DNA to protein!

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DNA to protein!
2014-09-25 19:59:14
Foundations fnd1 DNA tubberly

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  1. DNA helix types
    • B DNA is major form, right handed with open major groove
    • A DNA is minor form, right handed with smaller grooves
    • Z DNA is rare, left handed and probably regulatory
  2. RNA structure
    • Stems is where it is antiparallel base pairing
    • Loops are unpaired
  3. Bacterial genophore
    Not technically a chromosome, very active and always uncondensed
  4. Histones
    • Two H2A-H2B dimers and a H3-H4 tetramer form the octomer and have modifiable tails
    • Modification depends on how its modified and the site to form loose euchroatin and tight heterochromatin (more heterochromatin at telomeres and centromere and dispersed thoughout)
    • Heterochromatin can spread
    • H1 helps neutralize charge to package
  5. Repetitive DNA types
    • Highly repetitive is satelite DNA found near centromere
    • Middle repetitive tandem is multi copy genes, mini satelites (help ID people), microsatelites (even smaller)
    • Middle repetitive interspersed are SINES and LINES, which are retrovirus remnants that have gone in and out of genome throughout evolution. Some are just transposases to pop them in and out and might be important in development (they move around)
  6. rRNA genes
    Found in subtelomeric region of about 5 chromosomes
  7. tRNA genes
    Intersperesed thoughout chromosome on several chromosomes
  8. Telomere structure
    Shelterin complex protects against degradation
  9. mRNA gene organization
    • They can overlap
    • Genes can be in the introns of other genes
    • Often there are repeats throughout gene
    • Cluster in families for temporal regulation or if all part of same process
  10. miRNA genes
    • intergenic or intergenic clustered
    • intronic or intronic clustered
    • mitron (the intron is miRNA)
  11. lncRNA
    • long non-coding RNA
    • intergenic
  12. Nuclear organization
    • More heterochromatin in middle and outside
    • Chromosomes have territories
    • Centromeres and telomeres are randomly dispersed
    • High gene density chromosomes at center
    • Transcriptional processing complexes where genes that work together from different chromosomes are localized
  13. Eukaryotic initiation of replication
    • ORC which recruits cdc6 and MCM (helicase) to open
    • cdc6 is phosphorylated and released to initiate S phase
    • Helicase activity is ATP driven
    • Multiple origins
  14. Prokaryotic initiation of replication
    • Open complex with DnaA which recruits DnaG as helicase (ATP dependent helicase activity)
    • One origin of replication
  15. Replisome
    • Leading and lagging strand (always synthesize 5 to 3)
    • RNA primers
    • Leading strand uses pol alpha or episolon
    • Lagging strand uses pol alpha or delta
    • Epsilon and delta have 3-5 exonuclease and alpha doesn't
    • Polymerases are associated with helicase
  16. Things the replisome deals with
    • Supercoiling by toposiomerase1 works ahead of fork and only cuts one strand, toposisomerase2 works behind and cuts double strand when they get tangled
    • Hsitones by histone chaperones that mix and match old and new histones and conserve the pattern
    • RNA primers must be removed before pol can synthesize, then ligase ligates the nick
    • Sister chromatids don't float apart thanks to cohesin
    • Phosphorylation of ORC prevents second round of repliaction
    • Telomere lagging strand by telomerase with RNA template to extend leading strand and then make lagging strand
    • Anytime there is something else seriously wrong it stops and waits for it to be fixed then procedes
  17. Base Excision Repair
    • Single strand damage for single base
    • First (base)ylase removes just the base, then AP endonuclease and phosphodiesterase remove the sugar phosphate
    • Polymerase makes new base and ligase seals
    • This can be signaled by the transcription complex
  18. Nucleotide excision repair
    • single strand damage distorting helix
    • Nuclease cuts at either side some distance away
    • Helicase unwinds to free it
    • Polymerase and ligase synthesize and seal
    • This can be signaled by the transcription complex
  19. Mismatch repair
    Fixes mismatched bases form replication
  20. Non-homologous end joining
    • Double stranded damage before S phase
    • Nuclease chews back and ligase seals
  21. Homologous end joining
    • Double stranded damage after S phase
    • Special nuclease chews back, strand invasion occurs to synthesize using sister as template until sufficiently long for annealing, then polymerase and ligase synthesize and seal
    • Uses meiosis recomination machinery. This is also used in VDJ recombination
  22. Chromothripsis
    • Chromosome shattering and randomly put back together
    • Sometimes multiple chromosomes do this and get combined
    • Some chunks are left out
  23. DNA repair defect diseases
    • BRCA
    • Xeroderma pigmentosum
    • Heriditary nonpolyposis colorectal cancer
    • Werner syndrome (premature aging, helicase)
    • Bloom's syndrome (stunted growth, repair and helicase)
  24. RNA types and polymerase (eukaryote)
    • Pol I-rRNA
    • Pol II-mRNA, miRNA and most snRNA
    • Pol III-5S rRNA, tRNA snRNA and other small RNAs
  25. Transcription reading terms
    • DNA coding strand is same as mRNA
    • DNA template strand is antisense and "read"
    • T->A A->U G->C C->G
    • Downstream is the direction the replisome goes
    • Upstream is negative numbers
  26. Prokatyote promotion
    • Only 1 RNA polymerase with sigma factor to bind to promoter between -10 and -40ish with TATA box at -7 to -10
    • Activators bind
    • Sigma factor releases and synthesis begins
  27. Pol I promotion
    • There is an upstream binding factor UBF
    • One UBF binds to the core promoter element CPE and a second UBF binds to upstream control element UCE at -100. They form a dimer and recruit the selectivity factor 1 SL1 which recruits Pol I
  28. Pol II promotion
    • Promoter region with TATA box -20 to -30 to bind TBP and CAAT box, GC boxes -40 to -110
    • Highly regulated by promoter elements and sometimes (rare) there are downstream promoter elements
    • Regulatory proteins (activators or repressors) can bind to enhancers, whose orientation is unimportant and can be quite distant
    • Basal transcription complex with TBP and many TFIIA-H and coactivators is required
    • Mediator is a scaffold where the regulatory proteins bind (some modify histones and remodel chromatin good or bad)
    • Certain sites on the CTD (Pol II tail) are phosphorylated to bind processing factors for polyA, splicing, and capping
  29. Pol III promotion
    • Downstream promoter elements are common (in the gene) called Box A and Box B (tRNA)
    • DPE for 5S rRNA called Box A and Box C
  30. rRNA transcription and processing
    • Tandem repeats of transcription units with intergenic spacers inbetween
    • The transcription unit includes 18S 5.8S and 28S with ITS1 and ITS2 in between to be cleaved (not spliced though) after chemical modification of bases
    • Many Pol I go one after the other
    • 5S is transcribed by Pol III elsewhere
    • Trimming occurs in nucleolus and then nucleus
    • Assembly occurs in cytoplasm
  31. tRNA transcription and processing
    • Tandemly repeated clusters of various tRNAs
    • All processing in nucleus then exported
    • Intron (but not same as mRNA) removed to form anticodon loop
    • Bases are modified and ACC added to 3 prime
    • In cytoplasm tRNA synthase binds amino acid to ACC making it a charged tRNA
  32. hnRNA (heterogenous nuclear RNA) processing
    • All cotranscriptional
    • First the 7MG5 cap added with 5 prime to 5 prime triphosphate linkage
    • Spliceosome forms from U1-U6 snRNP to catalyze
    • U2 at branch site A doing nucleophilic attack on GU splice donor (U1) forms a lariat and U4-U6 bind. U5 binds to splice donor and splice acceptor to join
    • polyA tail signal in 3 prime non translated region causes snipping of last few bases of mRNA and addition of nontemplated polyA sequence by PolyA polymerase
    • Proteins bind to mRNA to protect it
    • Exported cap first and old proteins are removed and new ones are associated (checks for splicing)
  33. sn(and sc)RNA processing
    • For splicing, translation control, telomerase, rRNA base modification
    • Processed in the cytoplasm and most do work in the nucleus
  34. miRNA transcription and processing
    • There are different types but all get processed a bit in the nucleus and exported via EXPS
    • In cytoplasm bind to RISC which dices them to single strand (chooses 1)
    • Do work in cytoplasm or nucleus
  35. Gene rearrangement
    • Same mechanism that does homologous double strand repair
    • VDJ joins D and J genes then V to D and J
    • C attaches to J after splicing
  36. Gene amplification
    Cancers often do this, making small copies of a gene called "minutes" like tiny chromosomes
  37. Regulation of gene expression
    • Hormones can do this through various pathways by binding to receptors on plasma membrane or in nucleus
    • HSP90 increases gene expression
    • Protein kinase A increases target gene expression (when cAMP increases)
    • NFkB can activate target genes but it is inhibited by being bound to IkB, so this is phosphorylated to release (other IKK IKKK etc)
    • One factor can target many genes, which produce more regulatory proteins to activate even more genes making a transcription cascade
  38. Multiple promoters
    • Different promoters used can make different genes
    • Often tissue dependent
    • dystrophin
  39. Alternative splicing
    • Optional exons, optional introns, exclusive exons, internal splice site
    • Can result from mutation to cryptic splice site like beta thalassemia (usually get the normal protein at a small level)
    • Can result in alternative polyA tail signal
    • Which alternate you end up with can be regulated by miRNA degradation of one of the forms
  40. RNA editing
    • Edit bases in mRNA to make different proteins
    • Can make different stop codon UAG/UAA/UGA
    • ApoB100 liver v ApoB28 intestine
  41. Splicing factor titration
    Splicing is almost always done before export, so by changing levels of splicing factors you can change frequency of export of a protein
  42. Alternate start codon
    • 2 (or more I bet) different start codons to make different protein
    • Wilms tumor (also RNA editing and alternative splicing)
  43. Translational frameshifting
    • Viral, Gag or GagPol
    • Ribosome takes a step back 1 base at certain point or doesn't, resulting in 2 reading frame options
  44. Cap independent translation
    mRNA forms a stem loop structure to associate with 40S and initiate translation at a different site (also involves alternate start codon)
  45. Globin mRNA regulation
    • Heme regulates amount of globin
    • Heme binds to heme regulated inhibitor kinase (HRI) that phosphorylates eIF2, ceasing all translation
  46. Ferritin and transferrin receptor mRNA regulation
    • Iron starvation makes transferrin receptor
    • Excess iron makes ferritin
    • Cytosolic aconitase can bind to stem loops of these mRNAs unless it is bound by iron
    • Cytosolic aconitase binding to transferrin stabilizes it (prevents cleavage)
    • Binding to ferritin blocks translation
  47. Histone mRNA regulation
    • mRNA does has special stem loop binding protein (SLBP) to protect 3 prime intead of polyA tail
    • Phosporylation of SLBP causes release
  48. mRNA stability
    polyA tail is slowly degraded and at about 30As you can have decapping of 7MG5 and rapid 5 prime and 3 prime degradation
  49. Nuclear compartmentalization
    • ER is continuous with outer nuclear membrane and there are nuclear pore complexes (NPC) scattered across nuclear lamina
    • NPC does not penetrate, there is just a fold here, but either way it is super selective
    • Ran (GTPase) can regulate by changing the conformation of other proteins like nuclear import receptor molecules to make them go in and out
    • Ran in nucleus has GTP by guanine exchange factor (GEF)
    • Ran in cytoplasm has GDP by GAP
  50. 3 terms for genetic code
    • Specific (codon codes only 1 amino acid)
    • universal (same across species pretty much)
    • degenerate (multiple codons per amino acid)
  51. Prokatyotic initiation
    • 30S binds 3 initiation factors (IF1-3), GTP, and formylated methionine tRNA at P site
    • Binds to shine delgarno and IF 3 releases
    • 50S subunit recruited, hydolyzing GTP
  52. Eukaryotic initiation
    • Poly A tail and 7MG5 cap associate with some eIF4s (requires ATP)
    • eIF2 with GTP assocaites with methionine tRNA, bringing it to the 40S subunit which has other eIFs (eIF3 and 5)
    • This complex scans until the AUG in the Kozak sequence, where GTP hydrolyzes, allowing eIF2 to release, and Met tRNA is positioned in the P site
    • When eIF2 rleases the other eIFs release and 60S plus eIF58 bound to GTP
    • eIF58 GTP hydrolyzes and it releases
    • All of this regulation is for proofreading so you don't waste
  53. Elongation
    • Similar in prokaryotes and eukaryotes, uses slightly different elongation factors
    • Requries 2 GTP
    • 1st GTP with EF1alpha (EF-tu in prokaryotes) to get tRNA in A site
    • 2nd GTP with EF2 (EF-G in prokaryotes) to transfer polypeptide chain and shift both tRNA one site (translocation)
    • The other difference is prokaryotes do translation while they do transcription
  54. Translation termination
    Release factor binds to A site at stop codon UAG, releases polypeptide and ribosome disassembles
  55. Polyribosomes
    Multiple ribosomes work on same mRNA
  56. Ribosomes and ER
    • A certain signal sequence can be recognized by a signal recognition particle (SRP) that binds and halts translation, the signal recognition molecule binds to a receptor on the ER near a translocation channel
    • BIP chaperone in ER binds to polypeptide and helps and SRP leaves
    • This is for transmembrane proteins or proteins to be exported
    • The signal sequence is often cleaved because it stays in the membrane
    • You can have various start and stop transfer sequences later for multi pass proteins
  57. Protein modification
    • There are 162 identified ways to modify proteins
    • You can add a lipid group, cofactors like heme and flavin, small chemical groups like any -ylation you can think of
    • All these require enzymes and groups are usually added to conserved "consensus" sequences
    • Non enzyme modification can be glycation (just stick a sugar on it)
  58. Proteostasis
    • Unfolded--on pathway intermediates--native state
    • Chaperones are required between stages
    • Off pathway intermediates can be due to mutations or damage and can result in oligomers and aggregates
  59. Proteasome
    • Ubiquitin marks proteins for degradation and the enzymes that add ubiquitin are protein (or protein group) specific and add ubiquitin to certain lysine
    • E1 (~3 types, E2 specific) activates ubiquitin and transfers it to E2
    • E2 (various types, E3 specific) binds to E3 and transfers a ubiquitin chain to E3
    • E3 (various types, protein (group) specific) with E2 still bound ubiquitinates the protein and proteosome degrades
  60. Aggregates
    • Sometimes misfolded proteins aggregate rather than get degraded by ubiquitination
    • For some reason they can interact with dyneins and form an aggresome at the centrosome
  61. Lysosomes
    • In addition to proteasomes, degrade proteins from various sources like if you are recycling an organelle (macroautophagy)
    • Also can degrade aggresomes
    • Chaperones sometimes direct proteins to lysosomes
    • Microautophagy can degrade proteins selectively or just whatever is floating around
  62. Amyloid
    • Prions
    • Unique misfolding to make beta sheets into oligomers into long fibers
    • Once amyloid forms it can cause native state proteins to misfold and join
  63. Contemporaty epigenetics
    Mitotically or meiotically heritable changes that cannot be explained by DNA sequence changes. A cellular memory
  64. General epigenetics mechanism
    • Regulation of euchromatin and heterochromatin
    • Histone tail modification (trithorax-group (trxG) and polycomb-group (PcG) proteins
    • DNA methylation
    • Nuclear compartmentalization
  65. DNA methylation
    • 5th position of cytosine
    • There are many Dnmt proteins that do this, some are de Novo (Dnmt3a and 3b) and some are maintenance (Dnmt1)
    • CpG islands usually are hypomethylated and found near promoter regions (not pure CG but lots)
    • Imprinting!
  66. Imprinting and cancer
    • Insulin growth factor 2 (IGF2) is usually paternally expressed and regulated by maternally expressed H19
    • Male imprinting of both or female loss of imprinting or mutations can cause high IGF2 and low H19 to cause cell growth
    • Also Barr bodies are often reactivated in cancer
  67. HATs and HDACs
    • Certain histone residue modifications can recruit either histone acetyl transferases or histone deacetyltransferases
    • Usually acetyl group means relaxed
  68. HP1 and HMT
    • Depending on which lysine you methylate you can have condensation or relaxation
    • K4 (lysine 4) means relaxation
    • K9 (lysine 9) binds HP1, which binds HMT, which can methylate the next K9 lysine 9 and cause heterochromatin to spread
  69. Embryonic epigenetics
    • Condensing and relaxing proteins bound in stem cells, which chose one once they differentiate
    • X inactivation through Xist versus Tsix in humans b activating HDAC and HMT so it spreads
    • In mice there are two methods, sometimes the paternal imprinted X is completely inactivated and sometimes partially inactivated in the sperm, then at blastocyst both X copies are activated and then one is randomly deactivated in different lines