage. Not passed on, as opposed to inherited or germline mutations
THIIF is reponsible for what function during transcription?
Unwinding DNA double helix to create
Nonpolar/Aliphatic R groups:
MAGLIV (great lecturers value my awesome intellect) (methionine, alanine, glycine, leucine, isoleucine, valine) These are not very reactive, only hydrocarbon R groups (+methionine).
Chloramphenicol and erythromycin target what process?
Translation. Chloramphenicol inhibits the peptidyl transferase (blocks peptide bond formation), Erythromycin prevents translocation from A to P site
What are the four major families of DNA binding domains? (Trans-acting transcriptional activators aka transcription factors)
Homeodomain, zinc-finger, basic leucine zipper, helix-loop-helix
Craniosynostosis
Autosomal dominant mutation within Msx2 homeodomain. Binds too tightly, upregulates genes that encode for suture fusion. Infants have no fontanelle.
What does Swi/Snf do?
Swi/Snf is a DNA dependent ATPase that breaks the contacts between histones and DNA so the histones can move along the DNA.
HATs
Histone AcetylTransferases. Acetylate the lysine residues, which neutralizes their positive charge. Recruitment of HATs generally activates transcription. (Pattern of +,- charges recruit activators/repressors.)
HDACs
Histone DeACetylases. Remove the acetyl group from lysine residues on histone free ends, creating + charge. Recruitment of HDACs generally represses transcription. (Pattern of +,- charges recruit activators/repressors.)
Etiology of Rubinstein-Taybi syndrome
Mutation in CBP which is a transcriptional coactivator (HAT). Haploinsufficiency results in transcriptional changes (presumably decreased transcription).
Two Ways Transcriptional Activators and Repressors Work:
- Interact with general transcription factors/Polymerase II associated proteins to influence initiation of elongation of the primary transcript
- Interact with chromatin to regulate accessibility of DNA to Pol II transcriptional apparatus
Tamoxifen___
binds to estrogen receptor and prevents recruitment of HAT co-factors. (competitive estrogen antagonist at binding of estrogen receptor - some cancer cells require estrogen to grow)
Talk about scurvy, you dog.
Scurvy = lack of Vitamin C–>required for converting proline to hydroxyproline (an integral part of collagen). This is a form of post-translational modification 1/6
What does warfarin (Coumadin) do?
Prevents recycling of Vitamin K–>required for synthesis of carboxyglutamate (carboxylation of glutamate is a form of post-translational modification 2/6) which are important for blood clotting.
What is glycosylation? Give 2 common examples.
Attachment of a sugar to a protein. (Post translational modification 3/6) 1) N-linked (Asn) 2) O-linked (Ser/Thr)
Histone proteins undergo what type of post-translational modification? (4/6)
Acetylation of lysine. (Can also be methylated to regulate transcription)
What three AAs are commonly reversibly phosphorylated? (post-translational modification 5/6) Impt. in signaling pathways
Ser, Thr, Tyr
Chronic Myelogenous Leukemia (CML) is treated with ______, the trade name for which is _______, a drug that inhibits ________.
Imatinib; Gleevec, Tyrosine Kinase (Post-translational modification 5/6, phosphorylation)
What post-translational modification tags proteins for destruction?
Ubiquitination (6/6)
This drug is used to treat Multiple Myeloma_____. Its trade name is________, and it acts as a_______.
Bortezomib; Velcade; proteosome inhibitor
Where does methylation specifically occur on DNA? (often)
CG islands, 5’ Cytosine is methylated (think Fragile-X). This silences transcription of the downstream gene.
Interferon secreted by virally infected cells and does what?
eIF2alpha is responsible for starting translation (tRNA binding to ribosome). Interferon starts a cascade that causes phosphorylation shutting down this initiation factor to quench translation.
Rapamycin
4e binding proteins bind eIF4E, prevents binding to 5’ cap when they are dephosphorylated, blocking translation. When binding proteins are phosphorylated, translation proceeds as normal.
NFAT is a sequence specific binding protein (transcription factor for inflammatory genes). How is it regulated? What drugs target this pathway, and where?
High levels of Ca2+ activate calcineurin’s phosphatase activity. Calcineurin dephosphorylates NF-AT, exposing the NLS. NF-AT relocates to the nucleus, initiates gene transcription.
Cyclosporine and FK506 inhibit the phosphatase activity of calcineurin —-> immunosupressive drugs.
PDI
Protein Disulfide Isomerase. Helps form disulfide bonds.
Aspirin works by
NFkB is normally held in the cytoplasm by IkB which masks the NLS of NFkB. Phosphorylaton of IkB destroys it, unmasking the NLS of NFkB, which relocates to nucleus and initiates transx of inflammatory genes.
Asprin prevents phosphorylation of IkB.
Solubility of nucleotides (most soluble to least soluble)
pyrimidines > purines
nucleotides > nucleosides > bases
Nucleotides are polymerized by the _____ bond between the _____ of one and the ______ of another.
phospho-diester; 5’-phosphate of one nucleotide and the 3’-OH
ATP is an example of a _____ bond
phosphoanhydride
RNA Polymerase I
Ribosomal RNA
RNA Polymerase II
mRNA, snRNA, microRNA
RNA Polymerase III
tRNA
Eukaryotic DNA replication requires at least three DNA polymerases, Pol α, Pol δ, and Pol ε. What does each do?
DNA Pol α holoenzyme is a multi-protein complex that has both primase activity and DNA polymerase activity, although no proof-reading activity. DNA Pol α synthesizes the first ~20 deoxyribonucleotides after the RNA primer, and is then swapped for Pol δ and Pol ε for lagging and leading strand synthesis, respectively.
For example in BER, uracil glycosylase recognizes uracils in DNA that result from cytosine deamination, and 5- methylcytosine-DNA glycosylase recognizes 5-methylcytosine to initiate DNA demethylation. The glycosylase flips the altered base out from the double strand helix and then hydrolyzes the ____ (the bond within the nucleoside between the base and the sugar) to remove the damaged base, producing an apurinic or apyrimidinic (AP site) site. The AP site is then REMOVED by an AP-specific endonuclease and an AP lyase, and the resulting gap is filled by a DNA polymerase and the nick sealed by a DNA ligase.
N-glycosidic bond
Mismatch repair (MMR) fixes errors in nucleotide incorporation ______ . The mismatched base pair is recognized shortly after DNA synthesis by the _____ proteins in bacteria; their mammalian counterparts are _____ proteins.
made by DNA polymerase during DNA replication;
MutS and MutL;
MSH (MutS Homolog) and MLH (MutL Homolog)
_______ : protein enzymes that put the right amino acid on the right tRNA.
Aminoacyl tRNA synthetases
The Shine-Delgarno sequence is associated with _____?
Translation initiation in prokaryotes.
In eukaryotes, _____ is required to bind to the 7-methyl guanosine cap on the 5’ end of the mRNA. This leads to binding of many other eIFs (4G, 4A, 4B, etc.) and eventually to binding of the small ribosomal subunit, which itself is bound by several factors (eIF3, eIF1A, eIF1, eIF2, etc.). The ribosome then scans down the message to find the AUG start codon. At that point, the large subunit can join the small, the factors are released, and the goal of initiation has been achieved.
initiation factor (eIF) 4E
Many viruses use _____ to initiate translation after they shut down host cell cap-dependent synthesis.
IRESs
In eukaryotes, different start codons have different “strengths” depending on their ____ context. This process is associated with _____.
Kozak; translation
[AUGs with a weak Kozak context can be bypassed, allowing downstream AUGs to be used. The result would be two different proteins produced at certain amounts, from the same mRNA.]
In eukaryotes, the cap binding protein (eIF4E) can be bound by ____ that sequester it and blocks its function.
4E-binding proteins (4E-BPs)
[phopsphorylation of 4E-BPs inactivates them, generally they are phosphorylated so translation can proceed normally]
eIF2-alpha is critical for ______. When eIF2-alpha is _____, its activity is inhibited and this blocks initiation.
binding of the initiator tRNA to the ribosome; phosphorylated
[eIF2-alpha can be phosphorylated by several pathways. One is induced by interferon, which is produced when a cell is infected by a virus.]
What happens on the large vs small ribosomal subunits during translation?
The amino acids join to form the peptide chain in the peptidyl transferase center of the large subunit
The message is decoded on the small subunit
- Peptide bond formation results in a transfer of the nascent peptide from the P-site tRNA to the A-site tRNA.
- After the peptide bond is made, EF2 and GTP hydrolysis triggers movement of the mRNA and tRNAs exactly one codon in the 3’ direction.
- The result is the deacylated tRNA now in the E site, and the tRNA with the peptide chain attached in the P site.
- Now, the cycle can continue when a new aminoacylated tRNA, bound to EF1a, moves into the vacant A site.
**So, peptide is attached to the tRNA in the P site. That is where bond formation takes place. Somewhat confusing the way it is stated in the handouts.
True or false: during transcription, the RNA polymerase binds to closed DNA and unwinds it?
True.
What are the 5 functions of the mRNA cap? Where does each take place?
- splicing
- 3’ processing
- nuclear export
- translation
- degradation
4,5 cytoplasm. 1-3 nucleus
BRCA1 promotes ____.
HR (homologous recombination)
53BP1 promotes _____.
NHEJ
Compare and contrast Ulcerative Colitis with Crohns disease.
Crohns: Ileum (always) + colon, discontinuous. Fistulas common
UC: Rectum (always) + colon, continuous. Fistulas rare.
