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130 Cards in this Set
- Front
- Back
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pyrimidine bases:
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one ring
thymine, uracil, cytosine |
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purine bases:
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two rings
adenine, guanine |
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nucleotides vs nucleosides
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nucleotides: have phosphate bonds
nucleosides: don't have phosphate bound |
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chromatin:
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protein-DNA complex
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nucleosome
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200bp DNA wrapped around protein core
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octamer core
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2X four histone proteins
contain flexible N-terminal "tails" rich in lysine |
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histone H1
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linker histone that assists in higher order packing
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solenoid
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30nm fiber- formed by a bunch of nucleosomes in line
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________ of histones regulate transcription
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modifications of the tails
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examples of how histones are modified to regulate
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acetylation- pro-transcriptional
methylation- depends |
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acetylation of_______ causes_____
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lysine located in the tails leads to pro-transcription- the histones no longer bond DNA as tight and therefore a more loose conformation is present and transcription is upregulated
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How is DNA modified?
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methylated: DNa is irreversibly packaged into transcriptional silent chromatin
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which DNA found in our genome is highly methylated?
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repeat
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epigenetics
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the study of the patterns of methylation passed on
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general content of human genome: repeats, introns protein coating sequences
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63% repeats
25% introns 1.5% protein coding |
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interspersed repeats contain
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SINEs and LINEs, retroviral-like elements, and DNA transposon "fossils"
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SINEs
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used to identify us as individuals...DNa tests, paternity tests
Alu repeats, MIR, MIR3 |
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tandem repeats are also called
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satellite DNA
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satellite DNA
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found at centromeres
5-200bp humans, at least four diff. repeat types |
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mini-satellite DNA
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20kb in length with repeat units up to 25bp
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microsatellite DNA
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less than 150bp
repeat unit is 13bp or less found at telomeres, other locations used for DNA fingerprints |
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Trinucleotide repeat expansion
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increases the number of repeats present due to slippage of pol and continuous synthesis
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diesases from trinucleotide repeat expansion
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huntingtons diease
myotonic dystrophy |
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huntington disease
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increased number of glutamines in huntington protein- insoluble protein that aggregates in cytoplasm
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gross rearrangements can cause
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translocations, deletions, or transversions
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recombination events related to repeats could occur
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unequaly causeing an exon to be completely deleted
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an example of recombination events as a mutation caused by repeats:
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familial hypercholesterolemia
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origins of replication are marked by
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DNA/nucleosome modifications
Transcriptionally active regions are favored DNA-binding transcription factors may be involved |
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ORC
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origin recognition complex- binds to the ORI region in DNA during late M phase
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liscensing replication
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cdc6 and cdt1 required to recruit/load the MCM helicase complex onto the ORI during G1 phase
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cdc6 is removed:
while cdt1 is: |
from nucleus after licensing and is degraded
present only in G1 and S phase. it is degraded after S phase -this ensures replication will only occur during G1/S phase |
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geminin
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inhibitor of cdt1- present in S and G2
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timing of cell cycle is orchestrated by
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cyclin-dependent kinases (CDKs)
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MCM
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unwinds DNA
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after the DNA is unwound by MCM,
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it is coated by RPA which is a single stranded DNA binding
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RPA
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replication protein A
single stranded DNA binding protein |
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Primase enzyme
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DNA primase and DNA polymerase alpha
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DNA primase
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makes 10-15nt RNA primer
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DNA polymerase alpha
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adds 30nt of DNA onto RNA
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DNA polymerase needs
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RNA primer and DNA sequences inorder to add nucleotides during replication
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DNA polymerase enzyme
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copies DNA
requires a "clamp" to hold polymerase to DNA |
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PCNA
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"clamp" that holds DNA polymerase onto DNA
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topoisomerase I
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prevents overwinding of DNA ahead of replication fork
creates a single stranded nick |
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semi-discontinuous
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one of the strands is replicated in discontinuous way
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semi-conservative
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one strand from each parent in DNA replication
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topoisomerase is target for___
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cytotoxic drugs: antibiotics (quinolones) or anti-tumor (etoposide, doxorubicin)
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maturation of okazaki fragments
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DNA pol delta initiates at primer and elongates DNA to the next RNA
DNA pol delta displaces RNA primer endonuclease cuts off flap DNA ligase seals the nick (using ATP) |
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DNA mutations can be caused by
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-copying mistakes
- recombination - "genome mishandling" - physical/ chemical damage |
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Chromosome mis-segretation leads to
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aneuploidy- abnormal # of chromosomes
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chromosome rearrangement results from
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translocation
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base pair mutation
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results from a point mutation
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transition
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pyrimidine to pyrimidine or purine to purine
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transversion
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pyrimidine to purine or vice versa
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deletions and insertions
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can involve one or more base pairs
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Chemical DNA damage
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environmental, pharmacological, "benign" to active mutagens, DNA adducts, Free radicals, DNA alkylation, DNA cross-linking drug,
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DNA adducts
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adding chemical moiety to DNA can alter base pairing and produce double stranded breaks
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Free radicals
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chemical species that can react with DNA and cause damage
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DNA alkylation
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electrophilic agents transfer alkyl groups to DNA
EMS (ethane methyl sulfonate) is an example |
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DNA cross-linking drugs
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-this causes a failure of strand separation during replication
replication stalls and can produce double stranded breaks |
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Cisplatin
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example of a DNA cross-linking drug
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Physical mutagens
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UV light- produce thymidine dimers which cause DNA replication to stall or continue without basepairing correctly causing mutations
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Base excision repair
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abnormal base is removed and filled in by "repair" DNA polymerase
-no genetic defects observed in pathway |
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nucleotide excision repair
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larger, more severe lesions
helicase unwinds large region around lesion (XP genes) single strand of DNA removed. Filled in by DNA polymerase |
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xeroderma pigmentosum
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mutations in XP genes(helicases) cause this
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post-replication: mismatch pair
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errors during repliacation not fixed by proofreading.
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mismatch recognized by ______ and defective DNA excised and re-synthesized by _______
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MSH complex
DNA poly delta |
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Double strand break repair
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most serious DNA damage can be repaired by homologous recombination or non-homologous end joining
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homologous recombination
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use matching chromosome as a template to fix the break
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non-homologous end joining
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takes all broken chromosomes and puts them all together. This can potentially be very detrimental and translocations can contribute to cancer
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nick
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absence of phosphodiester bond in DNA backbone
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telomerase
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enzyme required to maintain telomere length in germline cells
chromosomes shorted with each cell division |
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hayflick limit
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approx, 50 cell divisions---senescense
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role of mRNA cap in translation initiation
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cap is the site of the CAP binding complex
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CAP binding complex contains
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three protein complex (eIF4F)
-eIF4A -eIF4G -eIF4E |
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translation initiation is regulated by
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avaliablility of eIF4E
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how is eIF4E regulated?
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4E binding proteins (4E-BPs) bind/sequester eIF4E.
phosphorylation of 4E-BPs causes them to release eIF4E and transcription occurs |
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P-site
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peptidyl tRNA site: peptide is found there and the growing chain is attached to tRNA
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A-site
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aminoacyl site: the aminoacyl-tRNA for next codon enters here and the peptide is briefly added to A-site and the ribosome is moved
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polysome
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polyribosomes: multiple ribosomes on a single mRNA
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direction of protein synthesis
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N-------to--------C
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fraction
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subpopulation of highly translated mRNA in a cell
expression of mRNA can be modulated by shifting into or out of polysome fraction |
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all_____ for an a.a. use ______
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isoacceptor tRNAs with amino acids by amino-acyl tRNA synthetases
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each aa-tRNA synthesis binds
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amino acids
ATP tRNA |
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which aa that is bound to the tRNA depends on
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lengths of stems and loops of tRNA and modifications of the tRNA
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eIF4F
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facilitates CAP binding to mRNA cap
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eIF2
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escorts methioyl-tRNA to ribosome
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eIF1 and eIF3
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binds small ribosomal subunit and interacts with cap binding complex to bring the ribosome to mRNA
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Once the start codon is recognized:
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eIF2 leaves and large ribosomal subunit combines with small subunit
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Kozak Sequence
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consecutive sequence before AUG
16s RNA binds to this sesquence and stalls the small ribosomal subunit and allows the large unit to bind translation can continue |
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regulation of translation by insulin
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insulin causes phosphorylation of 4E-BP which releases eIF4E and allows transcription to occur
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during stress:
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proteins fold incorrectly and can accumulate in the ER
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stress triggers:
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unfolded protein response which shuts down translation
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during the unfolded protein response:
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PERK kinase is activated
mechanisms to refold proteins are induced in continued stress, apoptosis is induced |
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PERK:
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phosphorylates eIF2 which cannot participate in initiation
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Mutation in PERK
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no longer has kinase activity- could be a reason for neonatal onset diabetes
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how antibiotics affect translation
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1/2 of all antibiotics target translation
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macrolide
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antibiotic that binds irreversible to 50s subunit
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Erythromycin
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inhibits translocation step where the peptide in the A site moves to the P site
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aminoglycoside
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binds 30s subunit and fixes the 30s and 50s complex to AUG
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snRNA
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small nuclear RNA- used for RNA splicing
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micro RNA
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gene "silencing"
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core promoter elements
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necessary for recruitment of TFs
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core promoter elements contain
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sequence information that directs transcription and is found essentially in all genes
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core promoter elements are located
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near the transcriptional start site
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distal regulatory elements
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affect the rate of the TF binding
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distal regulatory elements are located
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either near, far, upstream, downstream
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core promoter role in E Coli
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-10 and -35 sequences that are contact points for RNA polymerase proteins
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UP element
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part of the core promoter in E Coli that when bound, increases transcription
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Sigma factor of E coli
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component of RNA polymerase
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alpha subunit of RNA pol in E coli
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binds DNA
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beta subunit of RNA pol in E coli
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catalytic subunit
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beta prime subunit of RNA pol in E coli
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clamps DNA during transcription
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sigma subunit of RNA pol in E coli
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interacts with -10 and -35 (comes and goes)
ensures RNA pol binds stably to DNA only at promoters |
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four basic steps in gene transcription by eukaryotic RNA polymerase II
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1. loosen/open chromatin
2. general transcription factors bind core promoter 3. RNA pol II binds DNA 4. Phosphorylation of RNA pol II, promoter clearance, elongation |
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LOCUS control regions
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regulatory element that governs transcription of a GROUP of GENES at a single chromosomal locus- related to one function
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TFIIH
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phosphorylates RNA pol II
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TFIID
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TATA binding
nucleates "pre-initiation" complex can be affected by regulatory factors |
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chromatin remodeling complex
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contains 1 subunit of ATPase activity
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activators/repressors function by
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altering the rate of formation of pre-initiation complex
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What happens when a positive regulator proteins bind positive regulatory element
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it recruits histone acetylation enzymes and recruit chromatin remodeling complexes and contacts/recruits general transcription factors
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what happens when a negative regulator proteins bind negative regulatory elements
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they recruit histone deacetylases and this blocks the binding of general transcription factors
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c-myc
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an example of a transcriptional regulatory protein that can activate/repress transcription
-it is usually pro-growth and is a proto-oncogene |
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transcriptional regulators have
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DNA binding domain
transcriptional activation domain Dimerization and Ligand binding |
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the DNA binding domain of transcriptional regulators contain
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a zinc finger- which is a sequence of protein that binds DNA
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a zinc finger usually contains:
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two cys and two his and a Zn2+ usually maintains protein structure
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what usually binds the transcriptional activation domain
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HATs usually bind and this is also where TF will interact with
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when estradiol binds ER it forms _________and when tamoxifen binds ER is forms___________
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different structure
different structure |
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In breast cells: the change in structure from estradiol results in_________ and the change in structure from tamoxifen results in __________
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estradiol: binding of HAT - activation
tamoxifen: binding of HDAC- repression |
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in uterine cells: the change in structure from estradiol results in _______ and the change in structure from tamoxifen results in __________.
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estradiol: binding of HAT- activation
tamoxifen: binding of HAT- weaker activation |
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Why is there a difference in the binding of HAT and HDAC to tamoxifen bound ER in uterine cells and breast cells?
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the different tissues contain different concentrations of HDACs
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