Lecture 15 Flashcards
regulation methods
-transcriptional cascade
-splice
-polyadenylation, mRNA cap and translation regulation
-RNAi and miRNA
what determines mammal gender
transcriptional cascade bc it runs off certain transcription factors
what supports the theory of regulation
different somatic cells have the same genomic code so they must be expressing different regions
where is the enhancer
before the core promoter
what tells the enhancer what genes to turn on
insulators bc they can go in either direction and where they are means transcription is going the opposite way
ex: if insulator is upstream we are going to express genes downstream
what can block promoter access
sequence between enhancer and promoter (insulator)
what do insulators do to DNA
put it in loops and the enhancer will only bind to transcription activator on the loop that is being activated
-the other loop is not being expressed
CTCF
protein that bind insulator to DNA
-causes the loop
chip assay
identify DNA base pair sites, extract DNA with proteins still attached and then use antibodies to get the transcription factors of interest, selectively purify antibody and it will take anything bound to it, then sequence and map genome
one transcription factor has
thousands of sites and sequences
male vs female determined by
SRY TF
-when it is present it causes male development
-you can have an XY without SRY which will lead to a female XY individual
FGF9
boy
-production is stimulated by SOX9 and when FGF9 is produced it will inhibit WNT4
WNT4
girl
-inhibits FGF9 and then the female cascade begins
-typically wins
FGF9 and WNT4
-protein hormones
SOX 9
promotes by SRY to make FGF9 and inhibit WNT4
no SRY
WNT4 will win bc there is no FGF9 to inhibit itself
what happens when we block SRY
we get an XY female bc we cant inhibit WNT4
block WNT4
XX male bc female cant win
steroid hormones
activate nuclear hormone receptor transcription factors
-testosterone and estradiol (ovaries)
-a recceptor in cytoplasm will change to let it into the nucleaus where the transcription factors are
sex hormone
ligand binding to the transcription factors (like CAP or LUX R)
is the eukaryotic coding region continuous?
no
-it has introns and exons
non coding regions
-introns
-removed with splice
-NOT IN BACTERIA
-not in mRNA
splicesome
catalyze splice
-small ribonucleoprotein particles (snRNPs)
-form the lariat loop
-DONT CUT just hold the lariat folded
A in the splice
makes 3 bonds bc RNA has a 2 prime hydroxyl this is why DNA cant do the splice
-forms the lariat
splice
the lariat is removed and degrades and the 3 prime (donor) and 5 prime (acceptor) ends connect to make a continuous coding sequence
self splicing
-can self fold without proteins and use ribozymes which are RNA molecules that act like enzymes
-cuts lariant out itself
life theory
begain with catayltic RNA
splicing different exons determines?
the gene we encode
-ALTERNATIVE SPLICING
-one gene can encode many proteins depending on what we express
isoform
splice variant
-lets us make antibodies and recognize infection
DSCAM
how neurons detect each other and synapse
DOWNS
extra DSCAm which leads to neuro issues
-too many synapses
-neurons bump into each other
no DSCAM
neurons self synapse
why is each neuron different?
bc they are isoforms and all express different isoforms
-it knows its own DSCAM so it can detect others
mutant slicing machinery
retain introns, convert exons to introns, delete the wrong exons
treating DMD
-splice nucleic acid drug that will block 51 so 47 shifts to 52 and keeps translating
-results in a dystrophin protein missing an amino acid but it is still a funcitonal protein
DMD
deletion of exons 48- 50 which causes a framshift putting 47 next to 52 this causes a stop resulting in a useless truncated protein
what happens to the ends of premRNA
they get modified by hydrolytic enzymes
-also helps ribosome attach at 5 prime end
-this helps export mRNA and protect it
5 prime end
gets a guanine methyl cap
3 prime end
gets a poly A tail when polyadenylation signal is reached
-the cleave is after AAUAAA
-cleaved by an enzyme
translation initiation factor 4 (ELF4)
binds at 5 prime cap and gets the ribosome to the mRNA
-makes a complex with poly A binding protein which will make a loop that attracts the small ribosomal subunit
what does a cell do when it is starving?
4E-BP1 blocks the elf 4 from binding to PABP so we dont do translation and conserve energy
what happens when starved cell receives nutrients again
4e-bp1 is phosphorylated and translation can happen
3 prime UTR
determines the life span of mRNA bc of the nucleotides here
-can be transferred
eukaryotic mRNA lifespan. longer or shorter than prokaryot?
longer
transfer of long life UTR
-can transfer to short life mRNA to extend their lives
antisense mRNA
prevent translation by sending RNA that is complementary to the sequence being expressed so the ribosome cant access that sequence
-doesnt always work
-doesnt affect DNA
RNAi
-rna interference
-injecting dsRNA which stops translation
-ex: c elegan eating transgenic bacteria with dsRNA
-siRNA in mammal
-regulate lifespan
dsRNA in mammals
-dsRNA short hairpins (siRNA)
-short 22 nucleotide hairpins knock out translation of the gene and leave the rest unaffected
-only in mammals
does RNAi increase or decrease mRNA lifespan
decrease
-promotes RNA degradation
evolution of RNA
many viruses are dsRNA so maybe organisms developed to shut down at dsRNA so they dont get infected
c elegans have
4 larval stages
-L1-4
retarded
2 L1 phases
-lin mutant
-has lin 14 in the 3 prime UTR
precocious
skip L1
-lin mutant
lin 14 lf
mutant causing precosious
-recessive
-in coding region
lin 14 dominant
retared
- in untranslated region
-lin 14/ clock stays elevated for two long so organism doesnt move on
-also happens when there is no lin 4 present
lin 4
causes retarded
-encodes RNA sequence that is complementary to lin 14 in UTR (dominant)
-micro RNA/ miRNA
-inhibit/ reduce lin 14 that cuases retardation
double mutants
lin 14 and lin 4
-precocious
-therefore lin 4 effect is acting through lin 14 therefore retarded growth has to have normal lin 14
lin 14 gene
encodes a protein
-loss of function mutations are in the coding regions
clock
lin 14
-protein decreases as animal develops
lin 4 level
increases as animal develops
lin 4 miRNA model
-lin 4 makes 70 nucleotide precurser in hairpin that will unwind and bind to lin 14 which stops the translation of the lin 14 protein
-this is the mechanism of the dsRNA shutting down translation
miRNA
-hairpins that get processed and create dsRNA
-dicer
-RISC
dicer enzymes
cut dsRNA into 22 nucleotide pieces
-dsRNA is from the miRNA
why does 22 nt work better in mammals
skipping the first step
-we dont need dicer to work
RISC
-22 nt bind to RISC complex
-takes on strand from dsRNA and then guides remaining strand to the ssRNA that we want to shut down
-cuts that mRNA shutting it down
-no protein made
RNA dependant RNA pol
amplifies response of RNAi
-dont attach to RISC instead RdRP extends as a primer which makes a longer dsRNA for dicer to cut
-destroys mRNA but leaves 22 nt that can create a feedback loop and further shut down protein production
-can be started by a parent, some of the nt can be passed down to the offspring and retain the ability to shut down this protein
how to pass on change without changing DNA
recycle ds mRNA fragments/ 22 nt to offspring
-from RdRP loop
