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90 Cards in this Set
- Front
- Back
RNA polymerase changes what at initiation?
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size
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Several stages of initiation
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Holoenzyme (DNA Binding/Melting)
Open binary complex (abortive initiation?) Ternary Complex (release of sigma) RNA synthesis begins |
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Holoenzyme
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Stage 1 of initiation
closed binary complex (no txp bubble) binary = enzyme + DNA |
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Open Binary Complex
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txp bubble
Stage 2 irreversible step breaking of H bonds |
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Ternary complex
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enzyme, DNA, RNA
Stage 3 RNA synthesis begins |
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abortive initiation
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if somethings wrong, kicks off RNA (1st 9 nucleotides)
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Promoter has 3 components
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-35 seq
-10 seq TATAAT : highly conserved Startpoint (5-9 bp) |
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CAT
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most common start site in bacteria
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What protects the series of bonds against nuclease?
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protein
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E. coli has different sigma factors
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sig70: general
sigS: stress sig32: heat shock sig23: flagellar synthesis they operate by recognizin different promoters sigmas factors control specificity They control promoter recognition Sigmas has important conserved regions |
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BB' contacts...
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DNA/RNA
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Jacob/Monad
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studied how txp can be regulated
used trans-acting factor found they bind specific cis-acting sequences (DNA element) Cis: close to enes they control |
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Structural gene
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encodes protein
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regulator gene
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encodes protein that regulates
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Negative Regulation
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regulation in which the gene is always expressed, except when repressor is bound to an operator (DNA element); then it is off
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Positive regulation
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gene is always off, until trans-factor associates w/cis-acting sequence; then it is on
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phage
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lysogenic or lytic
reproduce in lytic development |
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lytic development
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is a regulatory cascade
sig factor: redirects RNA polymerase to specific promotors |
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anti-termination proteins
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transcribe past termination
early/middle/late late = phage component/structural genes |
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lambda has 2 lifestyles: early
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2 genes turned on early
cro (neg regulator) N (anti-terminator) |
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lambda has 2 lifestyles: delayed early
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cII, cIII : regulators
7 recombination enes 2 rep genes Q = antiterminator l |
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lambda has 2 lifestyles: delayed early --> middle (lysogenic)
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--> cI repressor activation ---> CI repressor activation (lysogenic)
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lambda has 2 lifestyles: late genes (lytic)
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late: 10 head, 10 tail, 2 lysis genes
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lambda genes are functionally clustered
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antiterminator N: in both lytic/lysogenic pathways
ex: lysogeny : cIII maintains cII |
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similar controls apply to sigma left and right txp
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pN = protein N
acts on terminators, shuts them down |
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Lambda cont'd: early
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N & cro transcribed from PL & PR
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Lambda cont'd: delayed early
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pN permits txp from same promoters to continue past N and cro
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Lambda cont'd: late
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txp initiates at PR (b/w Q & S) & pQ permits it to continue through all late genes
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lambda has a compact regulatory region
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PRM: transcribes cI (goes to left) --> lysogeny
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Maintain lysogeny:
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only need PRM promoter, all others are turned off
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cro protein binds to OL and OR
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ensures PR & PL produce longer transcripts
leads to Q gene expression Q gene: lead to late genes --> lysis |
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Repressor determines lysogeny & cro determines lytic cycle
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lytic: PRE active, PRM shut down
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Eukaryotic Txp I
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RNA polymerase has >10 subunits
3 RNA polymerases in eukaryotes: no ability to bind to DNA in sequence specific manner (no sigma factors) |
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pol I
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nucleolus, rRNA:18S, 28S
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pol II
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nuceoplasm: nucRNA
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pol III
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nucleoplasm: tRNA, 5sRNA, some snRNA
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a-amanitin
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effect mRNA genes (inhibition)
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factor v. element
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factor: protein
element: sequence |
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Factors
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basal factors
Upstream (housekeeping) gene regulatory factors (regulatable factors) |
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basal factors:
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general txp factors
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Upstream (housekeeping) factors
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DNA binding proteins
bind upstream of +1 site |
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Gene regulatory factors
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regulatable factors
determine when and where genes are transcribed bind upstream/downstream promoter specific (higher specificity) |
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elements
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promoter
reporter gene plasmid |
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promoter
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promoter assay: oocyte system (inject) --> use DNA for promoter
transfection system: not restricted like oocytes transgenic system: can do it in vivo (live organisms) transgenic organism cell free (in vitro) system |
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reporter gene plasmid
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promoter studies
reporter enzyme: CAT, lac G (b-gal) luciferase: produces light |
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promoter boundaries are defined by what?
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deletions
-deletion analysis -delete until promoter is reached |
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pol I cont'd
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promoters: have 2 sequence component
1: core promoter element 2: upstream promoter (control element) |
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upstream promoter control element (pol I)
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100 bp upstream of +1
UBF binds recruit RNA pol I holoenzyme SL (part of TBP) |
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pol III
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promoters may be downstream (have higher variability)
standard promoter: Oct, PSE, TATA downstream promoter: boxA/boxb TFIIIc; TFIIIb TFIIIa/c |
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TRIIIc
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internal/downstream use; recruits
(Pol III) |
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TFIIIb
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binds +1 & upstream
recruits RNA poll III |
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TFIIIa/c
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also recruits TFIIIb
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TBP also assists with...
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RNA txp
(general txp factor) TATA box binding protein |
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Pol II cont'd
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a minimal promoter has only 2 elements
TATA box: recruits TBP initiator element TATA-less core promoter: still need TBP DPE element possible (downstream) |
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other pol elements
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I- core promoter
II - housekeeping/upstream CAAT box GC box (spl) III - enhancer elements IV- silencer element |
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factors assemble into an initiation complex
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many more general txp factors
has bigger task: decides what gene to describe in 20,000 genes TF3: RNA pol III, TF2: RNApol II, etc |
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Txp is controlled by a promoter & an enhancer
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enhancer: regulatory elements
DNA sequence upstream/downstream & close/far to promoter (so location independent |
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Promoters have ____ modules
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mix and match
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Enhancer proximity is required for...
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activity
looping increases proximity to promoter |
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anti-enhancer
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silencer
similar properties, but turns of txp |
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UAS
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upstream activating sequence
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Polymerases bind via commitment factors
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ex: tbp is common in all 3 pols
not unique to pol II ex: TF2b: tbp + associated factors TATA box found at -20/30 : helps determine binding site |
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TFs/ preinitiation complex
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no TF2C
TF2d: first factor recruited binds to TATA b (has tbp +series of TAFs) |
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TAF
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tbp associated factors
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TF2h
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very large complex
nine subunits multiple roles: kinase activity- CTD DNA helicase activity: repair enzymes tail |
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TF2f
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associates off the DNA
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steps: initiation complex
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pre initiation: recruit of factors and RNA pol to promoter
initiation: when first phosphodiester bond is formed elongation: movement along gene/polA termination: stop synth. RNA |
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TBP surrounds DNA from...
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narrow groove site
2 related (40% identical) conserved domains: plant tbp was the key bending is important |
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TF2b
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helps position RNA pol II
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CTD tail
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carboxy terminal domain: heptapeptide repeat of aa's like tyrosine
number of repeats vary w/species: humans = 52, yeast = 26 on large subunit, has strong affinity for TF2d, phosphorylation --> elongates or affitinity decreases ---> elongation |
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CTD is phosphorylated at
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initiation.
CTD is required for modifying: involved in processing, recruitment for RNA processing factors |
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functional domains
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DBD
TAD NLS LBD (nuclear receptors, steriod receptors) |
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DBD
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zinc finger
helix-turn-helix (homeodomain) helix-loop-helix bzip |
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histone auto-transferase
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HAT activity
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histone d-autolase
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HDAC activity
histone more + turning off txp, on reg elements for genes that are off =corepressors |
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helix-turn-helix
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the homeodomain has three alpha helices
1&2 above DNA 3rd in major groove |
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helix-loop-helix
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longer loop instead of
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leucine-zipper (dimerization domain)
DBP: b zip |
basic zipper
leucine zippers dimerize |
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How do txp activators work?
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dimerization
turn on txp process by |
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TxpI termination
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Myc: expression regulated by txpI termination
RNA pol III: after series of u's at 3' pol II: 3' gives pre-RNA of multiple sizes cleaved |
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coactivators
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TAFS: tbp associated factors
non TAFs |
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RNA processing I
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eukaryotic mRNA is modified, processed and transported
txp end modification splicing (etc.) |
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steps RNA processin
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1- cap (5' mod)
2- poly-adenylation (3' mod) 3-removal coding regions (intron splicing) |
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capping
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GTP: not encoded: GCAP not encoded
GTP- removal of 2 phosphates: GMP in reverse addition: at 5' end: reverse orientation then, methylation |
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why cap?
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1- stabilization
2- translation 3- nuclear export |
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hnRNP
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hetero-nuclear (hn)
hn RNA (pre RNA) hnRNP proteins |
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hnRNP is organized in 40s particles
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coat pre-RNA
RNA is almost never naked: associated w/proteins ex: hnRNP + RNA |
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hnRNP
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analogous to DNAs ssb proteins
associate w/RNA factors Ex: splicing factors- nuclear export stabilization capping |
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3' end can be generated by cleavage
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cleavage essential for polyA addition
cleaving: need signal on preRNA polyAsignal: AAUAA |
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Factors involved in cleavage and 3'
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1: CF1/ CF2 (cleavage factors)
2: CSTF: binds to the GU' rich region 3: CPSF: recognize and binds to AAUAA 4: poly-pap: adds A residues |