Molecular Genetics Exam 3

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RNA polymerase changes what at initiation?

Back 1

size

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Several stages of initiation

Back 2

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

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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