Exam 3 Flashcards
(132 cards)
1
Q
exon / intron junct rule
A
in DNA: 5’ GT-AG 3’
in RNA: 5’ GU-AG 3’
when GT-AG is mutated, there is less RNA making it to the 40S (translation complex)
2
Q
components of the spliceosome
A
pre-mRNA, snRNPs (U snRNA and associating proteins), ATPase/Helicases
3
Q
5’ splice site
A
GU in RNA
4
Q
3’ splice site
A
AG in RNA
5
Q
branch point
A
A in RNA that helps facilitate the attack on the hydroxyl group to facilitate splicing, part of the BPS (branch point seq)
recog by U2
6
Q
splicing cycle: complex order
A
E > A > B > Bact > C > P
7
Q
splicing cycle: complex E
A
early complex, U1 snRNP recognizes the 5′ SS
8
Q
splicing cycle: complex A
A
U2 snRNP recruited to BPS
9
Q
splicing cycle: complex B
A
U4/6 & U5 tri-snRNP recruited
(U4 and U6 are always dimerized when outside of the spliceosome, are recruited together) (this is the only time all 5 snRNAs are together)
10
Q
splicing cycle: complex Bact
A
U1 e U4 snRNP displaced, spliceosome ready for catalysis
(still contains U2, U5, e U6)
11
Q
splicing cycle: complex C
A
2 transesterification reactions
complex contains U2, U5, e U6
12
Q
splicing cycle: complex P
A
post splicing
13
Q
features of snRNAs
A
have tri methyl cap specific to snRNAs (me3, not m7G)
U1, U2, U4, and U5, are transcribed pol II, U6 transcribed by pol III
14
Q
Sm ring
A
all snRNAs associate w the Sm ring
Decisive factors that determine snRNPs: 7 proteins that assemble into ring, form Sm group (that forms on Sm ring)
15
Q
what are the essential interactions between the snRNA and the pre-mRNA
A
U2 : Branch point sequence (BPS)
U6 : 5′ splice site (5′ SS)
U5 : 5′ exon
16
Q
which snRNAs make up the core of the sliceosome
A
triplex of U2, U5, and U6
17
Q
what does U4 do in the spliceosome
A
brings U6 to the core of the spliceosome, prevents it from forming the triplex too early
18
Q
what makes up the splicing active site
A
the intramolecular stem–loop (ISL) of U6 snRNA, helix I of the U2–U6 duplex, the associated Mg2+ ions and loop I of U5 snRNA
19
Q
what are the types of alternative splicing
A
alternative splice site selection (5’ or 3’) - exon-exon-intron-exon (5’) (or exon-intron-exon-exon (3’)) where the middle exon can be included/excluded
cassette-exon inclusion or skipping - exon-intron-exon-intron-exon where either both introns are removed and 3 exons expressed, or whole middle of intron-exon-intron is removed leaving only 2 exons expressed
intron retention - exon-exon-exon where the middle exon can be included/excluded
20
Q
how is alternative splicing regulated
A
by cis-elements (ESE, ESS, ISS, and ISE) and trans-acting splicing factors (SR proteins, hnRNP, and unknown factors)
they affect the recruitment of snRNPs (U1 and U2) to promote or repress splicing
SR and hnRNPs are ubiquitously expressed, but there are also tissue-specific splicing factors
21
Q
cis-elements that regulate splicing
A
ESE - exonic splicing enhancers
ESS - exonic splicing silencers
ISE - intronic splicing enhancers
ISS - intronic splicing silencers
22
Q
trans elements that regulate splicing
A
SR proteins (serine Arginine rich proteins)
hnRNPs (heterogenous ribonucleoprotein particles
23
Q
where are ISEs and ISSs found, what do they do
A
in the intron regions
ISEs promote splicing and ISSs repress splicing
24
Q
where are ESEs and ESSs found and what do they do
A
in the exon regions
ESEs promote splicing and ESSs repress splicing
25
what do SR proteins do
they help promote splicing, binding of SR to ESE promote the inclusion of that exon
26
what does hnRNP do
inhibits splicing, binding of hnRNP to ESS promotes the exclusion of that exon
usually binds silencingers
27
how do ESEs and ISEs affect splicing
promote splicing
promote the binding of U1 to the 5' ss and U2 to the 3' ss
28
how do ESSs and ISSs affect splicing
inhibit splicing
prevents the binding of U1 to the 5' ss and U2 to the 3' ss
29
how can you test for cis elements that regulated splicing
make a plasmid that encodes for GFP if an exon is kept/removed, measure GFP
30
HITS-CLIP assay
UV crosslink RNA to protein > Ab against protein of interest > pulldown protein > SDS-PAGE to isolate protein/RNA complex > purify RNA > make library / deep seq > what seq of RNA is bound to that protein?
have to control HITS-CLIP to relative abundance of RNA, if CLIP=relative abundance > probably background
Keys: (1) UV crosslinking is irreversible allows for RNA/protein complex to not come apart (need to digest protein to isolate RNA) (2) SDS-PAGE e transfer to membrane, RNA does not transfer only protein does, therefore only RNA that will be on the mem is that bound to proteins, reduces contaminants when purifying RNA
31
RNA Bind-n-Seq assay
Give RNA binding protein random pool, diff amounts of RBP, purify protein > which RNAs enriched by RNA binding proteins?
pure in vitro
pros: not need crosslinking or good Ab spec to protein of interest
32
back splicing
alt splicing that generates circular RNAs
has 5’ downstream e 3’ upstream, connects molecule 5’ to 3’ but backwards > circular RNA
Features: longer lived bc doesn’t have defined cap or polyA or translation med degradation
funct: RNA sponge, sequesters away RNABPs; miRNA sponge, has lots of binding sites for miR
33
functs of introns
encode lncRNAs, miR precursors ('miRtrons')
regulate yeast growth
34
components of the ribosome
RNA protein complex (RNP), full is 80S
large subunit: 60S, made up of 5S, 28S, and 5.8S + 47 RPs
small subunit: 40S, made up of 18S + 33 RPs
pol I transcribes 47S pre-rRNA which makes 18S, 28S, e 5.8S
pol III transcribes 5S rRNA
35
genome structure of ribosomal DNA
many copies throughout human genome
each individual can have diff number of ribosomal DNA, varies person to person
seq can vary between the copies on ea chr
hard to seq bc so many repeats
(normal genes only have 2 copies)
36
which genes funct as master regulators of rRNA transcription
p53, AKT, mTOR, c-Myc
in cancer, mut these genes > more ribosomes, multi-nucleolus e proliferation
37
ribosome assembly overview
rRNAs co-transcriptionally assemble w 200+ assembly factors around 80 snoRNAs e RPs as pre-90S ribosome (includes 47S pre-rRNA)
1st: cleave pre-60S and pre-40S (separately) by U3 snoRNP co-transcriptionally
added stepwise: assembly factor > fold e process (w RNases) > assembly factor > fold e process, repeat as it moves from nucleus > nucleoplasm > cytoplasm
folds from outside towards inside/middle
peptidyl transferase activity is last part folded
assembly factors prevent premature folding, stepwise association / dissociation of maturation control folding
38
functs of snoRNPs on rRNA
U3 snoRNP processes rRNA co-transcriptionally (NOT the same U3 from splicing)
put modifications on the rRNA, C/D box puts methylation e H/ACA box RNPs put pseudouridination
most mods (methylation e pseudouridination) conc at interaction between 60S e 40S
39
U3 snoRNP on rRNA
processes rRNA co-transcriptionally (not same U3 from splicing)
40
C/D box RNP funct
snoRNPs that mediate methylation of rRNA
imp for folding e funct
41
H/ACA box RNP funct
snoRNP that mediates pseudouridination
imp for folding e funct
42
where are most rRNA modifications
concentrated at interaction between 60S e 40S
imp for folding e funct
43
where do snoRNAs come from
mostly introns
44
order of ribosome folding
folds from outside towards inside/middle
the peptidyl transferase is processed last, do not want random peptide formation
assembly factors prevent premature folding, stepwise association and dissociation of maturation factors controls folding
ATPase recruitment is also stepwise
45
Rea1 funct
nuclear AAA-ATPase, funct in late rRNA processing / nuc export of 60S ribosomal subunits
has 'physical power' uses dynamin on MTs, causes ribosomal assembly factors to dissociate, imp for maturation e export
46
ATPases in ribosome biogenesis
ATPases also funct in stepwise manner, similar to assembly / maturation factors
ribosome biogenesis is E consuming process, uses GTPases, AAA-ATPases, ATPases, helicases, e kinases
ribosomes have long half-life, so very E heavy but last awhile (tho take several hours to make)
47
how is premature translation initiation blocked during ribosome biogenesis (40S subunit)
last step of ribosome biogenesis
block: subunit interface, platform, binding of essential translation iniation factors (elF3), block initator tRNA, entry channel, large subunit binding
TEST: can 60S join? does GTPase funct?
TEST: communication w 60S P-site?
TEST: GTPase site funct? A-site funct?
48
maturation and QC of 60S subunit
blocked: stalk, small subunit joining, exit tunnel
to mature: join w small subunit, move to cytoplasm, mature peptidyl transferase center
TEST: check if mature ribosome has ability to activate GTPase
if funct, then will be released and mark mature ribosome
opening exit tunnel is last step
49
diff in bacterial / eukaryotic / mammalian ribosomes
big size inc from bacteria > yeast > human
more rRNAs and proteins
larger proteins, inc in bp size
eukaryotes have 'tentacle' pieces (expansion segments) that stick out and give extra functs
50
expansion segments
eukaryote specific rRNA domains
basic ribosome structure is very conserved but expansion segments vary
varies between species, between individuals of same species, between cells of same individual, and between ribosomes in same cell
tissue spec translation programs
result of differences: diff levels of activity dep, preference for translating diff subsets of mRNAs
51
ribosome profiling
Cell lysate > treat w RNase > where ribosome bound, protected from RNase > RNA seq > looks at ribosome 'footprint'
Enrichment between start and stop codons
RNA seq will show whole transcript
Can see which transcripts are highly translated / have higher ribosome density
52
what is translation efficiency
number of RPFs (from ribosome profiling aka ribo-seq) / mRNA abundance (RNA seq)
which transcripts are highly translated / have higher ribosome density
53
naming conventions: eukaryotic translation proteins for initiation, elongation, e termination
initiation: elF (1, 1A, 2, 2, etc)
elongation: eEF (1A, 2)
termination: eRF (1, 3)
54
pioneer round of mRNA translation
used for quality checking mRNA
recog by CBC, which recruits the complex e begins scanning for start site
form of non-canonical translation initation (elF4E indep, canonical uses elF4E)
if EJC remains, mRNA degraded by NMD
55
IRES translation
internal ribosome entry site, used by viruses to initiate translation
Type I is elf4E indep, scans UTR to find AUG
Type II e III do not have scanning step, directly recruited 40S to start site (more similar to prokaryote)
Type IV is most primitive, has mimicking tRNA that can make codon-anticodon binding, starts translation directly from RNA, doesn’t use AUG
56
how do viruses repress cellular mRNA translation
inhibit the translation of capped mRNA, target elF4F, elF2, or elF3, leaves only IRES dep translation, reg global translation activity
57
transcription v translation on/off state
by default, transcription is more regulated, can be turned on or off while translation is always on
how can you turn translation off: integrated stress resp
58
integrated stress resp
reduce global translation by affecting translation initiation factors:
- methionine tRNA is required for initiation, the amount of it is reg by stress, affect ability to initiate
- phosphorylation of elF2alpha, diff stress activates diff kinases that all act on elF2alpha > suppresses global translation initiation
- aa depletion
by reducing global translation, you can turn on spec translation
59
translation activation of specific transcripts under ISR
in default: Gcn4 has 4 upstream ORFs, effectively sequestering ribosomes away from Gcn4
when aa depleted: less ternary complexes, ribosome has more space to bind > Gcn4 can be made
60
what is tunicamycin
Tm
ISR inducer
induces Gcn4 (yeast) / ATF4 (eukaryotes)
61
translation reg and neurodeg diseases
constant ISR activation leads to cell death > neurodeg
elF2alpha phos in AD
62
what is ISRIB
integrated stress response inhibitor
reverses the effect of elf2alpha-phos on translation (but doesn't affect the actual phosphorylation)
accelerates elF2B activity (mediates GDP to GTP for elF2alpha to consume) in the presence of elF2alpha-phos
represses Gcn4 (yeast) / ATF4 (eukaryotes)
63
what is RAN translation
repeat associated non-AUG translation, associated w neurodeg diseases,
non-canonical translation initiation (non-AUG) produces repeat derived small peptides > small peptides aggregate > toxic to cell > neurodeg
ISR required > PKR activation > RAN
PKR is activated by the repeat expansion, which can form hairpin structure to trigger PKR
presence of repeat not necessarily cause disease but big size can
64
mTOR translation
activates global translation at both initiation e elongation
2 functs: (1) mTORC1 phos 4EBP, dissociates it from elF4E, allows elF4E to be more free to bind mRNA, (2) mTORC1 phos S6K, inc active eEF2, promote elongation
mTORC1 activation priotritizes production of ribosome proteins, PABPs, translation e elongation factors (need to make ribosomes / translation to facilitate everything else, inc global translation)
inc polysomes, leads to immortalization
TOP motif - CT rich in translation start site, activated by mTORC1
imp in cancer e oncogenesis (and limb regen in axolotls, tho their mTOR is diff than ours)
65
polysome profiling
look at global translation efficiency
gradient > sep ribosomes by density > sep monsomes from poly somes > ea peak rep one unit on mRNA (1st peak = monosome, 2nd peak = 2 ribosomes on mRNA, etc)
the number of polysomes shows activity of translation
66
mTOR inhibitors
rapamycin
PP242
torin
67
how do you inc translation of spec transcripts
need global repression, then activation of spec transcripts
can use specialized ribosome or specialized transcript (w hairpin to affect scanning / initiation)
utilizes elF3, which is needed for global translation, can associate directly w spec transcripts to initiate them
imp for tissue spec translation patterns
68
EF1A funct
mediating the delivery of aminoacyl-tRNA to the ribosome's A-site during translation
69
co-translational n-term processing
not processed until start to exit from tunnel, recog by signal peptide if present, recruits SRP, brings to ER or mitochondria, translation resume
if no signal peptide, binds METAP1, which can remove initiation methionine, recruitment to chaperones to fold as being translated
HSP70 directly binds to coding center, reg translation speed, give more time to allow for proper folding > inc fold fidelity
70
co-post assembly
formation of one protein allows for the translation of another protein by binding the second protein co-translationally (as it is being translated)
71
co-co assembly
translation of two proteins are linked, both need to be translated at same time and bind ea other co-translationally
72
codon biases
two ways: tRNA abundance or codon usage
can affect:
- time spent on ea codon (diff codons that encode same aa, same aa will have diff amounts of tRNA for ea codon, allow reg of translation speed)
- speed of protein syn
- protein folding (slows translation, allow more time for folding)
- mRNA stability (can cause COMD)
73
how is tRNA abundance regulated
level of ea tRNA affected by the copy number of ea tRNA in the genome
amount of tRNA that has been aminoacylated by reg the aminoacyl transferase
can be tissue specific (ex: neurons express many diverse tRNAs), affected by stress > translation of diff transcripts
74
how do zygotes switch from maternal to zygotic transcription
diff in codon optimality between maternal and zygotic mRNA, maternal is selectively degraded by miR that selectively targets the 3' UTR of maternal
75
why does the speed of translation affect protein levels
faster > able to make more faster
ALSO, stalling causes collision, which can knock off ribosomes, 'crashing' is toxic to cells
can look at formation of disomes using ribosome profiling footprint, disomes will have larger nt steps than monosomes (disome is ~ dbl)
collision can be sensor trigger for ISR
76
ribosome collision
toxic to cells, result of ribosome stalling
stalling can be caused by non-optimal codons, aa starvation, damage in mRNA, rRNA, or tRNA
more collision > more disomes detected, if too many disomes / too much collision > apoptosis
collision triggers ZAKalpha-phos > apoptosis
ribosome collision can trigger ribosome quality control (RQC)
can also cause +1 frameshifting if NGD not fast enough > production of aberrant protein > imm activation / oncogenesis
77
ribosome quality control
ribosome stalls (NGD) > ribosome gets ub > cut mRNA (dont know if ribosome or mRNA is problem)
degrades mRNA, ribosome, e nascent peptide
if nascent peptide is short e no lys present, cannot be ub, undergoes template indep aa addition
78
template indep aa adition
Add aa indep of mRNA, recruits ala e thr tRNA, makes CAT (carboxyl-terminal alanine e theronine) tail on C-term, becomes target for aberrant protein degradation
79
complementarity: siRNA v miRNA
siRNA has 100% complementarity
miRNA is not perfect match, requires processing, most often binds at 3' UTR of mRNA not CDS
miRNA needs imperfect match in seed region, can inc stability of interaction w 3' end of miRNA supplementary pair but seed is most imp
80
antisense v dsRNA treatment effect on RNA levels
antisense causes some dec, dsRNA causes complete degradation of that mRNA > RNAi funct at RNA stability level and req dsRNA
exogenous dsRNA trigger causes targeted degradation of endogenous mRNA
81
paradigm shift from the nobel prize on RNAi
dsRNA causes global translation suppression in mammalian cells dsRNA is incapable of further specific base pairing
82
discovery of proteins needed for RNAi
- genetic screening: mut c elegans, treat w dsRNA that prevents reproduction, only those w mut RNAi pathway will not degrade the RNA, will be able to reproduce
discovery of Argonaute (Rde-1 in C elegans)
- biochem purification: Transfect drosophila cells w dsRNA > activate RNAi, degrade endogenous > lyse cells > if RNAi is working can add ssRNA, should be degraded
treat w micrococal nuclease (degrade DNA e RNA) or DNase I (degrade DNA) > show that need RNA for RNAi
discovery of RISC (RNA-induced silencing complex)
- column purifcation: column separate lysate, test ea fraction for ability to RNAi, columns that can do must have needed machinery, Ago-2 is present
- candidate testing: treat w known dsRNA enzymes, only Dicer can process long RNA to short RNA (22nt) > Dicer is req
83
components of RNAi
trigger: dsRNA
components: Dicer, siRNA, RISC (RNA-Induced silencing complex, contains Agonaute)
84
RNAi overview
dsRNA is processed by Dicer, small RNAs that match gene of interest loaded into RISC (contains argonaute), targets mRNA for degradation
85
Dicer
cuts dsRNA into 22nt size frag to be degraded by RISC/Ago
86
dsRNA sensors
MDA5, PKR, 2', 5' OAS
all can trigger dsRNA degradation
87
defining features of miRNA
- don't have 100% complementarity to target
- have phosphate e hydroxyl groups
- microRNAs belong to the same family tend to cluster
- precursor transcripts ALWAYS fold into hairpin struct
- mature miRNA can originate from either arm of the hairpin
- mature miRNA favors U at +1 position
- certain miRNA genes are conserved and differentially expressed in development and various tissues
- no complete antisense sequence found in the c. elegan’s genome for its miRNAs
causes degradation of gene they target
used to be called stRNA (small temporal) bc expressed at spec times
also uses Dicer / Ago, but they are imp for MAKING miRNA, which is why KO Dicer / Ago is same as removing miRNA
88
strategy for cloning miRNAs
purify from gel > enrich (needs to have end funct groups (phosphate e hydroxyl groups)
enzymes: T4 RNA ligase 2 adds 3' adaptor, T4 RNA ligase 1 adds 5' adaptor, RT, DNA pol to amplify, gel purify, illumina seq
89
miRNA biogenesis
pri-miRNA > micro-processor (has DROSHA, in nuc) > pre-miRNA (hairpins struct) > export via XPO5 > DICER cleavage > miRNA duplex > AGO recog > miRISC complex > miRISC recog mRNA > transport to P-body > CCR4-NOT mRNA degradtion (there is also translational repression)
miRNA starts in nuc, needs DROSHA e export via XPO5, siRNA is exogenous can start directly at DICER/AGO
90
RNAi components same / diff between siRNA e miRNA
common: DICER, AGO
diff: DROSHA (miR only)
siRNA starts at Dicer bc never enters nuc
91
what is meant by DNA rep being semi-conservative
post rep one strand is parent and one is daughter
92
leading strand pol
pol epsilon
use dNTP substrates
Cannot synthesize DNA de novo, *extend* primers in 5’ --> 3’ direction by incorporating dNMP’s
DNA is also a substrate
template-directed synthesis
primer 3’-end e template are critical elements of polymerase active site
not that processive, need enzymes to inc DNA binding
(e coli has one pol (pol III) for both leading and lagging)
can move through nucleomes, pol delta cannot
93
lagging strand pol
pol delta
very similar to pol epsilon
stopped by nucleosome, nucleosome sets size of lagging strand
coordinates w FEN1 for OF maturation
(e coli has one pol (pol III) for both leading and lagging)
94
levels of DNA pol fidelity
nt selectivity (DNA pol, 1:1000 mistakes)
exonucleolytic proofreading (DNA pol, 1:million mistakes)
mismatch repair ( MMR enzymes, 1:billion mistakes)
if add incorrect nt, addition of NEXT nt is also less efficient > more time to proofread / correct
95
how is dNMP inserted by DNA pol
DNA and dNTPs are substrates
dNTP bps w 1st unpaired template base
Divalent metals required (Mg2+), as well as carboxylic acid (glutamic or asp) to help hold dNTP in correct position and balance charges of the Mg2+
Primer 3’OH attacks alpha-phosphoryl group
dNMP is incorporated into DNA
Pyrophosphate is released (PP, comes from the dNTP)
DNA template is an integral part of active site – allows DNA pol to distinguish between 4 different dNTP substrates
rxn is SN2 like / tetrahedral like, orbitals need to be co-linear, will not work of 3'OH is not lined up
96
how is nt fidelity maintained during insertion
dep on the active site pocket, the correct bps have same geometry / shape, DNA pol (right hand shape) will not be able to close properly if wrong shape
note: some selectivity from H bonds (C:G will have 3 H bonds and A:T will have 2) but majority of selectivity is from the shape
if add incorrect nt, addition of NEXT nt is also less efficient > more time to proofread / correct
97
exonuclease proofreading
3' to 5', removes incorrect (and correct) nts
exonuc site sep from pol site (same enzyme in eukaryotes)
needs to melt DNA to remove wrong nts, easier to melt wrong than right nts
kinetics: if right, addition of next nt will be fast and exonuclease melting will be slow (sep and cumulative)
if wrong, addition of next nt will be slow and exonuclease melting will be fast (sep and cumulative)
speed of adding next nt more imp than speed of exonuclease activity
98
frameshift errors during DNA rep
Due to primer/template slippage or misalignment
Can result in insertions or deletions
Most often occur in homopolymeric runs and repetitive sequences
insertion / deletion happens when nt is added extrahelical, daughter strand has nt bulging from normal helix, during NEXT round of rep, indel will occur (need MMR or proofreading)
99
e coli polymerases: pol I v pol III
pol III is replicative pol (leading and lagging)
pol I for okazaki frag maturation, removes RNA primer and puts back DNA
100
major diff in e coli v eukaryote replication fork
primase only on lagging not leading, uses one pol for both lagging e leading, pol also has 3'>5' exonuc activity e flap exonuclease
okazaki frags are longer,
helicase (DnaB) acts as 'glue', everything tied to helicase via clamp loader, key to replisome; helicase moves 5' to 3',
clamp loader is dimer (eukaryote is trimer)
SSB is more 'spool' than 'caterpillar', only on lagging strand
primase/pol alpha is one enzyme
topoisomerase is type II, makes ds break bc post rep the 2 loops are intertwined
101
mutator polymerase generation / experiment
mut conserved Met in motif A (SLYPS), pol will make mistakes in a pattern, can cause transversion of A to T
mutations are strand spec, about 40x more likely to make TT than AA > use seq w A on top strand (5'>3') and T on bottom strand (3' > 5') > able to tell where pol is, if see TT transversion can assume it came from top strand
102
transversion v transition
transversion replaces a purine (AG) with a pyrimidine (CT) or pyrimidine with a purine
transition replaces a pyrimidine with pyrimidine or purine with purine, doesn't change size as much
103
URA3 assay
URA3 encodes enzyme to make uridine (UMP)
FOA (5-fluoroorotic acid) is converted to the toxic compound 5-fluorouracil (5- FU) by URA3
5-FU covalently modifies and inactivates thymidylate synthase which converts dUMP to dTMP
URA3+ cells are killed by FOA but URA3- cells are resistant
exp: screen for resistant mutations in URA3 and seq
make yeast that contain wt or transversion mut pol e URA3 gene in diff locations / orientations rel to ARS (ori for yeast) > pol epsilon mut makes TT mispairs on the strand it is copying > *discovery that epsilon syn DNA on leading strand*
104
RNA/DNA primer syn pol
primase / pol alpha
primase - DdRp, makes RNA primer from DNA, low processivity
pol alpha - extends RNA primer to make DNA primer (DNA pol alpha lacks 3' to 5' exonucleolytic proofreading, don't want to extend too long)
results in RNA/DNA hybrid primer for pol delta to extend
need FEN1 to replace RNA primer w DNA
105
fork protection proteins
AND-1 (mediates interaction between primase e CMG)
CLASPIN
TIM-TIPIN
106
Helicase activators
CDC45
GINS
107
helicase motor
MCM2-7
binds ssDNA, high processivity, moves 3' to 5' (in eukaryotes, all helicases are unidirectional)
uses a lot of ATP to drive translocation: ATP binds in loops between the 6 pt star, as ATP is hydrolyzed the loop moves up, repeat, 'walks' over the DNA, 1-2ATP per nt
displace complementary strand, proteins, 2' structures when translocating
108
exp to determine directionality of helicase
make artificial fork > add biotin bead on one side of DNA > see if DNA can be unwound or not
see which arm is needed: remove one arm, biotin other side, if can funct w/o arm on that side must use other side
109
holohelicase
CDC45, MCM2-7, GINS (CMG)
CMG is the active helicase, MCM2-7 is only the motor
forms holoenzyme w pol epsilon on leading strand
makes key contacts in the replisome
110
exp showing CMG is funct helicase
gel filtration e elution off column showing Cdc45 is forming complex w Mcm2-7 e GINS
purify the complex > use plasmid w primer w artificial fork > run on gel, small fragments where primer unwound from plasmid > fractions taht have CMG have helicase activity
111
clamp loader
RFC
has 5 finger-like subunits that interact between the 6 pt star of clamp, DNA sits in 'palm'
needs to load a clamp for every OF
req ATP to hydrolyze
112
sliding clamp
PCNA
inc processivity by encircling e binding DNA pol, tethering the pol to the DNA
makes 6-sided star shape, has IDCL (interdomain connecting loops) between the pts
long half life, req loader to put on bc so stable, e needs to be unloaded to be recycled
needs to be loaded at ea primer for ea OF
interacts w many proteins (~200)
may angle DNA to prevent it from sliding through?
113
exp to discover mech of sliding clamp in e coli
see when clamp is eluted from size exclusion model (elute earlier if in complex bc will be bigger)
clamp + loader + plasmid > clamp elute w plasmid
clamp + loader + plasmid + SmaI (linearizes plasmid) > clamp elute later, fell off of plasmid
clamp + loader + linear DNA w SSB on ends > elute w plasmid
where sliding clamp name comes from
114
DNA pol processivity
number of nts added per DNA binding event
DNA pol has weak binding > low processivity
115
alt clamp loader
CFT18-RFC
116
SSB
RPA
ssDNA binding proteins
inc the fidelity of DNA rep e repair by preventing re-annealing, 2' struct formation, e protect ssDNA from damage
high affinity for DNA AND highly dynamic
can move / slide on DNA
caterpillar / popcorn activity on DNA, has 4 domains that bind strong but each can pop on and off, allow it to 'inch' over the DNA (no directionality) > strong bc 4 domains, but ea domain comes on and off
does not block access to proteins from accessing DNA, can help recruit / interact w enzymes that act on DNA
SSB is e coli name
117
fork protection complex
CLASPIN, TIM-TIPIN, AND-1
118
okazaki fragments
~200bp in eukaryotes
main pathway: FEN1 for maturation, coordinates w pol delta to mature OFs, pol delta pushes 1-2 nt of RNA primer out, FEN1 cleaves the RNA, pol delta displaces more, FEN1 cleaves, repeat > mature OF
backup pathway: RNaseH2 can funct at early stages (before flap) but cannot remove last RMP, works if flap is too long (FEN1 cannot),
DNA2 can work if flap is bound to RPA, backup for uncontrolled flap
ligase can seal nicks (ligase cannot seal RNA:DNA nick, only DNA:DNA)
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ligase
joins 3'OH and 5' phosphate at nicks between OFs
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DNA rep inhibitor drugs
ara C - chemotherapy, leukemia
acyclovir - antiviral, herpes
AZT - antiviral, HIV
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Ctf4
glue that sticks helicase to primase, trimer that binds GINS and pol alpha (possibly 2 MCMs)
glues together the two replisomes at the fork
imp for protein:protein interactions
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topoisomerase
relax pos supercoils ahead of fork formed by unwinding
untangle DNA behind the fork (remember chr closed at ends)
type I - cut one strand, passes cut strand over other and then re-seals > undo 1 turn, more common ahead of fork (e during transcription)
type II - cut both strands, cross over > unwinds 2 turns at a time, more common behind fork, unwinds daughter from parent strand
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FEN1 v RNaseH2 v DNA2
FEN1 is major pathway, coordinates w pol delta to mature OFs, pol delta pushes 1-2 nt of RNA primer out, FEN1 cleaves the RNA, pol delta displaces more, FEN1 cleaves, repeat > mature OF
RNaseH2 can work before flap but cannot remove last RMP
DNA2 can cleave uncontrolled flaps that have bound RPA, make flap manageable for FEN1
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why does closed circular plasmids run further on EtBr gels
able to supercoil
nicked will run slower, even if same length
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movement of rep fork during DNA rep
the fork stays stationary and the DNA moves away from the replisomes, DNA feeds in and out
(DNA moves into replication factories)
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All components of rep fork
pol epsilon
pol delta
primase
helicase (CMG complex)
PCNA (sliding clamp)
RFC (clamp loader)
RPA (ssDNA binding protein)
Fen1 / RNaseH
nucleosome
Okazaki frag/termini
Ctf4
Topoisomerase
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nucleosome affect on DNA topography
causes -1 underwinding
ea neg turn opens about 10bp, helps transcription bc easier to open up
ability to remove nucleosomes during rep is enhanced by supercoiling
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components needed to orchestrate nucleosome assembly during DNA rep
histone chaperones needed
H2AH2B come off as dimer, H3H4 as tetramer
pol epsilon subunits direct (H3H4)2 to leading strand
MCM2 directs (H3H4)2 to lagging strand
random distribution between parent e daughter strands (keeps parental histone marks)
CAF-1 important for assembly of new nucleosomes
new nucleosomes have unique mark, readers/writers make mark to match old nucleosomes
methylation also maintained (DNMT1)
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exp for correlation between nucleosomes and OF length
deplete ligase (CDC9) > look at banding pattern
inhibiting ligase causes same size of DNA to be made as the length between nucleosomes
longer OF made in nucleosome-depleted regions
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clamp unloader
Elg1-RFC
can remove PCNA e sumolated PCNA
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telomerase
extends 3' ends of telomeres, uses RNA template to RT and make DNA
recog repetitive G rich seq at end of chr, can extend multiple times bc repetative
contains essential RNA (TER, Telomerase RNA) - temp for syn
contains RT protein (TERT, telomerase RT), catalyses syn
lagging strand machinery syn 5' strand after telomerase extends 3' ends (need OF maturation)
still has 3' overhand on end bc of RNA primer / pol cannot go to end > needs shelterin
shortening of telomeres > senescence
immortalized cells have re-activated telomerase
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shelterin
3 main functs: (1) protein chr ends from degradation, (2) distinguish chr ends from DNA damage, (3) reg telomere e 3' ssDNA overhang length
has components that bind dsDNA, binds ssDNA, reg telomere length / interact w telomerase, blocks damage resp
