Module 4 Flashcards
transcription start site differnce btw prokaryotes and eukaryotes
p- 5’UTR=10-30 nt
e- 5’UTR= 100nt
rna poly differnce btw pro/eur
p- one rna poly
- sigma factor allows for
- specificity of promtor
- RNA holoenzyme- 5 subunits + sigmafactor
- dna entry, exit and ddntp entry channel
dna unwind bubble is 17nt long and the rna-dna is 8-10nt
e- 3 rna poly
poly1 -rRNA most abundant 80%
poly 2- mRNA
- can be blocked by alpha-aminitin
RPB1 is largest subunit
30s is small subunit
50s is large subunit
-TATA
poly 3- tRNA
- TATA
even if they dont have TATA box they still will have TATA binding protein complex
Proofreading of bacterial RNA polymerase
kinectic proofreading
- creates a stall in RNA poly and uses pyrophosphoratlysis involve PPi
nucleophilic proofreading
- if not stalled it will fray and then backtraking and rna and dna will fray into the rNTP channel of RNA poly
- rna poly intrinsic nuclease will remove fray by H20
Prokaryottic promotor
transcriptional start site= downstream of promototr
-35 to -10 where sigma factor binds
-60 to -40 interact with alpja subunit of RNA poly and it AT rich
eukaryotic promotor
binding sites can be upstream or downstream
transwcription factors bind promoter region (+1)
what binds to the minor groove
tata binding protein
σ70 close and open complex
closed- n-terminus of sigma factor blocks dna entry
open- sigma factor unblocks site and bubble opens
what is the prokaryotic sigma factor that is for house keeping genes
σ70
what is abortive inititian
prokaryotes must have built 8-10 nt before it is stable
- or the RNA can dissociate from the DNA
using transcription factors for nuclear reprogramming
- induced pluripotency
- irect reprogramming
what is induced pluripotency
when a differnted stem cell is pushed back up the moutain to become a stem cell again
- so that it can rediffrentiate
what is direct reprogramming
changing the transcription factors to change a differnted cell to another differnented cell
intitation of transcription in prokaryotes
shine dalgarno sequence
- purine rich
- the 302 subunit has a 16s subunit that reconizes a shine dalgarno sequence
what is EMSA (electrophorectic mobility shift assay)
asseccing DNA binding to protein
- higher weight will go to top
- on native non-denaturing gel
initatiation of transcription in eukaryotes
kozak sequence
is 2-3 purine rich before the aug and G after the aug
interact with anticodon arm of Met-tRNAiMet
DNA footprinting steps
- amplify dna from one end radiolabeled (PCR)
- cleave DNA in the presence or absense of a dna binding protein
- one break per strand - separate strand by gel electrophorisis and then the spots that come empty mean that a dna binding protein was their
- products close to primer will fall first
DNA foot printing vs EMSA
dna footprinting tells where the dna is bound emsa tell if the dna binds
processing of pre-mRNA
- 5’ cap
-3’ tail - splicing
5’ capping methylated
that it prevents exoribonuclease activity from attacking the mRNA
- allow for circulation of mRNA- by elf4f
-enhance stability
- mediate binding of mRNA to the ribosome
- happens during transcription afte 20-30nt added
3’ poly a tail
prevent exoribonuxlease activity
- allow for circulation of mRNA- by elf4f
-added when their is a polyadenalation signal and gt rich signal
- recruite endonuclease to cut btw the two signals then PAP and PABP add A residues
splicing
allow for variation between genes but not chnage exons around just inclusion and exclusion
prokaryotes processing of mRNA
transcription and translation happen simultationously
what is not spliced out during splicing of premrna
5’utr and 3’utr
processing mRNA on quatifying gene expression
on RT and qPCR and RNA-seq
- can allow for more precess process since the exons are together can allow for more specific primers
on qpcr primer
- primer to span exon-exon so that it can exclude genomic dna and only inculde cdna
genetic code degenerate
61 amino acids codons with 20 aa
3 stop codons
robust genectic code allows
to absorb mutation= snps
Rules of the genetic code
non overlapping - tested by subsitution and it chnaged only one amino acid
- if not would effect 3 aa
read in triplets and no pauses- tested by insertion and deletion
linear
linear and c terminal- cterminal end was eaten first
3h and 14c were not linear
code mutations
- translation mutations
- pur -> pur or pyr->pyr - silent mutations
- change nt and but no aa change - missense mutations
- chnage in aa which chnages the aa can be deterious or non deterious - nonsense mutations
-codes for termination, causes non-funtionl/degragation of protein within first round of translation - non stop mutations
- remove termination
Nonsense mediated mRNA decay (NMD)
- detection of exon junction complex can tell it stoped premature
- decaping
- prone to exoribonuclease
- if pass this phase it can still be detected in final exon and trigger NMD at the 3’UTR
non stop mrnas detection
rna poly will read the poly a tail createing many lys= this triggers 3’-5’ exoribomnuclease to degrade the protein and the transcript
trna - features
small non-coding rna
cca amino acid arm and anticodon
what direction is the anticodon
5’-3’
their is ___ aminoacyl-tRNA synthetase for each amino acid
one
- 20
tRNA language
1. charged
2. uncharged
3. prokaryote met
4. prokaryote intitaition met
5. eukaryote met
6. eukaryote initiation met
- leu-tRNA^leu
- tRNA^leu
- tRNA^met
- tRNA^fmet
- tRNA^met
- tRNAi^met
fidility of charging is govered by
amino acyl-tRNA synthetase not the ribosome
what governs the fidility of codon anticodon parirting
the ribosome decoding center
subunits of prokaryotic ribosome and eukaryotiv ribosome
30s and 50s
making up the 70 s ribosome
40s and 60s
making up the 80s ribosome
wooble hypothesis rules
- codon 5’-3’ first wo bases must watson and crick
- anticodon 5’-3’ if C or A has one codon
- anticodon 5’-3’ if G or U has two codons
- I can be found on anticodon and it can wooble with C, A, U three codons
The ribosome
peptidyl transferase center and decoding center - 60% is rRNA and 40% is r protein
A site- aminoacyl tRNA entry on 3’ of mRNA
P site- growing peptide - transfer p sit to a site
E site- exit site for uncharged tRNA
prokaryotic intition
IF-1 blocks A site and ensure alignment of tRNA^fmet
IF3 stablize 30 s
IF2 is a chaperon which moves tRNA^fMet to the psite
what is the role of transformylase
to add formyl group to methonine preventing met from preforming in eleongation in a two step process(block N terminus)
what is polyscitronic mRNA
- it is in prokaryotes and a shine dalgarno seqeucne can be used mutiple times depending on the reading frame
- one transcript, multiple proteins
what is considered a poycistronic mRNA
prokaryotoes with overlapping genes
- shine-dalgarno sequnce can have overlapping start and stop to shift the reading frame
Fact: prokaryotes can also have non-overlapping so multiple SDS
eukaryotes have non-overlapping genes what does this mean
one mRNA will only have one protein
prokaryotic initition complex
tRNA^fmet with large and small ribosomal subunit and the intion factors dissociate uses GTP
eukaryotic intition factors
circulzation of mRNA by the 3’ poly a tail and the 5’ capping that will interact with elf4f
- multiple translation events on the same mrna
- polysome
what is a polysome
1 mrna multiple transcribing ribosomes
steps of elongation
- aminoacycl trna sythetase charges trna and EF-Tu brings into A site
- correct pairing causes ribosome A site (A1493-2/3 adensosine) to flip out and pair
- cause conformational change of 30s subunit opening the 16s subunit
- EF-Tu-GTP is hydrolyxed into EF-Tu-GDP + pi to provide enrgy for accomodation of tRNA
- if this was incorrect it will dissocaite
pt 2
- aa nucleophilic attact on fMEt (catalyzed by peptidyl transferase)
- tRNA to E site and growing chain onto A site then Asite opens up - translocation
what is EF-Tu
brings the charged trna to the a site for eleongation
what are aminoglycosides
they cause the flip out of the ribosome and reduce transitional fidelity
- kill bacteria through incorrect translation fidelity
- does not effect eukaryotes
termination
class 1
and class 2
class1: RF-1 and RF-2
reconzie stop and goes into A site mimic tRNA
class2: RF-3
removes RF1 and 2 by hydrolying to remove RF-3
lac operen rule
the lac operon is on when lactose is opresent and glucose is absent
- negative regulation
it is polyscietrnic mRNA
has a repressor (lac I) and operator (lac o)
- both have promotors
laz z - b galactoside to metabolize lactose
lac y- gaactside permease for lettting lactose into cell
lac a- modifies toxic galactosides
Lac repressor
seprate promoto and transcribed independently and consitituly
- binding of 2-3 operator sites tieing dna in a loop- on the major groove
- binds inverted repeats - since it is a heterodimer
what is the role of allolactose
is their when th lac operon is off but leaky and is broken down by lac z
- is a allosteric effector of the repressor and chnages its affintity to bind dna
- turns on the lac operon
The role of effectors in negative regulation
activation- bind to repressor to remove it from dna (on)
inhibition- bind to repressor to add the repressor (off)
The role of effectors in positive regulation
activation- bind to activator increase transcription ()
inhibition- bind to activator decrease transcription ()
positive regulation of the lac operon- catabolic repression
metabolism of lactose is blocked in the presence of glucose
what is the role of an activator
-they increase transcription dial up or down
differnce between negative and positive reghulation of the lac operon
negative regulation is dealing with on and off with the repressor
postitive is high or low and deals with the activator
CRP is a activator that works in the resence of cAMP which is a effector
when glucose is low cAMP is high (increase production, decrease export)
- increasing cAMP allows binding of CRP with increase lac operon expression
CRP structure
homodimer with cAMP as an effector
merodiploid cell
partially dipliod, haploid genome (lac operon) into a plasmid with an exisiting lacoperon
lac I properties
- repressor
- can be rescued with a working repressor lac operon
- if broken lac operon is always on
lac o - operator
- cant be rescued if broken the lac operon is always on
activator
in positive regulaton
-bind to dna and effect rna poly to turn on lac operon
-cAMP is its effector
effector
bind to repressor or activator to chnage its affinity for rna polymerase
repressor
in negative regulation
- bind to dna and effect rna poly to turn off lac operon
-allolactose is its effector
housekeeping genes
always being expressed since they are essential like the lac repressor
co-activator/co-repressor
they act as a bridge between activator and the rna poly
- co-activator with induce bindingn
-co repressor prevent binding
post transcriptional gene silencing (PTGS)
by mirna and sirna
- they will degrade mRNA after its been transcriped
how does mirna work
mirna is made with many self complementary parks and then exonucleases to cute the tail then exported out of the nucleus
- then they are recuirted by RISC and AGO and then a strand is descarded
- bind to mrna and is degraded since low complemaerity by AGO
how does sirna work
in the cytoplasm is is from virus, recruited by RISC and AGO and then a strnad is removed
- mrna is is cleaved when is bound
what is RISC
rna-induced silencing complec that cleaves dna that is complementart to the the match the rn its bound to
mirna and sirna bind? in pro and euk
p- the coding region
e- the 3’utr
reporter plasmid
- included a portion if mirna to see if its expressed
- the gene mrna will decrase glowing if more mirna is bound
more mirna= decrease in translation and increase in degragation of mrna
role of non-coding rna in mangment of mirna
such as incrna - they can compete for binding with mrna and then so that expression of mrna can happen
- remove saturation of mirna by having the 3’utr binding site
how to increase and decrease tanslation with mirna
increase Trans= decrase mirna
decrase trans= increase non-coding rna
