Transcription, Translation, and Regulation Flashcards
Hemophilia B-Leyden
X linked disorder that affect clotting Males have 1% of factor 9 until puberty due to mutation in DNA control element in Factor 9 promoter. At puberty, androgen receptor binds to same promoter and increase transcription so they have 60% of factor 9.
TFIIA
stabalizes TBP and TFIIB
Spinal muscular atrophy
due to SMN1 mutation can be rescued by alternative splicing of SMN2, which usually splices out exon 7
Basic Leucine Zipper (bZIP)
hydrophobic aa every 7 aa with helical domain in between. helical domain is responsible for DNA binding and dimerization C-fos C-jun
MDM2
ubiquitin ligase that marks p53 for degradation and inhibits it to make it a tumor promoter while MDM2 is bound.
Elongation of Translation
aa-tRNA enters the A-site (delivered by EF1A (euk) and EF-Tu (bac) –> anticodon base pairs with right codon in the mRNA –> peptdie bond is formed when protein chain moves from P site into the A site (requires ATP from tRNA charging) –> Translocation (GTP)
Where is Ferritin IRP located
before start codon
Amount of Activator/Repressor Regulation
target degradation by amount of protein that is available. APC, MDM2
Intron components
5’ GU 3’ AG A residue branch point pyrimidine rich segment near end of intron consensus seqeunce
Translation Initiation in Bacteria
Ribosome binds to start codon due to Shine Delgarno sequence upstream of start site. IF1 and IF3 bind to 30S –> mRNA bind tos 30S and SD sequence to place AUG in P site –> IF2 delivers initiation methionine tRNA to P site –> GTP hydrolysis on IF2 to lead to release of initiation factor and binding of 50S
Four RNA polymerases and functions
RNA POL I: rRNA in nucloeolus (busiest) RNA POL II: mRNA, snRNA, miRNA, lnc RNA RNA Pol III: tRNA Ecoli RNA: mRNA, tRNA, rRNA
Aspirin function
blocks IKB phosphorylation, so NFKB is stuck in the cysotol and transcription is not activated in an immune response
Kozak
Euk cells have different start codons that are of different strengths. Kozak sequences has a purine a couple Bs before AUG and a G directly behind it.
Iron Response Element (IRE)
RNA stem and loop found in mRNAs that bind to IRPs (Iron response proteins)
Mutations in ssDNA binding protein diseases..
1) Carinosynostosis 2) Androgen Insensitivity Syndrome 3) Waardenburg Syndrome Type II
cdc25 vs p53
p53 must be phosphorylated to be activates to repress cell cycle. Slow process because p21 needs to be made. cdc25 is normally an activator of cell cycle, so it is repressed with DNA damage. faster because nothing needs to be synthesized.
NFKB
associated with inflammation
sequesterd in cytoplasm by IKB that hides Nuclear localization signal. with inflammation, IKB gets phosphorylated and targeted for degradation. Now NFKB can move to nueclues
Id protein
a homodimer that is missing the basic domain of the Helix loop helix
IES Driven translation
cap independent IRE are structural RNA sound upstream of AUG and recruit 40S and IFs without using cap found in viruses ideal if you want to turn off protein synthesis, but not all
what energy source does Aminacyl tRNA synthetase use?
ATP to create an ester bond
CDKN1
cip/kip inhibit any CDK complex
High iron conditions
IRE-BPs are bound to FE and an’t bind to IRE-RNA since IREBP cant bidn to IRE, mRNA is no longer protected and transferring receptor creation is halted. mRNA proceeds and ferritin is created.
Calcenurin
Immune response protein
Removes phosphate group from NDAT, with uncovers NLS to move to nucleus to activate genes in immune system. if blocked, it is an immunosuppresive
CDKs
Cyclin Dependent Kinases (ser or thr) use ATP energy to phosphorylate regulator to activate or inhibit replication Present at all times, but is inactive unless bound to cyclin
Tamoxifen
anatogonist to estroben binds to Estrogen receptor without providing conformation change for dimerization - thus prevents binding to activation site on DNA and the recruitment of activators. Represses genes normally used in cell growth
Leukemia
fusion of HATS with other proteins to misdirect HATS to different region so chromosome and promote creation of proteins that arent supposed to be made
Polycisternic
more than one protein encoded on an mRNA
Common in bacteria to have multiple coding regions with multiple SD sequences convenient to make proteins that do similar function at same time ie. heat stress
Translocation
Movement of one site From A to P or from P to E Requires GTP
Heterochromatin
repressed because DNA is inaccesssible due to 1) hypoacetylation 2) DNA methylation 3) inaccessible to restriction enzymes and DNAse1 digestion 4) contains tandemy repeated segments 5) transcriptionally inactive.
Ferritin
sequester Fe outside of cell
Tetracycline
affacts aminacyl tRNA binding in A site
Histone DNA interactions
are electrostatic phosphodiester bonds in DNA associate with positively charged arginines located on histone folds
CDK7
phosphorylates Pol II C-Terminal Domain during promoter clearance
Modularity
of SS - DNA BP, modular and contain two major domains that can be switched and mixed to activate a variety of genes
E boxes
are bHLH show basic region binds to E box in DNA. when homodimerized by Id protein, but missing basic domain on one side so it can no longer bind to E box. Competative inhibitor to active E box.
What happens to cause transition from G2 to M phase?
cycB with CDK2
What happens to cause transition from S into G2 phase?
cycA activates and pairs with CDK2
Carinosynostosis
premature fusion of skull to lead to mishapen head and no room for brain to grow point mutation in Msx2 homeodomain from proline to histidine. causes tighter binding to DNA to transcribe genes more rapidly and cause premature fusion
mTOR
regulates translation initiation
Phosphorylates 4E-BP1 complex to release 4E so it can participate in initiation iwth eIF4F. under stress, 4E-BP is dephosphorylated and sequesters 4E Rapamycin mimics the actions of mTOR and phosphorylates 4E-BP
CDKN
CDK inhibitors CDKN2 (ink4) and CDKN1 (cip/kip)
Histone H1
binds to linker DNA to condense 10nm fiber into 30 nm fiber
Low iron conditions
IRE-BPs are free from Fe and can bind to IRE-RNA Since IREBP is bound to IRE, mRNA is protected and synthesis of transferrin receptor is created. IREBP binds to IRE to block access to the RNA to translate ferritin.
During G1 - CDK levels and pre-initation complex
Need to replicate
Low levels of active CDK 4,6 Allows for the building of Pre-RC but it is NOT activated
where is transferrin IRP located
after stop codon
What is RB?
Retinoblastoma protein 2 homologs p107 and p130 (you might have both or only one) When bound to E2F, it prevents transcription of genes involved in DNA replications. Activation of CDK by cyclin phosphorylates Rb, causing the release of E2F so it can not longer inhibit transcription.
what triggers phosphorylation of eIF2alpha?
oxidative stress
double stranded RNA (virus) heat sock UV amino acid depravation Interferon
Energy requirements for Translation
charge tRNA 2ATP –> 2AMP deliver aa-tRNA to A site –> GTP –> GDP Translocation GTP –> GDP
ATP dependent Chromatin Remodelers
Use ATP to break histone DNA contact and move histone along octamer ex. SWI/SNF
Translation Initiation Euk - Cap Indepenent
Internal Ribosomal entry sites drive the process Viruses use IREs to initiate translation after they shut down host cap-dependent synthesis some Euk use too when cells want to shut down protein synthesis, but need emergency response proteins made.
cdc25
is an activator of CDKs. Inhibition of it by Chk1 and Chk2 causes it is inhibit CDK action.
During S phase - CDK levels and pre-initation complex
Active replication HIhg levels of CDK activates Pre-RC but prevents further building
what blocks phosphorylation of IKB?
aspirin
Transferrin Rector
transports iron and transferrin into cell
protein binding to mRNA
3’ and 5’ UTR can be bound to proteins that affect mRNA stability, localization, degree that ribosomes bind
TATA binding Protein
binds to TATA box at minor groove helps direct assembly of initiation complex not highly specific and recognizes variations in TATA boces
Streptomycin
affect initiation and elongation in 30S
What happens when p53 is phosphorylated?
1) leads to the transcription of P21 (P53 is a TF) 2) p21 binds to whatever CDK is present to prevent from entering into S or M phase.
what CDKs are associated with entry into S phase?
CDK 2, 4, 6
Histone Code
distinct patterns or combinations of modifications at conserved regions of histone terminal domains that are important in the recruitment of specific chromatin-associated proteins.
Iron Response Protein
bind to Fe and regulate expression of Ferritin and TFR
how is binding specificity determined?
non-covalent interactions between atoms in an alpha helix in the DNA Binding domain and atoms at the edge of the base of the major groove of DNA (throguh H bonds and van der walls)
Histone Deacetylases
HDAC retain positive charge on terminal end (reove acetyl group) to maintain interaction with DNA and prevent access to promoter.
what is the goal for somatic cell cycle?
duplication of the genome in S phase followed by exact division of genome in M phase t produce two identical daughter cells
Reactions of RNA capping
1) triphosphatase (cut off triphosphate group) 2) guanylyltransferase (add GTP to form 5’ to 5’ triphsopahte bond. 3) guanin 7 methyl transferase (methylate 7th position)
What phase are the sister chromatids separated
Anaphase
What antibiotics affect peptidyl trasnferase in 50S?
Erythromycin, linconmysin, clidamycin, chloramphenicol
Rad17
senses DNA damage due to radiation (ds and ss breaks) Binds to DNA and recruits ATM and ATR
Elongation factor
proteins that deliver tRNA and move ribosome down mRNA
Marfan’s Syndrome
mutations that disrupt splicing of the fibrillin gene transpcript that is involved in the CT of walls of heart and blood vessels
CDKN 1 vs CDKN 2
CDKN2 is specific to CDK 4,6, while CDKN1 is for any CDK
Chk2 and Chk1
phosphorylated p53 and represses cdc25.
Estrogen Receptor
must homodimerzie to bind Estrogen binds to receptor to faciliate the homodimerization and binding of zinc finger to DNA. when bound to DNA requires HAT to recruit Pol and activate Transcription
Interferon
virus invades cell, cells make interferon in response to viral RNA and cell can either die or survive. Interferon is released form infected cells and activates those cells to synthesize viral proteins and turn off translation (phosphorylated eIF2Alpha) to protect them from virus infection.
how are sequence specific DNA binding proteins regulated?
1) altering protein conformation by binding to ligand 2) regulating entry into nucleus 3) regulating amount of TF in cell 4) regulating binding to DNA 5) phosphorylation of DNA BP
TFIIH
creates closed complex composed of 1) XBP 2) XPD 3) p44 4) CDK7
Translation Initiation Euk - Cap dependent
eIF4E binds to 5’ Cap –> binding brings in other eIF and small ribosomal subunit –> ribosome scans down the message for AUG –> At AGU, large subunit joins and IF release and initiation is achieved.
alpha-amanitin
non-competative inhibitor in mushrooms for RNA Pol II binds to bridge helix and block RNA elongation
U2
activates the 2’ OH at the branch point, which attaches a phosphodiester bond just past the GU 5’ splice site. this creates a free 3’ OH on the 5’ exon which attacks the AG 3’ splice site.
RNA Capping function
1) resistant to exonucleases 2) enhance subsequent maturation steps (splicing) 3) export from nuc 4) association with ribosome via eIF4E 5) removal signals mRNA degradation
ATM vs ATR
ATM specifically recognizes double stranded breaks ATR recognizes any kind of damage (Ds, stalled rep fork, DNA mismatch, NT damage) Activate Chk2 and Chk1
Steps of exit out of G1
growth factors accumulate in media to initiate signal cascade of proteins to signal that it is time to divide. (Ras, efos, jun) that lead to the synthesis of cycD1-3 –> cycD binds to CDK4, or 6 CDK4,6 activates cycE –> activates CDK2, which together inhibit RB protein to allow transition to S phase.
XBP
associated with TFIIH ATP dependent helicase activity near DNA start site.
modification that alters activity of SS DNA BP
CREB Ligand binds to G protein which phosphorylates CREB. p-CREB recruits CBP (a hat) and recruits RNA Pol II leading to trascription
Homeodomain Proteins
60 AA 3 alpha helices and a terminal domain Regulate development Hox, Pitl, Msx Affect many genes at once
DNA binding activity of Sequence Specific DNA BP inhibition
when DNA binding sequence has been altered so cannot activate transcription Id proteins with E boxes
Histone Tails
exposed site subject to modification that regulate chromatin structure and ability to bind to DNA
Rubinstein-Taybi Syndrome
gentic disorder, growth and metnal retardation, carniofacial dysmophism, broad thumbs Mutation in one copy of CREB binding protein that acts like a HAT
CDKN 2
Ink4 CDK inhibitor specific inhibitor to CDK 4,6 competative inhibitor with cyclin D. p16A is frequently mutated in Cancer, so cell cycle is always on.
Basic Helix loop Helix
Similar to leucine Zipper, with hydrophobic domian, but has loop in between. regulates muscle development myoD, myogenin, myf5
How is cycD synthesized?
Due to the accumulation of growth factors (Ras, efos, Jun) in the media during G1 t
Cell Cycle Checkpoints in G1
Size at restriction point (dependent on cyclin accumulation) DNA damage to assess whether you replicate this DNA.
Chromatin Remodeler Classes
1) ATP dependent 2) Histone Modifying enzymes
CD44
cell surface glycoprotein contributor to tumor metastasis and migration splice variants are used as diagnostic and prognostic markers
HAT
Histone acyetyltransferases Acetylate the N-termini Lycine of histone tail To neutralize positive lycine and eleminate electrostatic ineraction with DNA (co-Activator)
Fragile X syndrome
1:1500 males, mental retardation, dysmophic facial features, in normal: 6-50 CGC repeats at 5”end of FMRI gene to facilitate methylation of cytosine residues and transcription activation. in Fragile X, they have 200+ copies of CGC repeat and silence transcription
Promotor Proximal Elements
located 200 bp upstream of start site 20bp long regulates tran and binds to factors in cell type specific manners must be near start site
Cell Cycle Checkpoint in G2
Assess how DNA replication went. Time to fix the mistakes before proceeding to mitosis
Ribosome
Bacteria small is 30S Euk small is 40S 3 binding sites (A,P,E) small subunit decodes mRNA Large catalytic center of peptidyl transferase
E2F
binds to RB protein to be a protein inhibitor.
Examples of IRE driven tranlsation
polio HIV-1 HepA and C
APC
targets Beta-Catenin, a cell proliferation activator normally B-Catenin is in membrane and if excess exits it is degraded by APC In abnormal situations: WNT signaling leads to phosphorylation and inhibition of APC so B-Catanin cannot be degraded and builds up in cytoplasm. Build up leads to translocation tp nucleus to activate cell growth CANCER!
Alternative Splicing
1) exon skipping 2) mutation exclusive exons 3) exon truncation/extension at 5’ end by choosing alternative spice site at 3’ 4) exon truncation/extension at 3’ end 5) intron retainment or removal
Anisomycin
affect peptidyl transferase at 60S (cap dep)
Constitutive Heterochromatin vs. Facultative
faculative can change to euchromatin based on cell type or development stage
Histone Modifying Enzymes
Regulate chromatin structure by recruting Histone acytylases and histone deacyetylases
TFIIF
helps target POL II to promoter by interaction with TFIIB
Mutations in DNA contorl elements lead to these disease….
1) Thaiassemias 2) Hemophilia B-lyden 3) Fragile X
Cycloheximide
affect peptidyl transferase at 60S (cap dep)
ApoB
mRNA is edited by cytosine deamination to change CAA to UAA to make a premature stop codon in the bowel. Thus in the liver you have full length, but in the SI there is a truncated. both are functional, with different functions
Formation of Origin of Replication
Origin of Replication protein (Orc 1-6) binds to origin cdt1 and cdc6 bind and bring in DNA helicase (Mcm2-7) which forms ring around DNA.
BRCA2
familial tumor suppressor involved in DNA damage checkpoint. women get both breast and ovarian cancer with this mutation. see fusion of chromosomes in mice. in disease state, DNA damage persists and you get non-homologous recombination
Zinc Finger Domains
composed of either 4 cysteine or 2 cysteine and 2 Histine 2 anitparalle beta sheets, an alpha helix, all held together by a zinc ion Finger: DNA binding part Nuclear recpetors: estrogen, androgen, retinoic acid
Bridge Helix
Crab claw in RNA pol that flexes with each addition of rNTP
Cell cycle of somatic vs. Germ cells
Meiosis: start with diploid and undergo two rounds of meiosis to make 4 haploids. mitosis 2 2n cells Meiosis has 10,000x more recombination between non-sister chromatids than mitosis.
Somatic vs. ebryonic cell cycle replication
Somatic cells only have one origin of replication and it takes 8-10 hours. Embryo have multiple origins that are used and it only takes 30 minues
Overview of DNA checkpoint regulation
1) DNA senses DNA damage or replication block 2) they then send signals (kinase) to phosphorylate effectors. 3) effectors stop cell cycle until damage is repaired.
Transferrin
binds to iron - present when iron is lacking in cells
Restriction Point
in G1 point at which a cell must make a decision based on the presence of growth factors or hormones are there or whether cell is large enough to undergo DNA replication. and mitosis. most highly regulated step In embryonic cells this is bypassed exit point for cell differentiation
CDC7
activator of DNA replication activated when Rb is inhibited works with Dbf4
Poly A and termination of trascription
cleavage and PolyA signal to RNA polymerase to stop if you delete Poly A tail, RNA pol will continue working
Nucleosome
repeating unit of chromatin, 147 bp of DNA wrapped around octamer of histone proteins approx 1.7 times
what is the positive feedback loop during the transition to S phase?
Activated CDK 4,6 phosphorylates cycE to activate CDK2 Ireverisble once committed
Waardenburg syndrome Type II
deafness, pigmentation, anomalies in eyes, mutation in MITF gene, which encodes basic Helix loop helix
Kasugamycin
affects initiator tRNA binding in 30S subunit
Dbf4
activator of DNA replication activated when Rb is inhibited works with CDC7
Mutation in TFIIH
1) Xeroderma Pigmentosum 2) Cockaynes Syndrome 3) Trichothiodystrophy
Histone core complex
2 molecules each of H2A, H2B, H3 and H4 (octamer)
Significant features of RNA POL
1) four channels 2) Bridge Helix 3) Sliding clamp (PCNA) 4) C-Terminal Domain
TFIIB
binds to either side of TBP
Alternative Poly A sites
1) membrane and secreted cells of immunuglobulin are generated by altnernative Poly A site 2) can lead to cancer because it makes truncated proteins
Euchromatin
where genes reside, more accessible
C-Terminal Domain
On RNA pol composed of heptad repeats YSPTSPS reversibly phosphorylated binds to proteins to regulate elongation and processing of pre-mRNA
Transcription Factor
Activators or Repressors that are encoded on one gene and act on another gene to regulate transcription. Diffuse around nucleus and bind to DNA control elements
sequence specific DNA binding proteins - nuclear entry regulation
A receptor that is located in the cytosol until it binds to a certain protein or is sequestered by a certain protein, but a event triggers their release and it can freely enter into the nucleus (glucocorticoid and NFKB, Calcenurin)
Function of PolyA
Protection from degradation Nucleus export bound by PolyA binding protein to enhance translation
Androgen Insensitivity Syndrome
Feminization or undermasculinization of external genetalia at birth, abnormal 2°, infertility mutation in DNA binding domain or ligand binding domain of androgen receptor (zinc finger) makes them less responsive to androgen.
eIF2alpha phosphorylation
inhibits ternary complex formation eIF2 brings in t-RNA-Met into P site, so when phosphorylated this does not occur.
Termination Factors
end the process at the stop codon and dissociate the subunits to be used against
What are the classes of activators and repressors?
1) sequence specific DNA binding proteins 2) Co-Factors
Rapamysin
affects phosphorylation of 4EBP at mTOR (cap dependent)
Promoter
-70 to +30 control direction and frequency of transcription TATA box (-30 bp) promoter proximal elements (-30 to -1000 Bp) Enhancer region (-10,000 to -50,000 bps)
Histone Acetylation altered in disease
Leukemia Rubinstein-Taybi Syndrome
Translation Termination
Stop codon detected by release factor (not made out of tRNA) go in same space as tRNA but trigger termination of peptide chain and release of peptide and ribosomal subunits.
what happens when RB is inhibited?
CDC7 binds to Dbf4 which are both kinases needed for DNA replication
During mitosis - CDK levels and pre-initation complex
Replication is complete High levels of CDK2 to initiate mitosis prevents the building of pre-replication complex.
