exam 2 (ch 14,15) Flashcards
site specific recombination
- limited to specific sequences
- recombinase enzyme cuts them
- 20-200 bp recognition site (inverted repeats, unique core sequence, which are directional)
transposon vs retrotransposons
transposons: cut and paste ; replicate DNA
replicative: movement with duplication
cut and paste: movement without duplication
Retrotransposons: RNA intermediate
what determines the outcome of site recombination?
the orientation of unique core sequence
opposite vs same orientation of site specific recomb
opposite: inversion (flip; messes up the junction; can use this to turn genes on/off no matter how far away as long as can form the hairpin structure at some point)
same: deletion or insertion (usefull to remove or add a gene)
loxP
- recognition site
- location of crossover for P1 element
- 34 bp sequence made of 2 palindromic recognition sites separated by 8bp spacer that gives directionality to the sequence
recombinases
- act like restriction enzymes + DNA ligase (cut and rejoin)
- do not require ATP because phosphodiester bond energy is conserved in a DNA protein bond
- Tyr active site: cut one pair of strands at a time which produce HOlliday intermediate
- Ser active site: cut both pairs at same time, so no Holliday intermediate, but same overall result
phase shift
- to evade immune system
- Hin recombinase inverts promoter segment approx every 1000 generations
- in one orientation: FljB flagellin
- in other orientation FljC flagellin
Cre
Causes recombination –> cyclisation recombinase
only recombine 2 lox sites if they have the same spacer sequence
creates site-specific recomb
cells use for flagella
what happens when both loxP are oriented in opposite orientation?
recombination results in gene inversion
reversible
continual flipping means it’s not very useful for genetic manipulation
how do transposons vary in complexity?
1. insertion sequence: don’t carry foreign genes; inverted and transposase repeats
2. composite transposons (Tn5): can carry antibiotic resistance
3. complex transposons (Mu): can carry antibiotic resistant genes
what percentage of DNA is transposons?
50%
why are transposons referred to as selfish DNA?
can exist without any benefit to the host cell, very difficult to eliminate since they can replicate
control of tranposition
- excessive transposition can kill host
- production of inhibitor protein or short inhibitory RNA (RNAi) which accumulates and stops further transposition
what would happen if transposon copy number dropped?
inhibitor level falls –> limited (nonlethal) transposition until inhibitor levels increase
retrotransposons definition and examples
transpose via RNA intermediate copied into DNA by RT
human long interspersed elements (LINES): 1-6.5kb
human short intersperse elements (SINES): 150-500 bp
human LTR retrotransposons: 6-9 kb
LTR retrotransposons
- form virus-like-particles (VLP) which cannot leave the cell
- 8% of genome
- (like retro virus but never leave the cell)
- do NOT encode ENV envelope protein
what do retroviral infection require?
2 receptors: CD4, chemokine
retroviral infection examples
HIV, Covid, mouse sarcoma virus
what do some people with altered chemokine receptors have?
partial resistance to HIV
challenges in retroviral infection
1) promoter region is NOT transcribed into mRNA by RNA pol2 (when you transcribe a gene, dont transcribe the promoter)
2) one mRNA –> 3 types of proteins
pseudoknot controls reading frames, which produce RT
lower amounts of RT are needed than coat protein
retroviral infection proteins
gag: virion proteins
pol: reverse transcriptase, integrase
env: envelope proteins
Gag and pol region of mRNA removed by alt splicing to produce envelope proteins
insert genome longer than virus
RT error facts
- slow and error prone
- 4 hrs to copy 9 kb genome (0.6 bp/sec)
- 1 error per 10^4 -10^6 bases
- up to 1 mistake every time genome is copied
- DNA pol is about 100x better (error rate of 10^6 - 10^8)
speed of:
human DNA pol
E. coli DNA pol 3
RT
DNA pol: 50 bp/sec
DNA pol 3: 1000 bp/sec
RT: 0.6 bp/ sec (4 hours to copy the 9kb genome)
retroviral vectors
can infect but cant form viable virus particules
Rev transcriptase vs protease inhibitors
Rev: AZT; good target for retrovirus
Protease: Paxlovid; want it early when virus is still developing before it’s too late
How many types of Ab can we make with different binding specificities?
10^7
what is recombination mediated by?
RSS (recombination signal sequences)
mechanisms appears to have evolved from ancient transposon
terminal deoxynucleotide transferase adds a few RANDOM nucleotides to expose ends –> even more variations and diversity
why are only rearranged genes expressed?
- each region has a viable promoter (cant be expressed until recombined)
- requires an enhancer present in an intron near the constant region
- only after somatic recomb is the promoter close enough to enhancer to function
promoter far away- only when you recombine them get closer and ready to go
allelic exclusion
successful rearrangement that once it works it stops
dna vs rna pol difference
DNA pol: requires primer
RNA pol: doesnt
why does RNA use U and not T?
very hard to make thymine
DNA uses C to amminate to U, so wouldn’t be able to tell which bases are wrong when make mRNA
for mRNA encoding a protein, transcription ……….
never begins at start codon
extra RNA sequences are required upstream of start codon to:
5’ Untranslated Regions UTRs to:
1. load ribosomes
2. regulate translation efficiency
3. regulate RNA stability
transcription occurs on ……. strand of DNA
can occur on either strand of DNA as long as it goes 5’ –> 3’
how much DNA is unwound in RNAP
15 bp
RNA pol
- requires Mg2+ (active site, neutralizes - charge from phosphates)
- requires NTP
- bacteria have 1
- euk have 3
what are the types of RNA polymerases that eukaryotes have?
pol1: rRNA (RPC5/ RPC9, RPA1, RPA2, RPB6)
pol2: mRNA and microRNA (RPB3/RPB11, RPB1, RPB2, RPB6)
pol3: tRNA, 5S, rRNA (RPC5/RPC9, RPC1, RPC2, RPB6)
in what phage does RNA pol consist of only 1 polypeptie?
T7 and Sp6 RNA pol
Actinomycin D
inhibits RNA and DNA synthesis
a-amanitin
selectively inhibits RNA pol2
Bacterial RNA pol
4 core subunits (basic machine to make mRNA)
sigma factor (where to start and which direction to go)
Bacterial promoter
- closed complex
- -10 binding/specificity and initiation (those with -35 are generally weaker)
- some very strong promoters have “UP-element”: -60 to -40 region upstream and bidns RNAP a SU
what determines basal transcription rate?
divergence from consensus
RNA pol proofreading
pauses when mismatched bases- allows time to correct mistakes
RNA pol error rate: 10^-4 to 10^-5
DNA pol error rate: 10^-6 to 10^-8
proofreading during elongation phase
RNAP stalls at misincoporated base
pyrophosphorolysis- pyrophosphatase decreases chances RNAP stalls at misincorporated base (reverses)
hydrolytic editing- RNAP stalls and backsup, melting several DNA:RNA bp, intrinsic nuclease activity of RNAP promotes base hydrolysis
rho-independent termination
RNA pol pauses just after dyad hairpin RNA is synthesized (because the only things holding it together are the AU bp, which are weak, so do the hairpin)
UA bp after hairpin are weaker, so RNA is released
rho-dependent termination
- rut sites: CA-rich ss regions
- rho: DNA helicase that uses ATP to move along DNA
- rho extracts mRNA from pol: pushes pol forward and RNA gets released, pulls RNA out of the polymerase (extracts it), and cauess conformational change in the polymerase
what polymerazes require TBP?
ALL POL!!!! even tho euk dont have TATA box
RNA Pol1
- makes RNA
- precursor for 18S, 23S, and 5.8S rRNA
- SL1 contains TBP
Pol 3
- promoter elements are downstream from start site
- tRNA, 5SRNA, small rRNA
Pol2
makes mRNA and microRNAs
transcription at pol2A promoters
- TF2D: comes in with TBP, binds and bends the DNA, summoning everyone else
- A, B
- 2F + RNA pol2A
- 2E
- 2H
elongate vs initiate transcription???
elongate: phosphorylate (cant elongate until phosphorylated)
initiate: HEB comes off once initiation complex has been made
what does TBP do?
creates 80 degree bend in DNA (creates a platform for assembly of a pre-initiation complex)
CTD
- long tail in RNA2
- yeast has 26 copies; humans 52 (bacteria none)
- progressively modified during transcription
how does CTD serve as a scaffold for RNA processing?
- CTD modifications recruit appropriate enzymes at proper time
- Ser5-P early modification (binding to elongation) –> promoter clearance and recruitment of capping enzymes
- Ser2-P late modificaiotn (termination) –> recruits factors for termination and adding polyA tail
mediator complex
- general tf + enhancer binding activators are NOT enough for activated transcription
- intermediary between DNA binding tf and RNA pol
- regulates chromatin structure, RNA processing, and DNA repair
what must happen to mRNA in nucleus before translation can begin?
5’ cap, poly A tail, remove introns, transport into cytoplasm
what must happen for CTD to elongate?
phosphorylation of CTD during intitiation
what mRNAs dont have polyA tails?
mRNAs for histones
their 3’ end formation is different
what does guanylyltransferase do?
capping enzyme bound to CTD near mRNA exit channel
caps mRNA as it emerges from pol2
mRNA half life ranges
minutes to years
T or F: bacteria have introns
true
but they are rare and have a very short 1/2 life because mRNA is quickly degraded
7methyl G gap
- GTP attached “backwards” in a 5’-5’ linkage by capping enzyme Guanylyltransferase
- terminal G is then methylated using S-adenosylmethionine
- protects 5’ end from nucleases, cap-binding complex (CBC) recruits capped mRNAs to the ribosome for translation
what mRNAs have 7m G cap?
all eukaryotic mRNAs
7m G capping and termination
- capping enzyme is bound to CTD near mRNA exit channel
- mRNA gets capped as it emerges from pol2
- cap enzyme dissociates, CBC binds cap and also to CTD
- Ser2 phosphorylation
- pol2 transcribes polyA site AAUAAA
- polyA pol adds 80-250 A residues at 3’ end
- PAF bind polA signal, which inititates mRNA cleavage (but RNA pol2 continues)
-
Xrn2 nuclease binds 5’ end of continuing transcript (because end of transcript is no longer protected) and (moves 5’ to 3’) catches up to pol and terminates transcription (torpedo model) (pol is torpedoes by Xrn2, which chews up mRNA)
could be 1000s of bases downstream
mRNA is cut 10-30 ntds after AAUAAA
mRNA remains bound to CTD until ……
after pol2 transcribes the polyA addition site (AAUAAA)
how many introns does the average animal mRNA have?
8 and is 5-10x longer than coding sequence
but can have 20-30
how do we know which AAUAAA to cleave?
changing phosphorylation status
mRNA splicing statistics
adds important layers to gene expression
90% of human genes undergo alternate splicing
50% of human diseases are linked to splicing defects
how can splicing be regulated? example?
some alternate splice products are inhibitors of the active product since they have similar structures
ex: transposase and transpose are in inhibitors in drosophila
what is DMD?
lethal disorder in 1/5000 boys due to lack of dystrophin, which stabilizes muscle fibers and repairs microtears in muscles
x-linked
weakend muscles
Duchenne muscular dystrophy
what can be done about DMD?
Viltolarsen: (RNA analog) synthetic RNA that binds to exon53, causing it to be skipped
results in truncated but functional dystrophin protein
intron removal
- branch-point 2’-OH attacks 5’ splice site
- 5’ splice site attacks 3’ splice site
- intron is released from spliced mRNA as a lariat
- U5 holds exon1 and exon2 close togehter
- U2/6 catalyze nucleophilic attack of the 2’-OH branch point adenosine on 5’ splice site
- U2/6/5 catalyze joining of 5’ and 3’ splice site (5’ attacks 3’)
- lariat degraded
little homology between introns except
GU @ 5’ splice site
AG @ 3’ splice site
what controls mRNA splicing?
snRNPs
mRNA splicing steps
- U1 binds to 5’ and U2 binds to the branch point
- U4,6,5 bind and make loop
- 5’ end of intron cut and connected to A site (lariat)
- U1 displaced and U4 dissociates
- U5 binds end AG of exon1: A=U, G=U (wobble pairing); cut 3’
- U6 brings pieces together (??5’ holds exons togeth???)
- 5’ splice site and branch point brought closer together by U2/6 RNA:RNA interactions
which U identifies splice site?
U1
what marks spliced mRNA?
exon junction complexes
MAGOH, Y14, elF4AIII, MLN51
U6
- brings everyone together to do chemistry
- U4 is its regulator
how is mRNA exported to cytoplasm?
through nuclear pores
exported via TREX-2 complex
1. associates with CTD
2. binds 5’ cap of nascent mRNA via CBC
3. 5’ –> 3’ mRNA export via nuclear pore complex
most noncoding RNA is transported through nuclear pores via …..
importins and exportins
powered by GTP hydrolysis
(Ran protein + GTP)
nuclear pre-mRNA splicing
very common, used for most euk genes
2 transesterificaiton reactions; branch site A
major and minor spliceosomes
Group 2 introns snRPS
rare; some euk from organelles, prokaryotes
structure looks like SNRPs
mechanism is same as pre-mRNA (2 transesterificaition reactions, branch site A)
RNA enzyme encoded by intron (ribozyme)
self-splicing
produce a lariat like snRNPs
Group 1 RNA splicing
rare; genes in nunclear rRNA in some euk, organelle , and a few prokaryotic
2 transesterification; branch site G
self-splicing introns require G but dont need energy
mRNA edit before translation
trypanosomes: extensive!!!! (African sleeping sickness)
mammals v rare!!!: by adenosine deaminase acting on RNA (ions on brain w ADAR)
cytosine deaminase (ApoB in cholesterol transport to block expression in intestine)
guide RNA changes it
RNA degradation in euk
- allows response to changing env conditions
- exosomes have 3’ to 5’ exonucleases that degrade mRNA
- P bodies in cytosol have exonuclease 3’ –>5’ that degrade mRNA
mRNA degradation steps
- polyA removal
- 5’ cap removal in P-bodi
how can RNA binding proteins regulate turnover of specific mRNA?
differentially stabilizing P body or ribosome binding
RNA blot
cant tell level of expression
approaches to approximate actual changes in transcription
- label nascent mRNA with a pulse of 4-thioU, biotinylate, and purify with streptavidin beads
- run-on transcription: blocks new transcription with detergent sarkosyl, allow pol that had started transcription before you added sarkosyl to continue with labeled NTP, affinity purify with Br-U Ab
transcription rate vs mRNA half life
high transcription rate = low mRNA half life (low stability)
can be altered by biological stimuli
transcription buffering
balance between mRNA synthesis and degradation can either:
1. work in opposite direction (ex: compensate for mutations in key components)
2. work in the same direction, higher expression/faster degradation or lower/lower
how fast does RNA pol move?
50-100????
how wide is RNA pol?
60 bp
